Biomedical subjects

H Richard Koerber

Publications and source records attributed to H Richard Koerber.

11 recordsLinked to original sources

PubMed2006

Glial cell line-derived neurotrophic factor family members sensitize nociceptors in vitro and produce thermal hyperalgesia in vivo.

Nerve growth factor (NGF) has been implicated as an effector of inflammatory pain because it sensitizes primary afferents to noxious thermal, mechanical, and chemical [e.g., capsaicin, a transient receptor potential vanilloid receptor 1 (TRPV1) agonist] stimuli and because NGF levels increase during inflammation. Here, we report the ability of glial cell line-derived neurotrophic factor (GDNF) family members artemin, neurturin and GDNF to potentiate TRPV1 signaling and to induce behavioral hyperalgesia. Analysis of capsaicin-evoked Ca2+ transients in dissociated mouse dorsal root ganglion (DRG) neurons revealed that a 7 min exposure to GDNF, neurturin, or artemin potentiated TRPV1 function at doses 10-100 times lower than NGF. Moreover, GDNF family members induced capsaicin responses in a subset of neurons that were previously insensitive to capsaicin. Using reverse transcriptase-PCR, we found that artemin mRNA was profoundly upregulated in response to inflammation induced by hindpaw injection of complete Freund's adjuvant (CFA): artemin expression increased 10-fold 1 d after CFA injection, whereas NGF expression doubled by day 7. No increase was seen in neurturin or GDNF. A corresponding increase in mRNA for the artemin coreceptor GFRalpha3 (for GDNF family receptor alpha) was seen in DRG, and GFRalpha3 immunoreactivity was widely colocalized with TRPV1 in epidermal afferents. Finally, hindpaw injection of artemin, neurturin, GDNF, or NGF produced acute thermal hyperalgesia that lasted up to 4 h; combined injection of artemin and NGF produced hyperalgesia that lasted for 6 d. These results indicate that GDNF family members regulate the sensitivity of thermal nociceptors and implicate artemin in particular as an important effector in inflammatory hyperalgesia.

Animals↗

PubMed2006

Artemin overexpression in skin enhances expression of TRPV1 and TRPA1 in cutaneous sensory neurons and leads to behavioral sensitivity to heat and cold.

Artemin, a neuronal survival factor in the glial cell line-derived neurotrophic factor family, binds the glycosylphosphatidylinositol-anchored protein GFRalpha3 and the receptor tyrosine kinase Ret. Expression of the GFRalpha3 receptor is primarily restricted to the peripheral nervous system and is found in a subpopulation of nociceptive sensory neurons of the dorsal root ganglia (DRGs) that coexpress the Ret and TrkA receptor tyrosine kinases and the thermosensitive channel TRPV1. To determine how artemin affects sensory neuron properties, transgenic mice that overexpress artemin in skin keratinocytes (ART-OE mice) were analyzed. Expression of artemin caused a 20.5% increase in DRG neuron number and increased the level of mRNA encoding GFRalpha3, TrkA, TRPV1, and the putative noxious cold-detecting channel TRPA1. Nearly all GFRalpha3-positive neurons expressed TRPV1 immunoreactivity, and most of these neurons were also positive for TRPA1. Interestingly, acid-sensing ion channel (ASIC) 1, 2a, 2b, and 3 mRNAs were decreased in the DRG, and this reduction was strongest in females. Analysis of sensory neuron physiological properties using an ex vivo preparation showed that cutaneous C-fiber nociceptors of ART-OE mice had reduced heat thresholds and increased firing rates in response to a heat ramp. No change in mechanical threshold was detected. Behavioral testing of ART-OE mice showed that they had increased sensitivity to both heat and noxious cold. These results indicate that the level of artemin in the skin modulates gene expression and response properties of afferents that project to the skin and that these changes lead to behavioral sensitivity to both hot and cold stimuli.

Acid Sensing Ion Channels↗

PubMed2006

Phenotypic characterization of gastric sensory neurons in mice.

Recent studies suggest that the capsaicin receptor [transient receptor potential vanilloid (TRPV)1] may play a role in visceral mechanosensation. To address the potential role of TRPV1 in vagal sensory neurons, we developed a new in vitro technique allowing us to determine TRPV1 expression directly in physiologically characterized gastric sensory neurons. Stomach, esophagus, and intact vagus nerve up to the central terminations were carefully dissected and placed in a perfusion chamber. Intracellular recordings were made from the soma of nodose neurons during mechanical stimulation of the stomach. Physiologically characterized neurons were labeled iontophoretically with neurobiotin and processed for immunohistochemical experiments. As shown by action potential responses triggered by stimulation of the upper thoracic vagus with a suction electrode, essentially all abdominal vagal afferents in mice conduct in the C-fiber range. Mechanosensitive gastric afferents encode stimulus intensities over a wide range without apparent saturation when punctate stimuli are used. Nine of 37 mechanosensitive vagal afferents expressed TRPV1 immunoreactivity, with 8 of the TRPV1-positive cells responding to stretch. A small number of mechanosensitive gastric vagal afferents express neurofilament heavy chains and did not respond to stretch. By maintaining the structural and functional integrity of vagal afferents up to the nodose ganglion, physiological and immunohistochemical properties of mechanosensory gastric sensory neurons can be studied in vitro. Using this novel technique, we identified TRPV1 immunoreactivity in only one-fourth of gastric mechanosensitive neurons, arguing against a major role of this ion channel in sensation of mechanical stimuli under physiological conditions.

Animals↗

PubMed2006

Synaptic plasticity in the adult spinal dorsal horn: the appearance of new functional connections following peripheral nerve regeneration.

Peripherally regenerated fibers were impaled in the dorsal columns. Each impaled fiber's adequate stimulus was determined and the fiber was activated by passing brief (200 ms) current pulses through the microelectrode. Cord dorsum potentials (CDPs) elicited by fiber stimulation were recorded at 8 sites, and then the fiber was injected with Neurobiotin (NB). In the same preparations, dorsal horn cells were impaled and their receptive fields (RFs) mapped; areas of skin from which the most vigorous responses were elicited were noted. Needle electrodes inserted into these cutaneous "hot spots" were used to electrically activate minimal numbers of peripherally regenerated fibers while simultaneously recording the resulting CDPs and any intracellular EPSPs. This allowed determination of connectivity between regenerated fibers and dorsal horn cells with overlapping RFs. In agreement with findings in intact animals, NB revealed long-ranging collaterals which were not seen using intraaxonally injected horseradish peroxidase (HRP). Although there was no qualitative difference in their morphology compared to those seen in controls, the correlation between spatial distribution of boutons and amplitudes of the monosynaptic CDPs of peripherally regenerated fibers revealed significant shifts in the functional efficacy of many central connections. Transcutaneous electrical stimulation revealed a significantly higher incidence of connectivity between regenerated fibers and cells with overlapping RFs at 9-12 months (86%) than at 5-6 months (34%). Although there was no obvious anatomical reorganization of afferent projections in the dorsal horn, the observed functional changes with time following transection show the formation of new functional central connections.

Afferent Pathways↗

PubMed2006

Glial cell-line-derived neurotrophic factor expression in skin alters the mechanical sensitivity of cutaneous nociceptors.

Neurons classified as nociceptors are dependent on nerve growth factor (NGF) during embryonic development, but a large subpopulation lose this dependence during embryonic and postnatal times and become responsive to the transforming growth factor beta family member, glial cell line-derived growth factor (GDNF). To elucidate the functional properties of GDNF-dependent nociceptors and distinguish them from nociceptors that retain NGF dependence, the cellular and physiologic properties of sensory neurons of wild-type and transgenic mice that overexpress GDNF in the skin (GDNF-OE) were analyzed using a skin, nerve, dorsal root ganglion, and spinal cord preparation, immunolabeling, and reverse transcriptase-PCR assays. Although an increase in peripheral conduction velocity of C-fibers in GDNF-OE mice was measured, other electrophysiological properties, including resting membrane potential and somal action potentials, were unchanged. We also show that isolectin B4 (IB4)-positive neurons, many of which are responsive to GDNF, exhibited significantly lower thresholds to mechanical stimulation relative to wild-type neurons. However, no change was observed in heat thresholds for the same population of cells. The increase in mechanical sensitivity was found to correlate with significant increases in acid-sensing ion channels 2A and 2B and transient receptor potential channel A1, which are thought to contribute to detection of mechanical stimuli. These data indicate that enhanced expression of GDNF in the skin can change mechanical sensitivity of IB4-positive nociceptive afferents and that this may occur through enhanced expression of specific types of channel proteins.

Acid Sensing Ion Channels↗

PubMed2005

From innervation density to tactile acuity 2: embryonic and adult pre- and postsynaptic somatotopy in the dorsal horn.

We tested the hypothesis that dorsal horn laminae III-IV cell receptive fields (RFs) are initially established in three steps: cutaneous axons penetrate the dorsal horn near their rostrocaudal (RC) levels of entry into the spinal cord. Their terminal branches distribute mediolaterally (ML) according to their relative distoproximal RF locations on the leg, and form nonselective synapses with nearby dorsal horn cell dendrites, establishing the initial dorsal horn cell RFs. Rootlet axon RFs in adult cats were used to approximate the RC entry levels of hindlimb skin input. Cord dorsum recordings of monosynaptic field potentials evoked by electrical skin stimulation provided the RC distributions of synaptic input. These were in close agreement. Simulated projections of all 22,000 hindlimb axons were similar to projections predicted from EPSP distributions, and with the observed projections of dorsal roots, cutaneous nerves, and individual axons. The simulated terminals were connected nonselectively to nearby dendrites of 135,000 simulated lamina III-IV cells whose dendritic surface area distributions were based on intracellularly stained cells. There was an overall similarity among pre- and postsynaptic embryonic and adult somatotopies, with a progressive transformation of RF angular location as a function of RC, ML dorsal horn location from an initial embryonic presynaptic concentric pattern to an adult postsynaptic radial one. The initial embryonic dorsal horn cell RF assembly hypothesis was supported by the simulations, as was the additional hypothesis that further refinement of connections would be necessary to establish sufficient selectivity to account for observed adult RFs and somatotopy.

Afferent Pathways↗

PubMed2004

Nociceptors lacking TRPV1 and TRPV2 have normal heat responses.

Vanilloid receptor 1 (TRPV1) has been proposed to be the principal heat-responsive channel for nociceptive neurons. The skin of both rat and mouse receives major projections from primary sensory afferents that bind the plant lectin isolectin B4 (IB4). The majority of IB4-positive neurons are known to be heat-responsive nociceptors. Previous studies suggested that, unlike rat, mouse IB4-positive cutaneous afferents did not express TRPV1 immunoreactivity. Here, multiple antisera were used to confirm that mouse and rat have different distributions of TRPV1 and that TRPV1 immunoreactivity is absent in heat-sensitive nociceptors. Intracellular recording in TRPV1(-/-) mice was then used to confirm that TRPV1 was not required for detecting noxious heat. TRPV1(-/-) mice had more heat-sensitive neurons, and these neurons had normal temperature thresholds and response properties. Moreover, in TRPV1(-/-) mice, 82% of heat-responsive neurons did not express immunoreactivity for TRPV2, another putative noxious heat channel.

Amino Acid Sequence↗

PubMed2004

From innervation density to tactile acuity: 1. Spatial representation.

We tested the hypothesis that the population receptive field representation (a superposition of the excitatory receptive field areas of cells responding to a tactile stimulus) provides spatial information sufficient to mediate one measure of static tactile acuity. In psychophysical tests, two-point discrimination thresholds on the hindlimbs of adult cats varied as a function of stimulus location and orientation, as they do in humans. A statistical model of the excitatory low threshold mechanoreceptive fields of spinocervical, postsynaptic dorsal column and spinothalamic tract neurons was used to simulate the population receptive field representations in this neural population of the one- and two-point stimuli used in the psychophysical experiments. The simulated and observed thresholds were highly correlated. Simulated and observed thresholds' relations to physiological and anatomical variables such as stimulus location and orientation, receptive field size and shape, map scale, and innervation density were strikingly similar. Simulated and observed threshold variations with receptive field size and map scale obeyed simple relationships predicted by the signal detection model, and were statistically indistinguishable from each other. The population receptive field representation therefore contains information sufficient for this discrimination.

Animals↗

PubMed2003

Widespread projections from myelinated nociceptors throughout the substantia gelatinosa provide novel insights into neonatal hypersensitivity.

Skin sensory neurons have long been thought to undergo major changes in anatomy and physiology over the first few weeks of postnatal life. Low-threshold mechanoreceptors (LTMRs) are believed to project extensively throughout superficial dorsal horn laminas initially and provide the afferent limb for hyperactive nocifensive reflexes. However, our recent studies revealed that neonatal LTMRs do not project into "pain-specific" regions; instead, they exhibit adult-like anatomy shortly after birth. We sought to determine whether the same might be true for myelinated high-threshold mechanoreceptors (HTMRs). We used an intact, ex vivo somatosensory system preparation from neonatal mice to allow intrasomal recording and neurobiotin labeling of individual sensory neurons characterized via natural skin stimuli. Neonatal HTMRs displayed a number of key hallmarks of their adult counterparts; relative to LTMRs, they exhibited broader, inflected somal spikes and higher mechanical thresholds and/or responded in an increasingly vigorous manner to incrementally graded forces in a manner capable of encoding stimulus intensity. Two types were discerned on the basis of central anatomy: one subset projected to superficial laminas (I/II); the other gave rise to diffuse, dorsally recurving collateral arbors extending throughout the entire dorsal horn (I-V). The latter represent a novel cutaneous afferent morphology that persists in older animals. These studies reveal that inputs from myelinated afferents to superficial pain-specific laminas in neonates arise from HTMRs and not LTMRs as commonly thought. This frequently overlooked population is in a position, therefore, to contribute substantially to paradoxical nocifensive behaviors in neonates and various pain states in adults.

Action Potentials↗

PubMed2002

Glial cell line-derived neurotrophic factor is a survival factor for isolectin B4-positive, but not vanilloid receptor 1-positive, neurons in the mouse.

Most, if not all, nociceptor sensory neurons are dependent on nerve growth factor (NGF) during early embryonic development. A large subpopulation of these sensory neurons loses NGF dependency between embryonic day 16 and postnatal day 14 and become responsive to glial cell line-derived growth factor (GDNF), a member of the transforming growth factor beta (TGF-beta) family. To examine the survival and phenotypic effects of GDNF on sensory neurons in vivo, we generated transgenic mice that overexpress GDNF in the skin. GDNF-overexpresser mice had increased numbers of small unmyelinated sensory neurons that express the tyrosine kinase receptor Ret and bind the plant isolectin B4 (IB4). Surprisingly, in wild-type and transgenic mice, few ( approximately 2%) IB4-positive neurons expressed the vanilloid receptor VR1, a heat-sensitive receptor expressed by many IB4-positive neurons of the rat. Thus, in mouse, GDNF-dependent IB4-positive neurons must use a non-VR1 heat receptor. In addition, the behavior of GDNF-overexpresser animals to noxious heat or mechanical stimuli was indistinguishable from wild-type animals, indicating that, on a behavioral level, peripherally applied GDNF does not alter the sensitivity of the somatosensory system.

Animals↗

PubMed2002

Comprehensive phenotyping of sensory neurons using an ex vivo somatosensory system.

In adults, primary sensory neurons exhibit pronounced diversity in many phenotypic traits including peripheral response properties, somal spike shape, neurochemical content, and laminar distribution of projections in the spinal dorsal horn. While these traits are correlated with sensory submodality type, single traits are not sufficient to accurately characterize individual neurons. We have recently developed a novel mouse ex vivo somatosensory preparation that allows the examination of multiple traits for individual cutaneous sensory neurons. Here we describe the results from our initial studies of adult and neonatal mice employing this preparation. Adult mice were anesthetized and perfused with chilled oxygenated artificial cerebrospinal fluid (aCSF). The spinal cord, dorsal root ganglia (DRGs), several attached dorsal cutaneous nerves (DCNs), and skin were isolated and placed in a chamber with a circulating bath of oxygenated aCSF. Sensory neuron somata were impaled, their cutaneous mechanical response properties were determined, and then one soma/ganglion was injected with Neurobiotin (NB). Spinal cord sections containing the cell's central projections were reacted with horseradish peroxidase-conjugated avidin and visualized using diaminobenzidene. DRG sections were reacted with primary antisera for calcitonin gene-related peptide (CGRP), and the NB-stained somata were visualized using avidin-FITC. In these initial studies, neonatal cutaneous sensory neurons are found to be virtually miniature replicas of the their adult counterparts. These findings challenge long-held beliefs that the laminar distribution of the central projections and somal spike properties of cutaneous low-threshold mechanoreceptors (LTMRs) undergo delayed maturation during the first 2-3 weeks of postnatal development.

Animals↗