Alan R. Light

α2-Adrenergic receptors in human spinal cord : specific localized expression of mRNA encoding α2-adrenergic receptor subtypes at four distinct levels

Abstract α2-Adrenergic receptor (AR) subtype mRNA (α2a, α2b, α2c) neuronal localization in human spinal cord has not been described. We therefore performed in situ hybridization to identify cell bodies at four levels of human spinal cord (cervical, thoracic, lumbar, sacral) containing α2AR subtype specific mRNA. α2AR mRNA is present in gray matter only (ventral > dorsal; sacral > cervical > thoracic = lumbar). In addition to α2AR mRNA in cell bodies in thoracic and lumbar intermediolateral (sympathetic) and sacral intermediate (parasympathetic) cell columns (lamina VII), all levels in dorsal horn laminae I, II, V, and ventral horn lamina IX, we demonstrate α2AR mRNA in dorsal horn laminae III and IV, and dorsal nucleus of Clarke, where α2ARs have not been described. Previously unreported heterogeneity in α2AR subtype distribution (α2a and α2bAR mRNA present, α2cAR mRNA virtually absent) is found at all sites of α2AR mRNA expression in human spinal cord, including locations known to mediate effects of α2AR agonist drugs on nociception, autonomic function and motor tone. Cervical spinal cord demonstrates a predominance of α2a mRNA signal, while thoracic, lumbar, and sacral spinal cord demonstrate an increasing predominance of α2bAR mRNA. If confirmed at a protein level, these findings have profound implications for therapeutic strategies in managing human pain.

The effect of stimulus duration on noxious-stimulus induced c-fos expression in the rodent spinal cord

C-fos is a proto-oncogene that is expressed within some neurons following depolarization. The protein product, fos, has been proposed as an anatomical marker for neuronal activity following noxious peripheral stimulation. However, the literature on noxious-stimulus induced fos expression contains several puzzling observations on the time course and laminar distribution of neuronal labeling within the spinal cord. This study has analyzed the effect of stimulus duration on the expression of fos-like immunoreactivity (FLI) within the spinal cord of anesthetized rats. In order to examine the time course of fos expression following brief periods of stimulation, we required a type of stimulus that was intense enough to activate nociceptors but that did not produce tissue damage. We have therefore employed pulsed, high intensity electrical stimulation, with stimulus durations ranging from 3 s to 24 h. The results indicate that stimulus duration has a profound effect upon the number of labeled cells, the intensity of neuronal labeling, the laminar pattern of FLI, and the time course of fos expression. Brief stimulation periods induce relatively few and relatively lightly labeled neurons, located predominantly within the most superficial laminae of the dorsal horn. Maximal immunoreactivity appears approximately 2 h after stimulation has ceased, and disappears within hours. Continuous stimulation produces many more labeled cells, darker labeling, and FLI within both dorsal and ventral laminar regions. Maximal FLI is seen after approximately 4.5 h of continuous stimulation, with reduction in the number of labeled cells thereafter. These data indicate that the results of any study employing c-fos as a marker for neuronal activity may be affected by the duration of the exciting stimulus.

Microglial reactions after subcutaneous formalin injection into the rat hind paw

Microglia in primary afferent projection territories are activated and proliferate after peripheral nerve injury. However, it is not known whether stimulation of peripheral nerves by noxious stimuli applied to their receptive fields activates microglial cells in the spinal cord. This study was designed to investigate the response of microglia in the lumbar spinal cord and in the brainstem to a tonic noxious stimulus. Thirty-two male Sprague-Dawley rats received subcutaneous injections of 5% formalin (50 microliter) into the plantar surface of the right hind paw, and 24 rats were injected with 50 microliter saline as a control. The lumbar spinal cord and brainstem were evaluated for immunoreactivity (IR) to complement receptor C3bi (monoclonal antibody OX-42) and major histocompatibility complex class II (monoclonal antibody OX-6) on postinjection hours 0, 2, 4 and 8 and days 1, 3, 7, 14 and 28. A qualitative and quantitative increase of OX-42-IR microglial cells were observed in the medial portion of the dorsal horn and in the gracile nucleus of the brainstem on the side ipsilateral to the formalin injection, starting on days 1-3 and peaking on day 7 postinjection. OX-6-positive cells were scattered both in gray and white matter, but no difference was detected between the two sides of the spinal cord or between formalin-injected and control animals. This is the first study that reports that subcutaneous injection of formalin into the rats hind paw induces microglial activation in the spinal cord as well as in the brainstem. Although we have not determined whether these responses result from nociceptor activity, peripheral inflammation, or degeneration of primary afferents and/or central neurons, this method provides a simple, effective and stable animal model that will permit the future study of the mechanisms that contribute to microglial activation and its pathophysiological consequences.

Differential termination of large-diameter and small-diameter primary afferent fibers in the spinal dorsal gray matter as indicated by labeling with horseradish peroxidase

Abstract Primary afferent fibers, collaterals, and terminations in the spinal dorsal horn were labeled by the orthograde transport of horseradish peroxidase. Lesions of fine afferent fibers comprising the lateral division of dorsal rootlets resulted in the staining of almost no boutons in the marginal zone and substantia gelatinosa but of many boutons in the nucleus proprius. Lesions of large-diameter fibers comprising the medial division restricted staining to a very few boutons in the nucleus proprius while many were stained in the marginal zone and the substantia gelatinosa.

The ultrastructure of group Ia afferent fiber synapses in the lumbosacral spinal cord of the cat

Ia synapses in laminae VI and IX of the cats spinal cord were examined in the electron microscope following iontophoretic injection of horseradish peroxidase (HRP) into single, identified, Ia afferent fibers from gastrocnemius muscles. Ia boutons contacting motoneuron dendrites in lamina IX contained spherical synaptic vesicles and generally contacted only one postsynaptic profile. The Ia boutons were often postsynaptic to smaller P-type axonal terminals. Consequently Ia boutons may be classified as S-boutons with axo-axonic contacts.

Relationship between nociceptor activity, peripheral edema, spinal microglial activation and long-term hyperalgesia induced by formalin

To determine whether initial nociceptive inputs caused by subcutaneous injection of formalin into the hindpaw are necessary and/or sufficient for allodynic behavior and microglial activation observed at one week following behavior, we examined Sprague-Dawley rats under five test conditions. Test condition 1. Formalin alone group (six rats), 5% formalin was injected subcutaneously into the dorsal side of the right hind paw. Test condition 2. Bupivacaine/Formalin group (six rats), bupivacaine was injected into the ankle area and into the site of formalin injection 10 min before formalin injection. Test condition 3. Saline/Formalin group (six rats), saline was injected 10min before formalin in the same manner as bupivacaine. Test condition 4. Formalin/Bupivacaine group 1 (six rats), bupivacaine was injected 10 min after formalin. Test condition 5. Formalin/Bupivacaine group 2 (six rats), bupivacaine was injected similarly 1h after formalin. The magnitude of paw edema and paw withdrawal thresholds to mechanical stimuli applied to the plantar surface of the injected paw and on the dorsal surface of the contralateral side were evaluated prior to and one week after formalin injection. The lumbar spinal cord was immunohistochemically processed at one week to assess the expression of a marker for activated microglia. The results showed: (i) pre-treatment with bupivacaine blocked both phases of formalin-evoked pain behaviors and the mechanical allodynia that developed one week post-formalin injection, but did not block microglial activation; (ii) treatment with bupivacaine 1h after formalin injection reduced paw edema and prevented skin ulceration, but one week allodynia and microglial activation were still present; and (iii) prolonged spinal microglial activation was not dependent on acute formalin-induced nociceptor activity, but was strongly associated with the amount of tissue destruction. Our studies suggest that: (i) the central sensitization associated with the phase II of formalin-evoked behaviors and spinal microglial activation are both necessary to permit the development of the long-term hyperalgesia produced by the subcutaneous administration of formalin into the rats hindpaw; and (ii) acute nociceptive inputs following formalin injection are not necessary for central microglial activation that may be triggered by nerve damage or prolonged signals from peripherally inflamed tissue

The ultrastructure and synaptic connections of serotonin-immunoreactive terminals in spinal laminae I and II.

In order to study the synaptic relationships of serotonin (5-HT)-containing axons, boutons in laminae I and II of the cat spinal cord were labeled for serotonin with peroxidase-antiperoxidase immunocytochemistry. Labeled boutons were examined with the light microscope and recut into serial ultrathin sections for examination with the electron microscope. Labeled axons exhibiting boutons were sagittally oriented, and were most numerous in lamina I and outer lamina II (IIo) and least numerous in inner lamina II (IIi). Two types of labeled boutons were observed ultrastructurally. A relatively rare, large, scalloped or egg-shaped bouton, which contained many mitochondria and dense core vesicles, was found in laminae I and IIo. A smaller dome-shaped bouton, which contained fewer dense core vesicles and round or pleomorphic, clear vesicles, was found throughout laminae I and II. Both types commonly established symmetrical synaptic contacts with the distal portion of a dendritic tree, rarely with proximal portions or cell somas, and never with axon terminals. The results suggest that there are heterogeneous serotonergic systems that may selectively modify different inputs postsynaptically to functionally different types of neurons in the superficial dorsal horn of the spinal cord.

Physiological and Morphological Characteristics of Spinal Neurons Projecting to the Parabrachial Region of the Cat

Neurons in the lumbosacral, superficial spinal dorsal horn in the cat were recorded extra- and intracellularly, using dorsal root stimulation as a search stimulus. Isolated neurons were tested for antidromic activation from the contra- and ipsilateral parabrachial region. Seventy-one nociceptive-specific neurons, 11 innocuous cooling neurons, and 8 multireceptive neurons were antidromically activated from the lateral parabrachial region. The receptive fields and response properties were typical of other lamina I and lamina II neurons, in that the receptive fields were usually discrete and relatively small, and the responses ranged from sluggish and decrementing to brisk and augmenting with afterdischarge. The conduction velocity to the parabrachial region averaged 3.7 m/sec for the nociceptive-specific neurons, 3.9 m/sec for the innocuous cooling neurons, and 13.5 m/sec for the multireceptive neurons. Intracellularly labeled neurons were mostly medium to large Waldeyer-like neurons in lamina I. Some had axon collaterals that distributed varicosities in laminae I, II, and V. These data indicate that a slowly conducting nociceptive-specific and thermoreceptive pathway exists between the superficial dorsal horn and the parabrachial region at the pontine-midbrain junction.

Electrical stimulation in the medullary nucleus raphe magnus inhibits noxious heat-evokedfos protein-like immunoreactivity in the rat lumbar spinal cord

Noxious heat applied to the footpad evokes the expression of fos protein-like immunoreactivity in the rat spinal cord lumbar dorsal horn. Electrical stimulation in the medullary nucleus raphe magnus (NRM) while not evoking fos-like immunoreactivity itself, reduced significantly the number of neurons (to 50% of control) in the dorsal horn demonstrating fos protein-like immunoreactivity in response to noxious heating of the footpad. Thus descending projections from the medullary NRM appear to have direct inhibitory effects on dorsal horn neurons that receive nociceptive input from cutaneous thermal nociceptors.

Sensory experiences in man evoked by intraneural electrical stimulation of intact cutaneous afferent fibers

SummaryThe response of slowly conducting myelinated and unmyelinated afferent units to natural types of cutaneous stimuli was recorded extracellularly with tungsten microelectrodes from intact human skin nerves. Seven fibers had characteristics of C-polymodal units (conduction velocity: 0.75–1.2 m/s) and two fit descriptions of myelinated high-threshold mechanoreceptors (conduction velocity of one: 19 m/s).Attempts were made to evoke a sensation in a subject by stimulating the impaled fascicles directly with electrical pulses of controlled amplitude, duration and frequency so as to try to correlate responsive properties of the sensory units and sensory experience. The subjective sensations evoked by natural stimuli to the skin surface were used as criteria for comparison.