João M. Bráz

Restriction of Transient Receptor Potential Vanilloid-1 to the Peptidergic Subset of Primary Afferent Neurons Follows Its Developmental Downregulation in Nonpeptidergic Neurons

Primary afferent “pain” fibers (nociceptors) are divided into subclasses based on distinct molecular and anatomical features, and these classes mediate noxious modality-specific contributions to behaviors evoked by painful stimuli. Whether the heat and capsaicin receptor transient receptor potential vanilloid-1 (TRPV1) is expressed heterogeneously across several sensory populations, or is selectively expressed by a unique nociceptor subclass, however, is unclear. Here we used two lines of Trpv1 reporter mice to investigate the primary afferent expression of TRPV1, both during development and in the adult. We demonstrate, using Cre-induced lineage tracing, that during development TRPV1 is transiently expressed in a wide range of dorsal root ganglion neurons, and that its expression is gradually refined, such that TRPV1 transcripts become restricted to a specific subset of peptidergic sensory neurons. Finally, the remarkable sensitivity that is characteristic of these reporter mice revealed an innervation of central and peripheral targets by TRPV1+ primary afferents in the adult that is considerably more extensive than has previously been appreciated.

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.

Forebrain GABAergic Neuron Precursors Integrate into Adult Spinal Cord and Reduce Injury-Induced Neuropathic Pain

Neuropathic pain is a chronic debilitating disease characterized by mechanical allodynia and spontaneous pain. Because symptoms are often unresponsive to conventional methods of pain treatment, new therapeutic approaches are essential. Here, we describe a strategy that not only ameliorates symptoms of neuropathic pain but is also potentially disease modifying. We show that transplantation of immature telencephalic GABAergic interneurons from the mouse medial ganglionic eminence (MGE) into the adult mouse spinal cord completely reverses the mechanical hypersensitivity produced by peripheral nerve injury. Underlying this improvement is a remarkable integration of the MGE transplants into the host spinal cord circuitry, in which the transplanted cells make functional connections with both primary afferent and spinal cord neurons. By contrast, MGE transplants were not effective against inflammatory pain. Our findings suggest that MGE-derived GABAergic interneurons overcome the spinal cord hyperexcitability that is a hallmark of nerve injury-induced neuropathic pain.

Injured sensory neuron-derived CSF1 induces microglial proliferation and DAP12-dependent pain

Although microglia have been implicated in nerve injury–induced neuropathic pain, the manner by which injured sensory neurons engage microglia remains unclear. We found that peripheral nerve injury induced de novo expression of colony-stimulating factor 1 (CSF1) in injured sensory neurons. CSF1 was transported to the spinal cord, where it targeted the microglial CSF1 receptor (CSF1R). Cre-mediated sensory neuron deletion of Csf1 completely prevented nerve injury–induced mechanical hypersensitivity and reduced microglial activation and proliferation. In contrast, intrathecal injection of CSF1 induced mechanical hypersensitivity and microglial proliferation. Nerve injury also upregulated CSF1 in motoneurons, where it was required for ventral horn microglial activation and proliferation. Downstream of CSF1R, we found that the microglial membrane adaptor protein DAP12 was required for both nerve injury– and intrathecal CSF1–induced upregulation of pain-related microglial genes and the ensuing pain, but not for microglial proliferation. Thus, both CSF1 and DAP12 are potential targets for the pharmacotherapy of neuropathic pain.

Differential ATF3 expression in dorsal root ganglion neurons reveals the profile of primary afferents engaged by diverse noxious chemical stimuli

&NA; Although transgenic and knockout mice have helped delineate the mechanisms of action of diverse noxious compounds, it is still difficult to determine unequivocally the subpopulations of primary afferent nociceptor that these molecules engage. As most noxious stimuli lead to tissue and/or nerve injury, here we used induction of activating transcription factor 3 (ATF3), a reliable marker of nerve injury, to assess the populations of primary afferent fibers that are activated after peripheral administration of noxious chemical stimuli. In wild‐type mice, hindpaw injections of capsaicin, formalin, mustard oil or menthol induce expression of ATF3 in distinct subpopulations of sensory neurons. Interestingly, even though these noxious chemicals are thought to act through subtypes of transient receptor potential (TRP) channels, all compounds also induced ATF3 in neurons that appear not to express the expected TRP channel subtypes. On the other hand, capsaicin failed to induce ATF3 in mice lacking TRPV1, indicating that TRPV1 is required for both the direct and indirect induction of ATF3 in sensory neurons. By contrast, only low doses of formalin or mustard oil failed to induce ATF3 in TRPA1 null mice, indicating that injections of high doses (>0.5%) of formalin or mustard oil recruit both TRPA1‐ and non‐TRPA1 expressing primary afferent fibers. Finally, peripheral injection of menthol, a TRPM8 receptor agonist, induced ATF3 in a wide variety of sensory neurons, but in a TRPM8‐independent manner. We conclude that purportedly selective agonists can activate a heterogeneous population of sensory neurons, which ultimately could contribute to the behavioral responses evoked.

Transneuronal tracing of diverse CNS circuits by Cre-mediated induction of wheat germ agglutinin in transgenic mice

Systems neuroscience addresses the complex circuits made by populations of neurons in the CNS and the cooperative function of these neurons. Improved approaches to the neuroanatomical analysis of CNS circuits are thus of great interest. In fact, significant advances in tract-tracing methods have recently been made by using transgenic mice that express transneuronal lectin tracers under the control of neuron-specific promoters. The utility of those animals, however, is limited to the CNS circuit influenced by the particular promoter. Here, we describe a new transgenic mouse that can be used for transneuronal tracing analysis of circuits in any region of the brain or spinal cord. The transgene in these mice results in expression of LacZ in neurons throughout the CNS. Excision of the LacZ gene by Cre-mediated recombination initiates expression of the lectin, wheat germ agglutinin (WGA). To illustrate the diverse uses of these ZW (LacZ-WGA) mice, we triggered WGA expression either by crossing the mice with two Cre-expressing transgenic mouse lines or by microinjecting a Cre-expressing adeno-associated virus into the cerebellum or cerebral cortex. Both approaches resulted in extensive WGA expression in the cell bodies and dendrites of neurons in which the recombination event occurred, as well as anterograde and transneuronal transport of the lectin to second and third order neurons. Because the lectin can be induced in developing and adult animals, and in all regions of the brain and spinal cord, these ZW may prove extremely valuable for numerous studies of CNS circuit analysis.

Dorsal Horn Parvalbumin Neurons Are Gate-Keepers of Touch-Evoked Pain after Nerve Injury

SUMMARY Neuropathic pain is a chronic debilitating disease that results from nerve damage, persists long after the injury has subsided, and is characterized by spontaneous pain and mechanical hypersensitivity. Although loss of inhibitory tone in the dorsal horn of the spinal cord is a major contributor to neuropathic pain, the molecular and cellular mechanisms underlying this disinhibition are unclear. Here, we combined pharmacogenetic activation and selective ablation approaches in mice to define the contribution of spinal cord parvalbumin (PV)-expressing inhibitory interneurons in naive and neuropathic pain conditions. Ablating PV neurons in naive mice produce neuropathic pain-like mechanical allodynia via disinhibition of PKCγ excitatory interneurons. Conversely, activating PV neurons in nerve-injured mice alleviates mechanical hypersensitivity. These findings indicate that PV interneurons are modality-specific filters that gate mechanical but not thermal inputs to the dorsal horn and that increasing PV inter-neuron activity can ameliorate the mechanical hypersensitivity that develops following nerve injury.

Inputs to serotonergic neurons revealed by conditional viral transneuronal tracing.

Descending projections arising from brainstem serotonergic (5HT) neurons contribute to both facilitatory and inhibitory controls of spinal cord “pain” transmission neurons. Unclear, however, are the brainstem networks that influence the output of these 5HT neurons. To address this question, here we used a novel neuroanatomical tracing method in a transgenic line of mice in which Cre recombinase is selectively expressed in 5HT neurons (ePet‐Cre mice). Specifically, we injected the conditional pseudorabies virus recombinant (BA2001) that can replicate only in Cre‐expressing neurons. Because BA2001 transports exclusively in a retrograde manner, we were able to reveal a subset of the neurons and circuits that are located upstream of the Cre‐expressing 5HT neurons. We show that diverse brainstem regions differentially target the 5HT neurons of the dorsal raphe (DR) and the nucleus raphe magnus of the rostroventral medulla (RVM). Among these are several catecholaminergic and cholinergic cell groups, the periaqueductal gray, several brainstem reticular nuclei, and the nucleus of the solitary tract. We conclude that a brainstem 5HT network integrates somatic and visceral inputs arising from various areas of the body. We also identified a circuit that arises from projection neurons of deep spinal cord laminae V–VIII and targets the 5HT neurons of the NRM, but not of the DR. This spinoreticular pathway constitutes an anatomical substrate through which a noxious stimulus can activate 5HT neurons of the NRM and in turn could trigger descending serotonergic antinociceptive controls. J. Comp. Neurol. 514:145–160, 2009.

Genetically expressed transneuronal tracer reveals direct and indirect serotonergic descending control circuits.

Despite the evidence for a significant contribution of brainstem serotonergic (5HT) systems to the control of spinal cord “pain” transmission neurons, attention has turned recently to the influence of nonserotonergic neurons, including the facilitatory and inhibitory controls that originate from so‐called “on” and “off” cells of the rostroventral medulla (RVM). Unclear, however, is the extent to which these latter circuits interact with or are influenced by the serotonergic cell groups. To address this question we selectively targeted expression of a transneuronal tracer, wheat germ agglutinin (WGA), in the 5HT neurons so as to study the interplay between the 5HT and non‐5HT systems. In addition to confirming the direct medullary 5HT projection to the spinal cord we also observed large numbers of non‐5HT neurons, in the medullary nucleus reticularis gigantocellularis and magnocellularis, that were WGA‐immunoreactive, i.e., were transneuronally labeled from 5HT neurons. FluoroGold injections into the spinal cord established that these reticular neurons are not only postsynaptic to the 5HT neurons of the medulla, but that most are also at the origin of descending, bulbospinal pathways. By contrast, we found no evidence that neurons of the midbrain periaqueductal gray that project to the RVM are postsynaptic to midbrain or medullary 5HT neurons. Finally, we found very few examples of WGA‐immunoreactive noradrenergic neurons, which suggests that there is considerable independence of the monoaminergic bulbospinal pathways. Our results indicate that 5HT neurons influence “pain” processing at the spinal cord level both directly and indirectly via feedforward connections with multiple non‐5HT descending control pathways. J. Comp. Neurol. 507:1990–2003, 2008.

Primary Afferent and Spinal Cord Expression of Gastrin-Releasing Peptide: Message, Protein, and Antibody Concerns

There is continuing controversy relating to the primary afferent neurotransmitter that conveys itch signals to the spinal cord. Here, we investigated the DRG and spinal cord expression of the putative primary afferent-derived “itch” neurotransmitter, gastrin-releasing peptide (GRP). Using ISH, qPCR, and immunohistochemistry, we conclude that GRP is expressed abundantly in spinal cord, but not in DRG neurons. Titration of the most commonly used GRP antiserum in tissues from wild-type and GRP mutant mice indicates that the antiserum is only selective for GRP at high dilutions. Paralleling these observations, we found that a GRPeGFP transgenic reporter mouse has abundant expression in superficial dorsal horn neurons, but not in the DRG. In contrast to previous studies, neither dorsal rhizotomy nor an intrathecal injection of capsaicin, which completely eliminated spinal cord TRPV1-immunoreactive terminals, altered dorsal horn GRP immunoreactivity. Unexpectedly, however, peripheral nerve injury induced significant GRP expression in a heterogeneous population of DRG neurons. Finally, dual labeling and retrograde tracing studies showed that GRP-expressing neurons of the superficial dorsal horn are predominantly interneurons, that a small number coexpress protein kinase C gamma (PKCγ), but that none coexpress the GRP receptor (GRPR). Our studies support the view that pruritogens engage spinal cord “itch” circuits via excitatory superficial dorsal horn interneurons that express GRP and that likely target GRPR-expressing interneurons. The fact that peripheral nerve injury induced de novo GRP expression in DRG neurons points to a novel contribution of this peptide to pruritoceptive processing in neuropathic itch conditions.