Kate Skinner

Innocuous, not noxious, input activates PKCgamma interneurons of the spinal dorsal horn via myelinated afferent fibers.

Protein kinase C γ (PKCγ), which is concentrated in interneurons of the inner part of lamina II of the dorsal horn, has been implicated in injury-induced allodynia, a condition wherein pain is produced by innocuous stimuli. Although it is generally assumed that these interneurons receive input from the nonpeptidergic, IB4-positive subset of nociceptors, the fact that PKCγ cells do not express Fos in response to noxious stimulation suggests otherwise. Here, we demonstrate that the terminal field of the nonpeptidergic population of nociceptors, in fact, lies dorsal to that of PKCγ interneurons. There was also no overlap between the PKCγ-expressing interneurons and the transganglionic tracer wheat germ agglutinin which, after sciatic nerve injection, labels all unmyelinated nociceptors. However, transganglionic transport of the β-subunit of cholera toxin, which marks the medium-diameter and large-diameter myelinated afferents that transmit non-noxious information, revealed extensive overlap with the layer of PKCγ interneurons. Furthermore, expression of a transneuronal tracer in myelinated afferents resulted in labeling of PKCγ interneurons. Light and electron microscopic double labeling further showed that the VGLUT1 subtype of vesicular glutamate transmitter, which is expressed in myelinated afferents, marks synapses that are presynaptic to the PKCγ interneurons. Finally, we demonstrate that a continuous non-noxious input, generated by walking on a rotarod, induces Fos in the PKCγ interneurons. These results establish that PKCγ interneurons are activated by myelinated afferents that respond to innocuous stimuli, which suggests that injury-induced mechanical allodynia is transmitted through a circuit that involves PKCγ interneurons and non-nociceptive, VGLUT1-expressing myelinated primary afferents.

Immunoreactive TRPV-2 (VRL-1), a capsaicin receptor homolog, in the spinal cord of the rat.

The vanilloid receptor‐like 1 protein (VRL‐1, also called TRPV2) is a member of the TRPV family of proteins and is a homolog of the capsaicin/vanilloid receptor (VR1, or TRPV1). Although VRL‐1 does not bind capsaicin, like VR1 it is activated by noxious heat (>52°C). Unlike VR1, however, VRL‐1 is primarily expressed by medium‐ and large‐diameter primary afferents, which suggests that nociceptive processing is but one of the functions to which VRL‐1 contributes. To provide information on the diverse spinal circuits that are engaged by these VRL‐1‐expressing primary afferents, we completed a detailed immunocytochemical map of VRL‐1 in rat spinal cord, including light and electron microscopic analysis, and generated a more comprehensive neurochemical characterization of VRL‐1‐expressing primary afferents. Consistent with previous reports, we found that VRL‐1 and VR1 are expressed in different dorsal root ganglion (DRG) cell bodies. Almost all VRL‐1‐expressing cells labeled for N52 (a marker of myelinated afferents), consistent with VRL‐1 expression in Aδ and Aβ fibers. EM analysis of the DRG and dorsal roots confirmed this and revealed two categories of neurons based on the intensity of immunolabeling. The densest VRL‐1 immunoreactivity in the spinal cord was found in lamina I, inner lamina II, and laminae III/IV. This is consistent with the expression of VRL‐1 by myelinated nociceptors that target laminae I and IIi and in nonnociceptive Aβ fibers that target laminae III/IV. Dorsal rhizotomy reduced, but did not eliminate, the immunostaining in all dorsal horn laminae, which indicates that VRL‐1 expression derives from both DRG cells and from neurons intrinsic to the brain or spinal cord. Spinal hemisection reduced immunostaining of the ipsilateral dorsal columns in segments rostral to the lesion and in the dorsal column nuclei, presumably from the loss of ascending Aβ afferents, but there was no change caudal to the lesion. Thus, supraspinal sources of dorsal horn VRL‐1 immunoreactivity are likely not significant. Although we never observed VRL‐1 immunostaining in cell bodies in the superficial dorsal horn, there was extensive labeling of motoneurons and ventral root efferents—in particular, in an extremely densely labeled population at the lumbosacral junction. Finally, many ependymal cells surrounding the central canal were intensely labeled. These results emphasize that VRL‐1, in contrast to VR1, is present in a diverse population of neurons and undoubtedly contributes to numerous functions in addition to nociceptive processing. J. Comp. Neurol. 470:400–408, 2004.

Anatomical and functional analysis of aquaporin 1, a water channel in primary afferent neurons.

Abstract Aquaporin 1 (AQP1) is the archetypal member of a family of water channel proteins that contribute to water homeostasis in kidney, lung, and other tissues. Although there is limited evidence that aquaporins are expressed in the nervous system, AQP4 is expressed in glia and AQP9 is present on some neuronal and glial mitochondria. In the present study, we used immunohistochemistry to show that AQP1 is heavily expressed in a population of small diameter primary sensory neurons of dorsal root, trigeminal, and nodose ganglia. AQP1 immunoreactivity is abundant in DRG cell bodies and in both the peripheral and central branches of primary afferent neurons, and colocalizes with markers of nociceptors, notably substance P and IB4. AQP1 expression in DRG is first detectable at embryonic day 15.5, which corresponds to the developmental stage when the majority of fine cutaneous afferents penetrate the dorsal horn. Electron microscopy revealed dense membrane labeling of unmyelinated axons, a few fine diameter myelinated axons, and synaptic terminals in the superficial dorsal horn. Because this restricted and dense expression suggested that AQP1 contributes to nociceptive processing, we studied behavioral responses of wildtype and AQP1 −/− mice in a comprehensive battery of acute and persistent pain tests. We also used in vivo electrophysiology in wildtype and mutant mice to measure the responses of wide dynamic range neurons in lamina V of the dorsal horn to thermal stimulation before and after noxious stimulus‐induced sensitization. To date we have not detected a differential phenotype suggestive of a functional contribution of AQP1 to nociceptive processing.

Co‐localization of endomorphin‐2 and substance P in primary afferent nociceptors and effects of injury: a light and electron microscopic study in the rat

Endomorphin‐2 (EM2) is a tetrapeptide with remarkable affinity and selectivity for the mu‐opioid receptor. In the present study, we used double‐fluorescence and electron microscopic immunocytochemistry to identify subsets of EM2‐expressing neurons in dorsal root ganglia and spinal cord dorsal horn of adult rats. Within the lumbar dorsal root ganglia, we found EM2 immunoreactivity mainly in small‐to‐medium size neurons, most of which co‐expressed the neuropeptide substance P (SP). In adult rat L4 dorsal root ganglia, 23.9% of neuronal profiles contained EM2 immunoreactivity and ranged in size from 15 to 36 µm in diameter (mean 24.3 ± 4.3 µm). Double‐labelling experiments with cytochemical markers of dorsal root ganglia neurons showed that approximately 95% of EM2‐immunoreactive cell bodies also label with SP antisera, 83% co‐express vanilloid receptor subtype 1/capsaicin receptor, and 17% label with isolectin B4, a marker of non‐peptide nociceptors. Importantly, EM2 immunostaining persisted in mice with a deletion of the preprotachykinin‐A gene that encodes SP. In the lumbar spinal cord dorsal horn, EM2 expression was concentrated in presumptive primary afferent terminals in laminae I and outer II. At the ultrastructural level, electron microscopic double‐labelling showed co‐localization of EM2 and SP in dense core vesicles of lumbar superficial dorsal horn synaptic terminals. Finally, 2 weeks after sciatic nerve axotomy we observed a greater than 50% reduction in EM2 immunoreactivity in the superficial dorsal horn. We suggest that the very strong anatomical relationship between primary afferent nociceptors that express SP and EM2 underlies an EM2 regulation of SP release via mu‐opioid autoreceptors.

Cholecystokinin and enkephalin in brain stem pain modulating circuits

NEURONS in rostral ventromedial medulla and the periaqueductal gray modulate dorsal horn nociceptive transmission. Endogenous peptides implicated in this modulation include enkephalin (ENK), which is antinociceptive, and cholecystokinin (CCK), which has anti-opioid effects. In this study double-label fluorescence immunocytochemistry demonstrated somata and terminals with ENKor CCK-like immunoreactivity in these regions. Although the distribution of CCKand ENK-immunoreactive terminal fields overlapped significantly, co-localization was rare. Furthermore, CCK- and ENK-immunoreactive somata had different morphologies and distinct distributions. The overlap of CCKand ENKimmunoreactive terminals arbors provides a morphological substrate for an antagonistic interaction of CCK and ENK within brainstem pain modulating circuits, as has been demonstrated in the spinal cord.

GABA-immunoreactive boutons contact identified OFF and ON cells in the nucleus raphe magnus.

The pontomedullary raphe magnus (RM) contains two physiologically defined types of neurons that participate in the opioid‐induced modulation of dorsal horn nociceptive messages: OFF cells, which decrease, and ON cells, which increase their discharge rates when reflex behavior is evoked by noxious pinch or heat. Because both types of neuron have inhibitory inputs and because there is evidence that γ‐aminobutyric acid (GABA) inhibitory mechanisms within RM contribute to the antinociceptive action of opioids, we have sought anatomical evidence for a direct GABAergic input to OFF and ON cells. In this study, cells of each type located in the RM were electrophysiologically defined and intracellularly filled with horseradish peroxidase or Neurobiotin. One cell of each type was labeled in the cat, and 2–3 cells of each type were labeled in the rat. Thin sections were labeled by a postembedding immunogold procedure by using an antibody directed against glutaraldehyde‐conjugated GABA. GABA‐immunoreactive (GABA‐ir) boutons contained small, round, clear vesicles and made symmetrical synapses with identified dendrites. GABA‐ir boutons were apposed to soma and to proximal and distal dendrites of both cell types in both species. These findings demonstrate direct GABAergic input to identified OFF and ON cells in the RM. J. Comp. Neurol. 378:196–204, 1997.

Noradrenergic input to nociceptive modulatory neurons in the rat rostral ventromedial medulla.

Within the rostral ventromedial medulla (RVM), there are two classes of putative pain modulation neurons: ON cells and OFF cells, which respectively burst or pause prior to withdrawal reflexes elicited by noxious stimulation. Alpha‐adrenergic agonists injected into the RVM produce changes in the latency of spinal nocifensive reflexes and, when iontophoretically applied, alter the firing of RVM ON but not OFF cells. To provide further information about the contribution of norepinephrine to RVM neuron function, we analyzed the distribution of tyrosine hydroxylase immunoreactive (TH‐ir) appositions upon RVM ON and OFF cells.

Gaba immunoreactive synapses onto physiologically identified neurons in the cat rostral ventromedial medulla

Weinstein), Depts of lNeurology, 9Anatomy and 3Physiology, UCSF, and r ACC Hall E Abs No 866 4Dept. of Gerontology, Mt Zion Hospital, San Francisco, CA 94143 USA. Neurons in the rostra1 ventromedial medulla(RVM), including cells in the nucleus raphe magnus, are important in the suppression of nociceptive transmission produced by systemic morphine. Neurons that are inhibited during a withdrawal reflex evoked by noxious stimulation and excited by morphine administration are termed off-cells; these neurons are hypothesized to inhibit nociceptive transmission in the dorsal horn. Since the direct effects of opiates are typically inhibitory, it is possible that the opiate excitation of offcells is a result of disinhibition. Microinjection studies have demonstrated that both opioid agonists and GABA antagonists in the RVM suppress nociceptive transmission. Since iontophoretic bicucculine blocks the inhibition produced by noxious stimulation, it is possible that the opioid excitation of off-cells is produced by opioid blockade of GABA-mediated inhibition. In order to test this hypothesis, post-embedding, electron microscopic (EM) immunocytochemistry was combined with intracellular recording and labeling to determine if RVM off-cells receive GABA synapses. Off-cells were physiologically characterized and then intracellularly labeled with horseradish peroxidase (HRP) in the lightly anesthetized cat. Sections were then osmicated, embedded in plastic and the somatodendritic arbor of each cell was reconstructed. Post embedding staining for immunoreactive GABA was performed using a colloidal gold procedure. GABA immunoreactive terminals were found in contact with off-cells. Symmetrtcal synapses were observed between GABA terminals and HRP labeled off-cells. These results demonstrate an anatomical substrate for the GABAergic control of off-cells shown by iontophoresis. In addition, the present results are consistent with the hypothesis that the opioid excitation of offcells is mediated through a disinhibitory mechanism.