Thermo-sensitive TRP channels in peripheral nerve injury: a review of their role in cold intolerance.

One of the sensory complications of traumatic peripheral nerve injury is thermal intolerance, which manifests in humans mainly as cold intolerance. It has a major effect on the quality of life, and adequate therapy is not yet available. In order to better understand the pathophysiological background of thermal intolerance, we focus first on the various transient receptor potential (TRP) channels that are involved in temperature sensation, including their presence in peripheral nerves and in keratinocytes. Second, the role of thermo-sensitive TRP channels in cold and heat intolerance is described showing three different mechanisms that contribute to thermal intolerance in the skin: (a) an increased expression of TRP channels on nerve fibres and on keratinocytes, (b) a lower activation threshold of TRP channels and (c) the sprouting of non-injured nerve fibres. Finally, the data that are available on the effects of TRP channel agonists and antagonists and their clinical use are discussed. In conclusion, TRP channels play a major role in temperature sensation and in cold and heat intolerance. Unfortunately, the available pharmaceutical agents that successfully target TRP channels and counteract thermal intolerance are still very limited. Yet, our focus should remain on TRP channels since it is difficult to imagine a reliable treatment for thermal intolerance that will not involve TRP channels.

Spinal glycinergic and GABAergic neurons expressing C-fos after capsaicin stimulation are increased in rats with contralateral neuropathic pain.

There is increasing evidence that pain transmission on one side of the body is influenced by a painful state on the other side. We have investigated this phenomenon by studying the activation pattern (using C-fos labeling) of spinal glycinergic and GABAergic (Gly/GABA) neurons after capsaicin injection in the ipsilateral hind paw of rats that were preconditioned with an acute or chronic pain stimulus in the contralateral hind paw or rats that were not preconditioned (control). For this purpose, fluorescent in situ hybridization with GlyT2 and GAD67 mRNA probes was combined with fluorescent C-fos immunohistochemistry. Rats were preconditioned with acute (capsaicin, Complete Freund's Adjuvant (CFA) 1.5 h), chronic inflammatory (CFA 20 h and 4 days), neuropathic (spared nerve injury (SNI) 2 weeks), or control pain stimuli (saline 20 h and 4 days; sham-SNI 2 weeks). We found that after capsaicin injection in rats preconditioned with CFA inflammation (4 days), sham-SNI or with SNI neuropathic pain, the numbers (27 ± 3, 21 ± 2, and 21 ± 2, respectively) and percentages (55% ± 4, 43% ± 2, and 42% ± 2, respectively) of C-fos activated neurons that were Gly/GABA increased significantly as compared with control (10 ± 1 and 25% ± 2). The increase in the total number of C-fos activated Gly/GABA neurons was present primarily in the superficial dorsal horn (laminae I and II; control: 9%; CFA 4 days: 56%; SNI 2 weeks: 42%). This increase in C-fos activation of Gly/GABA neurons occurred without significant changes in the total number of C-fos activated neurons, and without any significant changes in the mechanical thresholds in the hind paws after capsaicin injection. The results showed that one-sided chronic pain, especially inflammation, significantly increases the C-fos activation pattern of spinal Gly/GABA neurons on the other side of the spinal cord. This further underlines the existence of a dynamic interaction between ipsi- and contralateral spinal neurons in the processing of nociceptive information.

Intrathecal injection of GDNF and BDNF induces immediate early gene expression in rat spinal dorsal horn.

Glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) are potent trophic factors for dorsal root ganglion cells. In addition, these factors are produced in subsets of dorsal root ganglion cells and transported anterogradely to their terminals in the superficial dorsal horn of the spinal cord, where they constitute the only source of GDNF and BDNF. We investigated the effect of 10 mug GDNF and BDNF injected by lumbar puncture on the expression of the immediate early gene (IEG) products c-Fos, c-Jun, and Krox-24 in the adult rat dorsal horn. In the dorsal horn of S1 spinal segments, GDNF and BDNF induced a strong increase in IEG expression, which was most pronounced in laminae I and II (2.9- to 4.5-fold). More distal from the injection site, in the dorsal horn of L1/L2 spinal segments, the increase in IEG expression was less pronounced, suggesting a concentration-dependent effect. In order to explain the effects of intrathecally injected GDNF, we investigated whether lumbo-sacral dorsal horn neurons expressed RET protein, the signal-transducing element of the receptor complex for GDNF. It was found that several of these neurons contained RET immunoreactivity and that some of the RET-labeled neurons had the appearance of nociceptive-specific cells, confirming their presumed role in pain transmission. Additionally, using double-labeling immunofluorescence combined with confocal microscopy, it was found that after intrathecal GDNF injection 35% of c-Fos-labeled cells were also labeled for RET. These results demonstrate that intrathecally administered GDNF and BDNF induce IEG expression in dorsal horn neurons in the adult rat, supposedly by way of their cognate receptors, which are present on these neurons. We further suggest that the endogenous release of GDNF and BDNF, triggered by nociceptive stimuli, is involved in the induction of changes in spinal nociceptive transmission as in various pain states.

CuZn superoxide dismutase (SOD1) accumulates in vacuolated mitochondria in transgenic mice expressing amyotrophic lateral sclerosis-linked SOD1 mutations.

Cytosolic Cu/Zn superoxide dismutase (SOD1) is a ubiquitous small cytosolic metalloenzyme, which catalyses the conversion of superoxide anion to hydrogen peroxide. Mutations in the SOD1 gene cause a familial form of amyotrophic lateral sclerosis (fALS). The mechanism by which mutant SOD1s cause the degeneration of motor neurons is not understood. Transgenic mice expressing multiple copies of fALS-mutant SOD1s develop an ALS-like motor neuron disease. Vacuolar degeneration of mitochondria has been identified as the main pathological feature associated with motor neuron death and paralysis in several lines of fALS-SOD1 mice. Using confocal and electron microscopy we show that mutant SOD1 is present at a high concentration in vacuolated mitochondria, where it colocalises with cytochrome c. Mutant SOD1 is also present in mildly swollen mitochondria prior to the appearance of vacuoles, suggesting that the leakage or translocation of mutant human SOD1 in mitochondria may be the primary event triggering their further degeneration. Vacuolated mitochondria containing SOD1 also occur in transgenic mice expressing a high concentration of wildtype human SOD1. In sum, our data suggest that both fALS-mutant and wild-type SOD1 may cross the mitochondrial outer membrane, and by doing so induce the degeneration of these mitochondria.

Human Cu/Zn superoxide dismutase (SOD1) overexpression in mice causes mitochondrial vacuolization, axonal degeneration, and premature motoneuron death and accelerates motoneuron disease in mice expressing a familial amyotrophic lateral sclerosis mutant SOD1.

Cytosolic Cu/Zn superoxide dismutase (SOD1) is a ubiquitous small cytosolic metalloenzyme that catalyzes the conversion of superoxide anion to hydrogen peroxide (H(2)O(2)). Mutations in the SOD1 gene cause a familial form of amyotrophic lateral sclerosis (fALS). The mechanism by which mutant SOD1s causes ALS is not understood. Transgenic mice expressing multiple copies of fALS-mutant SOD1s develop an ALS-like motoneuron disease resembling ALS. Here we report that transgenic mice expressing a high concentration of wild-type human SOD1 (hSOD1(WT)) develop an array of neurodegenerative changes consisting of (1) swelling and vacuolization of mitochondria, predominantly in axons in the spinal cord, brain stem, and subiculum; (2) axonal degeneration in a number of long fiber tracts, predominantly the spinocerebellar tracts; and (3) at 2 years of age, a moderate loss of spinal motoneurons. Parallel to the development of neurodegenerative changes, hSOD1(WT) mice also develop mild motor abnormalities. Interestingly, mitochondrial vacuolization was associated with accumulation of hSOD1 immunoreactivity, suggesting that the development of mitochondrial pathology is associated with disturbed SOD1 turnover. In this study we also crossed hSOD1(WT) mice with a line of fALS-mutant SOD1 mice (hSOD1(G93A)) to generate "double" transgenic mice that express high levels of both wild-type and G93A mutant hSOD1. The "double" transgenic mice show accelerated motoneuron death, earlier onset of paresis, and earlier death as compared with hSOD1(G93A) littermates. Thus in vivo expression of high levels of wild-type hSOD1 is not only harmful to neurons in itself, but also increases or facilitates the deleterious action of a fALS-mutant SOD1. Our data indicate that it is important for motoneurons to control the SOD1 concentration throughout their processes, and that events that lead to improper synthesis, transport, or breakdown of SOD1 causing its accumulation are potentially dangerous.

Neurotrophins: peripherally and centrally acting modulators of tactile stimulus-induced inflammatory pain hypersensitivity.

Brain-derived neurotrophic factor (BDNF) is expressed in nociceptive sensory neurons and transported anterogradely to the dorsal horn of the spinal cord where it is located in dense core vesicles in C-fiber terminals. Peripheral inflammation substantially up-regulates BDNF mRNA and protein in the dorsal root ganglion (DRG) in a nerve growth factor-dependent fashion and results in novel expression of BDNF by DRG neurons with myelinated axons. C-fiber electrical activity also increases BDNF expression in the DRG, and both inflammation and activity increase full-length TrkB receptor levels in the dorsal horn. Sequestration of endogenous BDNF/neurotrophin 4 by intraspinal TrkB-Fc fusion protein administration does not, in noninflamed animals, change basal pain sensitivity nor the mechanical hypersensitivity induced by peripheral capsaicin administration, a measure of C fiber-mediated central sensitization. TrkB-Fc administration also does not modify basal inflammatory pain hypersensitivity, but does block the progressive hypersensitivity elicited by low-intensity tactile stimulation of inflamed tissue. BDNF, by virtue of its nerve growth factor regulation in sensory neurons including novel expression in A fibers, has a role as a central modulator of tactile stimulus-induced inflammatory pain hypersensitivity.

Depletion of GDNF from primary afferents in adult rat dorsal horn following peripheral axotomy.

Glial cell line-derived neurotrophic factor (GDNF) is produced in a subset of adult rat spinal ganglion neurons and anterogradely transported to the superficial dorsal horn. In this study the effect of sciatic nerve axotomy on the expression of GDNF protein in the dorsal horn was investigated, using immunocytochemistry. Image analysis showed a 44% decrease relative to the non-transected side after 5 days survival, progressing to more than 80% decrease after 10 days and remaining so for at least 100 days. This rapid and strong decrease suggests active down-regulation of GDNF protein after peripheral axotomy. The observed down-regulation of GDNF is compared with changes observed for other substances in primary afferents after peripheral axotomy and is discussed in light of its presumed trophic or transmitter role in nociception.

Immunocytochemical localization of GDNF in primary afferents of the lumbar dorsal horn.

Immunocytochemistry was used to identify glial cell line-derived neurotrophic factor (GDNF) in rat spinal cord. Strong GDNF labeling was found in fibers and terminals in laminae I and II (outer) and to a lesser extent in the remaining laminae. A few spinal ganglion cells also contained GDNF. After dorsal root transection GDNF disappeared from the dorsal horn and after dorsal root ligation there was accumulation of GDNF only on the ganglion side of the ligation. These findings demonstrate anterograde transport of GDNF within primary afferent fibers, which constitute the only source of GDNF labeling in the dorsal horn. The strong presence of GDNF in the superficial dorsal horn may indicate that GDNF has a role in pain transmission in the adult rat spinal cord.

Light microscopic and ultrastructural investigation of the dopaminergic innervation of the ventrolateral outgrowth of the rat inferior olive.

The ventrolateral outgrowth of the inferior olive is involved in the control of compensatory eye movement responses to optokinetic stimuli about the horizontal axis that is perpendicular to the ipsilateral anterior semicircular canal. Combining immunocytochemistry with retrograde tracing of WGA-BSA-gold, we demonstrated in the present study that this olivary subnucleus receives a substantial dopaminergic input, and that the prerubral parafascicular area and its surrounding regions form the sole source of this input. In addition, we investigated the postsynaptic distribution of the dopaminergic terminals in the inferior olive at the ultrastructural level. About a third (32%) of the dopaminergic terminals was found to make synaptic contacts in the olivary neuropil. The majority (81%) of these boutons terminated on cell bodies or extraglomerular dendrites, while the remaining terminals contacted dendritic spines inside glomeruli. In contrast, GABAergic terminals in the inferior olive formed more frequently (66%) synaptic contacts and they terminated more frequently (38%) in glomeruli. Thus, the ventrolateral outgrowth receives a dopaminergic input from the mesodiencephalic junction, and the postsynaptic distribution of this input reveals a characteristic pattern.

A differential and time-dependent decrease in AMPA-type glutamate receptor subunits in spinal motoneurons after sciatic nerve injury.

After sciatic transection a strong decrease in immunoreactivity occurred, starting at 2 days. After 6, 10, 14, and 20 days survival only 5% of the sciatic motoneurons were strongly labeled for GluR2/3 against 80% in the control situation. From Day 20, GluR2/3 labeling started to increase again, reaching near normal levels at Day 80 after sciatic transection. In contrast, after sciatic crush, the decrease in GluR2/3 labeling in motoneurons was less pronounced and returned to normal in 30 days. In all animals, the GluR1 and GluR4 labeling of motoneurons remained unchanged after sciatic transection or crush. It is concluded that sciatic nerve injury leads to a strong, time-dependent decrease in the expression of GluR2 and 3 subunits in the corresponding motoneurons. As a consequence, AMPA receptors with a different subunit composition may be assembled, leading to a change in the functional properties of these receptors. Moreover, if they lack the GluR2 subunit, they may become calcium permeable.

Correlating sheet plastinated slices, computed tomography images and magnetic resonance images of the pelvic girdle: a teaching tool.

Sheet plastination is currently used to produce anatomical slices of different body structures, allowing one to study and teach their topography in an anatomically correct state. Correlation with computed tomography (CT) and magnetic resonance imaging (MRI) techniques gives more insight into their anatomy. Using two female cadaver pelvises CT and MRI were performed. One pelvis was used to prepare 2-mm-thick coronal plastinated slices according to the technique described by von Hagens. We found a good overall correlation between plastinated slices, CT and MRI images. This combined approach provides a unique anatomical insight and is a valuable addition to other teaching tools used by medical students, radiologists and anatomists.

Distribution of dopamine immunoreactivity in the rat, cat and monkey spinal cord.

In the present study, the distribution of dopamine (DA) was identified light microscopically in all segments of the rat, cat, and monkey spinal cord by using immunocytochemistry with antibodies directed against dopamine. Only fibers and (presumed) terminals were found to be immunoreactive for DA. Strongest DA labeling was present in the sympathetic intermediolateral cell column (IML). Strong DA labeling, consisting of many varicose fibers, was found in all laminae of the dorsal horn, including the central canal area (region X), but with the exception of the substantia gelatinosa, which was only sparsely labeled, especially in rat and monkey. In the motoneuronal cell groups DA labeling was also strong and showed a fine granular appearance. The sexually dimorphic cremaster nucleus and Onuf's nucleus (or its homologue) showed a much stronger labeling than the surrounding somatic motoneurons. In the parasympathetic area at sacral levels, labeling was moderate. The remaining areas, like the intermediate zone (laminae VI-VIII), were only sparsely innervated. The dorsal nucleus (column of Clarke) showed the fewest DA fibers, as did the central cervical nucleus, suggesting that cerebellar projecting cells were avoided by the DA projection. In all species, the descending fibers were located mostly in the dorsolateral funiculus, but laminae I and III also contained many rostrocaudally oriented fibers. It is concluded that DA is widely distributed within the spinal cord, with few differences between species, emphasizing that DA plays an important role as one of the monoamines that influences sensory input as well as autonomic and motor output at the spinal level.

Induction of c-Jun immunoreactivity in spinal cord and brainstem neurons in a transgenic mouse model for amyotrophic lateral sclerosis.

Transgenic mice carrying amyotrophic lateral sclerosis (ALS)-linked superoxide dismutase 1 (SOD1) mutations develop a motoneuron disease resembling human ALS. c-Jun is a transcription factor frequently induced in injured neurons. In this study we have examined the distribution of c-Jun-immunoreactivity in the brainstem and spinal cord of transgenic SOD1 mice with a glycine 93 alanine (G93A) mutation. In non-transgenic littermates c-Jun immunostaining was predominantly situated in motoneurons. The number of c-Jun immunoreactive motoneuron was reduced in SOD1(G93A) mice due to pronounced loss of motoneurons. In SOD1(G93A) mice, however, c-Jun-immunoreactivity was strongly induced in neurons in the intermediate zone (Rexed's laminae V-VIII and X) of the spinal cord and throughout the brainstem reticular formation. These findings are of interest since increased levels of c-jun also have been found in the intermediate zone of the spinal cord of ALS patients. This c-Jun may be involved in the neurodegenerative processes both in ALS and in motoneuron disease in SOD1(G93A) mice.

Localization of dopamine D2 receptor in rat spinal cord identified with immunocytochemistry and in situ hybridization.

In the present study the distribution of dopamine D2 receptors in rat spinal cord was determined by means of immunocytochemistry using an anti-peptide antibody, directed against the putative third intracellular loop of the D2 receptor and in situ hybridization (ISH) using a [35S]UTP labelled anti-sense riboprobe. With the immunocytochemical technique, labelling was confined to neuronal cell bodies and their proximal dendrites. Strongest labelling was present in the parasympathetic area of the sacral cord and in two sexually dimorphic motor nuclei of the lumbosacral cord, the spinal nucleus of the bulbocavernosus and the dorsolateral nucleus. Moderately labelled cells were present in the intermediolateral cell column, the area around the central canal and lamina I of the dorsal horn. Weak labelling was present in the lateral spinal nucleus and laminae VII and VIII of the ventral horn. Except for the two sexually dimorphic motornuclei of the lumbosacral cord labelled motoneurons were not encountered. With the ISH technique radioactive labelling was present in many neurons, indicating that they contained D2 receptor mRNA. The distribution of these neurons was very similar to the distribution obtained with immunocytochemistry, but with ISH additional labelled cells were detected in laminae III and IV of the dorsal horn, which were never labelled with immunocytochemistry. The present study shows that the D2 receptor is expressed in specific areas of the rat spinal cord. This distribution provides anatomical support for the involvement of D2 receptors in modulating nociceptive transmission and autonomic control. Our data further indicate that D2 receptors are not directly involved in modulating motor functions with the exception, possibly, of some sexual motor functions.

Inhibitory synaptic inputs to the oculomotor nucleus from vestibulo-ocular-reflex-related nuclei in the rabbit.

Studies of the pathways involved in the vestibulo-ocular reflex have suggested that the projection from the superior vestibular nucleus to the ipsilateral oculomotor nucleus is inhibitory, whereas the medial vestibular nucleus, the abducens nucleus and the contralateral superior vestibular nucleus most likely exert excitatory effects on oculomotor neurons. In order to determine directly the termination pattern and the neurotransmitter of these afferents, we studied their input to the oculomotor nucleus in the rabbit at the light microscopic level with the use of anterograde tracing of Phaseolus vulgaris-leucoagglutinin combined with retrograde tracing of horseradish peroxidase from the extraocular muscles, and at the ultrastructural level with the use of anterograde tracing of wheatgerm-agglutinated horseradish peroxidase combined with GABA and glycine postembedding immunocytochemistry. The general ultrastructural characteristics of the neuropil and the types of boutons observed in the rabbit oculomotor nuclei are in general agreement with the descriptions for the oculomotor complex of other mammals. The superior vestibular nucleus projected bilaterally to the superior rectus and inferior oblique subdivisions, and ipsilaterally to the inferior rectus and medial rectus subdivision; the medial vestibular nucleus projected bilaterally to the medial rectus, inferior oblique, inferior rectus and superior rectus subdivisions with a strong contralateral predominance. The abducens nucleus projected contralaterally to the medial rectus subdivision. More than 90% of all the anterogradely labeled terminals from the ipsilateral superior vestibular nucleus were GABAergic. These terminals were characterized by flattened vesicles and symmetric synapses, and they contacted somata, as well as proximal and distal dendrites of motoneurons. All terminals derived from the medial vestibular nucleus the abducens nucleus and the contralateral superior vestibular nucleus were non-GABAergic. These non-GABAergic terminals showed spherical vesicles and asymmetric synapses, and they contacted predominantly distal dendrites. None of the anterogradely labeled terminals from the studied vestibular nuclei or abducens nucleus were glycinergic. The present study provides the first direct anatomical evidence that most, if not all, of the synaptic input from the superior vestibular nucleus to the ipsilateral oculomotor nucleus is GABAergic, and that the medial rectus subdivision is included in the termination area. Furthermore, it confirms that the projections from the medial vestibular nucleus, the abducens nucleus and the contralateral superior vestibular nucleus are exclusively non-GABAergic.

GABA and glycine frequently colocalize in terminals on cat spinal motoneurons.

In this ultrastructural study the colocalization of gamma-amino butyric acid (GABA) and glycine in terminals within cat lumbar motoneuronal cell groups was investigated and the frequency of this colocalization was determined. For this purpose the post-embedding immunogold technique was applied on serial sections, using antibodies directed against either GABA or glycine. Analysis of all labelled terminals in a random area of cat motoneuronal cell groups showed that 25 +/- 5% were labelled for GABA only, 29 +/- 6% were labelled for glycine only and 46 +/- 9% were labelled for both GABA and glycine, meaning that nearly two out of every three GABA-labelled terminals were also labelled for glycine and vice versa. Based on these results and on other data suggesting a high frequency of colocalization, it is concluded that in cat motoneuronal cell groups colocalization of GABA and glycine is the rule rather than the exception.

Colocalization of GABA and glycine in the rabbit oculomotor nucleus.

In the present study we examined the possible colocalization of the inhibitory neurotransmitters glycine and GABA in the oculomotor nucleus of the rabbit. Serial sections were processed alternately for glycine and GABA postembedding immuno-cytochemistry. Ultrastructural analysis revealed that all terminals that showed glycine-positive immunoreactivity were also GABA positive; up to 5% of the GABA-positive terminals were also glycine positive.

A glycinergic projection from the ventromedial lower brainstem to spinal motoneurons. An ultrastructural double labeling study in rat.

In the present study it was determined whether glycine was present in the descending brainstem projections to spinal motoneurons in the rat. For this purpose injections of wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) were made in the ventromedial part of the lower brainstem at the levels of the rostral inferior olive and the caudal facial nucleus. After perfusion, WGA-HRP histochemistry was performed, followed by the postembedding immunogold technique with an antibody against glycine. Electron microscopical examination of the lumbar motoneuronal cell groups showed that 15% of the WGA-HRP labeled terminals, derived from the ventromedial reticular formation, were also labeled for glycine. The majority (91%) of these double labeled terminals were of the F-type (containing many flattened vesicles), while the remaining 9% were of the S-type (containing mostly spherical vesicles). Many of the double labeled terminals established a synapse, mostly with proximal and distal dendrites. The present data, combined with our previous findings that 40% of the projections from the same ventromedial brainstem area to lumbar motoneurons contained gamma-aminobutyric acid (GABA), indicate that over 50% of these brainstem projections contain GABA and/or glycine, exerting a direct inhibitory effect on spinal motoneurons. The possibility that the glycinergic fibers within these projections play an important role in producing muscle atonia during rapid eye movement (REM) sleep is discussed.