Márk Kozsurek

Cocaine- and amphetamine-regulated transcript peptide (CART) is present in peptidergic C primary afferents and axons of excitatory interneurons with a possible role in nociception in the superficial laminae of the rat spinal cord

Cocaine‐ and amphetamine‐regulated transcript peptides (CART) have been implicated in the regulation of several physiological functions, including pain transmission. A dense plexus of CART‐immunoreactive fibres has been described in the superficial laminae of the spinal cord, which are key areas in sensory information and pain processing. In this study, we used antibody against CART peptide, together with markers for various types of primary afferents, interneurons and descending systems to determine the origin of the CART‐immunoreactive axons in the superficial laminae of the rat spinal cord. Calcitonin gene‐related peptide (CGRP), a marker for peptidergic primary afferents in the dorsal horn, was present in 72.6% and 34.8% of CART‐immunoreactive axons in lamina I and II, respectively. The majority of these fibres also contained substance P (SP), while a few were somatostatin (SOM)‐positive. The other subpopulation of CART‐immunoreactive boutons in lamina I and II also expressed SP and/or SOM without CGRP, but contained vesicular glutamate transporter 2, which is present mainly in excitatory interneuronal terminals. Our data demonstrate that the majority of CART‐immunoreactive axons in the spinal dorsal horn originate from peptidergic nociceptive primary afferents, while the rest arise from excitatory interneurons that contain SP or SOM. This strongly suggests that CART peptide can affect glutamatergic neurotransmission as well as the release and effects of SP and SOM in nociception and other sensory processes.

Subpopulation-specific patterns of intrinsic connectivity in mouse superficial dorsal horn as revealed by laser scanning photostimulation

•  Sensory neurons that detect painful and non‐painful stimulation of body tissues have axons that project to the dorsal horn of the spinal cord, where their terminations are partially segregated into superficial (I–II) and deep (III–IV) dorsal horn laminae, respectively. •  The dorsal horn contains many excitatory and inhibitory interneurons whose axons synapse on other dorsal horn neurons to enhance or suppress sensory transmission. •  This study used a localized stimulation technique (laser scanning photostimulation) for high‐resolution mapping of synaptic connections between dorsal horn interneurons, in an in vitro‘slice’ preparation of the mouse lumbar spinal cord. •  Some neurons in superficial layers of the dorsal horn have long dendrites that extend ventrally into deeper layers of the dorsal horn, and these neurons can receive excitatory or inhibitory synaptic input from neurons in the deeper layers. •  These interlaminar connections may be involved in interactions between transmission of signals underlying painful versus non‐painful sensations.

Nonselective innervation of lamina I projection neurons by cocaine- and amphetamine-regulated transcript peptide (CART)-immunoreactive fibres in the rat spinal dorsal horn.

Cocaine‐ and amphetamine‐regulated transcript (CART) peptides have been implicated in spinal pain transmission. A dense plexus of CART‐immunoreactive fibres has been described in the superficial laminae of the spinal cord, which are key areas in sensory information and pain processing. We demonstrated previously that the majority of these fibres originate from nociceptive primary afferents. Using tract tracing, multiple immunofluorescent labelling and electronmicroscopy we determined the proportion of peptidergic primary afferents expressing CART, looked for evidence for coexistence of CART with galanin in these afferents in lamina I and examined their targets. Almost all (97.9%) randomly selected calcitonin gene‐related peptide (CGRP)‐immunoreactive terminals were substance P (SP)‐positive (+) and CART was detected in approximately half (48.6%) of them. Most (81.4%) of the CGRP/SPergic boutons were galanin+ and approximately half (49.0%) of these contained CART. Many (72.9%) of the CARTergic boutons which expressed CGRP were also immunoreactive for galanin, while only 8.6% of the CARTergic terminals were galanin+ without CGRP. Electron microscopy showed that most of the CART terminals formed asymmetrical synapses, mainly with dendrites. All different morphological and neurochemical subtypes of spinoparabrachial projection neurons in the lamina I received contacts from CART‐immunoreactive nociceptive afferents. The innervation density from these boutons did not differ significantly between either the different neurochemical or the morphological subclasses of these cells. This suggests a nonselective innervation of lamina I projection neurons from a subpopulation of CGRP/SP afferents containing CART peptide. These results provide anatomical evidence for involvement of CART peptide in spinal pain transmission.

Secretagogin-dependent matrix metalloprotease-2 release from neurons regulates neuroblast migration

Significance Persisting proliferative capacity and regeneration in the adult brain are confined to a minority of regions. In the murine brain, the olfactory system is supplied by thousands of newly born neuroblasts daily to support sensory plasticity. Here, we reveal a neuronal scaffold that resides within and alongside this migratory route [termed the “rostral migratory stream” (RMS)] to guide forward migration of newly born neuroblasts. These neurons can externalize the enzyme matrix metalloprotease-2 to loosen the extracellular matrix, thus producing permissive corridors for migrating neuroblasts. This mechanism is likely phylogenetically conserved because it exists in the RMS equivalent in human fetal brains. This inducible mechanism might be pharmacologically targeted for therapeutic benefit. The rostral migratory stream (RMS) is viewed as a glia-enriched conduit of forward-migrating neuroblasts in which chemorepulsive signals control the pace of forward migration. Here we demonstrate the existence of a scaffold of neurons that receive synaptic inputs within the rat, mouse, and human fetal RMS equivalents. These neurons express secretagogin, a Ca2+-sensor protein, to execute an annexin V-dependent externalization of matrix metalloprotease-2 (MMP-2) for reconfiguring the extracellular matrix locally. Mouse genetics combined with pharmacological probing in vivo and in vitro demonstrate that MMP-2 externalization occurs on demand and that its loss slows neuroblast migration. Loss of function is particularly remarkable upon injury to the olfactory bulb. Cumulatively, we identify a signaling cascade that provokes structural remodeling of the RMS through recruitment of MMP-2 by a previously unrecognized neuronal constituent. Given the life-long presence of secretagogin-containing neurons in human, this mechanism might be exploited for therapeutic benefit in rescue strategies.

Inhibition of α2A-Adrenoceptors Ameliorates Dextran Sulfate Sodium-Induced Acute Intestinal Inflammation in Mice.

It has been hypothesized that α2-adrenoceptors (α2-ARs) may be involved in the pathomechanism of colitis; however, the results are conflicting because both aggravation and amelioration of colonic inflammation have been described in response to α2-AR agonists. Therefore, we aimed to analyze the role of α2-ARs in acute murine colitis. The experiments were carried out in wild-type, α2A-, α2B-, and α2C-AR knockout (KO) C57BL/6 mice. Colitis was induced by dextran sulfate sodium (DSS, 2%); alpha2-AR ligands were injected i.p. The severity of colitis was determined both macroscopically and histologically. Colonic myeloperoxidase (MPO) and cytokine levels were measured by enzyme-linked immunosorbent assay and proteome profiler array, respectively. The nonselective α2-AR agonist clonidine induced a modest aggravation of DSS-induced colitis. It accelerated the disease development and markedly enhanced the weight loss of animals, but did not influence the colon shortening, tissue MPO levels, or histologic score. Clonidine induced similar changes in α2B- and α2C-AR KO mice, whereas it failed to affect the disease activity index scores and caused only minor weight loss in α2A-AR KO animals. In contrast, selective inhibition of α2A-ARs by BRL 44408 significantly delayed the development of colitis; reduced the colonic levels of MPO and chemokine (C-C motif) ligand 3, chemokine (C-X-C motif) ligand 2 (CXCL2), CXCL13, and granulocyte-colony stimulating factor; and elevated that of tissue inhibitor of metalloproteinases-1. In this work, we report that activation of α2-ARs aggravates murine colitis, an effect mediated by the α2A-AR subtype, and selective inhibition of these receptors reduces the severity of gut inflammation.

Glial cell type-specific changes in spinal dipeptidyl peptidase 4 expression and effects of its inhibitors in inflammatory and neuropatic pain

Altered pain sensations such as hyperalgesia and allodynia are characteristic features of various pain states, and remain difficult to treat. We have shown previously that spinal application of dipeptidyl peptidase 4 (DPP4) inhibitors induces strong antihyperalgesic effect during inflammatory pain. In this study we observed low level of DPP4 mRNA in the rat spinal dorsal horn in physiological conditions, which did not change significantly either in carrageenan-induced inflammatory or partial nerve ligation-generated neuropathic states. In naïve animals, microglia and astrocytes expressed DPP4 protein with one and two orders of magnitude higher than neurons, respectively. DPP4 significantly increased in astrocytes during inflammation and in microglia in neuropathy. Intrathecal application of two DPP4 inhibitors tripeptide isoleucin-prolin-isoleucin (IPI) and the antidiabetic drug vildagliptin resulted in robust opioid-dependent antihyperalgesic effect during inflammation, and milder but significant opioid-independent antihyperalgesic action in the neuropathic model. The opioid-mediated antihyperalgesic effect of IPI was exclusively related to mu-opioid receptors, while vildagliptin affected mainly delta-receptor activity, although mu- and kappa-receptors were also involved. None of the inhibitors influenced allodynia. Our results suggest pathology and glia-type specific changes of DPP4 activity in the spinal cord, which contribute to the development and maintenance of hyperalgesia and interact with endogenous opioid systems.