Zic2-dependent axon midline avoidance controls the formation of major ipsilateral tracts in the CNS.

In bilaterally symmetric organisms, interhemispheric communication is essential for sensory processing and motor coordination. The mechanisms that govern axon midline crossing during development have been well studied, particularly at the spinal cord. However, the molecular program that determines axonal ipsilaterality remains poorly understood. Here, we demonstrate that ipsilateral neurons whose axons grow in close proximity to the midline, such as the ascending dorsospinal tracts and the rostromedial thalamocortical projection, avoid midline crossing because they transiently activate the transcription factor Zic2. In contrast, uncrossed neurons whose axons never approach the midline control axonal laterality by Zic2-independent mechanisms. Zic2 induces EphA4 expression in dorsospinal neurons to prevent midline crossing while Robo3 is downregulated to ensure that axons enter the dorsal tracts instead of growing ventrally. Together with previous reports, our data reveal a critical role for Zic2 as a determinant of axon midline avoidance in the CNS across species and pathways.

Pruriceptive spinothalamic tract neurons: physiological properties and projection targets in the primate.

Itch of peripheral origin requires information transfer from the spinal cord to the brain for perception. Here, primate spinothalamic tract (STT) neurons from lumbar spinal cord were functionally characterized by in vivo electrophysiology to determine the role of these cells in the transmission of pruriceptive information. One hundred eleven STT neurons were identified by antidromic stimulation and then recorded while histamine and cowhage (a nonhistaminergic pruritogen) were sequentially applied to the cutaneous receptive field of each cell. Twenty percent of STT neurons responded to histamine, 13% responded to cowhage, and 2% responded to both. All pruriceptive STT neurons were mechanically sensitive and additionally responded to heat, intradermal capsaicin, or both. STT neurons located in the superficial dorsal horn responded with greater discharge and longer duration to pruritogens than STT neurons located in the deep dorsal horn. Pruriceptive STT neurons discharged in a bursting pattern in response to the activating pruritogen and to capsaicin. Microantidromic mapping was used to determine the zone of termination for pruriceptive STT axons within the thalamus. Axons from histamine-responsive and cowhage-responsive STT neurons terminated in several thalamic nuclei including the ventral posterior lateral, ventral posterior inferior, and posterior nuclei. Axons from cowhage-responsive neurons were additionally found to terminate in the suprageniculate and medial geniculate nuclei. Histamine-responsive STT neurons were sensitized to gentle stroking of the receptive field after the response to histamine, suggesting a spinal mechanism for alloknesis. The results show that pruriceptive information is encoded by polymodal STT neurons in histaminergic or nonhistaminergic pathways and transmitted to the ventrobasal complex and posterior thalamus in primates.

Hypothalamospinal oxytocinergic antinociception is mediated by GABAergic and opiate neurons that reduce A-delta and C fiber primary afferent excitation of spinal cord cells.

Recent results implicate a new original mechanism involving oxytocin (OT), as a mediator via descending fibers of the paraventricular hypothalamic nucleus (PVN), in antinociception and analgesia. In rats electrical stimulation of the PVN or topical application of OT selectively inhibits A-delta and C fiber responses in superficial dorsal horn neurons, and this inhibition is reversed by a selective OT antagonist. However, little is known about the mechanisms and the spinal elements participating in this phenomenon. Here we show that topical application of bicuculline blocks the effects produced by PVN electrical stimulation or OT application. PVN electrical stimulation also activates a subpopulation of neurons in lamina II. These PVN-On cells are responsible for the amplification of local GABAergic inhibition. This result reinforces the suggestion that a supraspinal descending control of pain processing uses a specific neuronal pathway in the spinal cord in order to produce antinociception involving a GABAergic interneuron. Moreover, the topical administration of naloxone or a mu-opiate receptor antagonist beta-funaltrexamine only partially blocks the inhibitory effects produced by OT application or PVN electrical stimulation. Thus, this OT mechanism only involves opiate participation to a minor extent. The OT-specific, endogenous descending pathway represents an interesting mechanism to resolve chronic pain problems in special the neuropathic pain.

Glutamine uptake contributes to central sensitization in the medullary dorsal horn.

Mustard oil application to tooth pulp produces central sensitization in rat medullary dorsal horn (MDH) nociceptive neurons, which has been implicated in persistent pain mechanisms. We found that superfusion onto MDH of methylaminoisobutyric acid, a competitive inhibitor of the neuronal system A transporter for presynaptic uptake of glutamine (a glutamate precursor released from astroglia), significantly depressed development of mustard oil-induced central sensitization in rat MDH nociceptive neurons. This finding indicates that the system A transporter is required for the expression of central sensitization and confirms the important roles of astroglia, glutamine and presynaptic modulation of glutamate release in the development of central sensitization.

Medullary dorsal horn neurons providing axons to both the parabrachial nucleus and thalamus.

It has often been suggested that the trigemino- and spino-thalamic pathways are highly implicated in sensory-discriminative aspects of pain, whereas the trigemino- and spino-parabrachial pathways are strongly implicated in affective/emotional aspects of pain. On the other hand, the superficial laminae of the spinal dorsal horn, where many nociceptive neurons are distributed, have been reported to contain projection neurons innervating both the parabrachial nucleus (PBN) and thalamus by way of axon collaterals (Hylden et al., 1989). For the medullary dorsal horn (caudal subnucleus of spinal trigeminal nucleus: Vc), however, the existence of such neurons has not been reported. Thus, in the present study, we examined whether the Vc might contain projection neurons sending their axons to both the thalamus and PBN. Dual retrograde labeling with fluorescence dyes was attempted. In each rat, tetramethylrhodamine-dextran amine and Fluoro-gold were stereotaxically injected into the PBN and thalamic regions, respectively. The proportion of the dually labeled Vc cells in the total population of all labeled Vc cells was about 20%. More than 90% of the dually labeled neurons were distributed in lamina I (marginal zone), less than 10% of them were located in lamina II (substantia gelatinosa), and only a few (about 1%) were found in lamina III (magnocellular zone). The results indicate that some Vc neurons in the superficial laminae mediate nociceptive information directly to the PBN and thalamus by way of axon collaterals and that the vast majority of them project to the ipsilateral PBN and contralateral thalamus.

Effects of electro-acupuncture on the expression of c-jun and c-fos in spared dorsal root ganglion and associated spinal laminae following removal of adjacent dorsal root ganglia in cats.

This study evaluated the plastic changes of c-jun and c-fos in the right sixth lumbar dorsal root ganglion (L6 DRG), Rexed's lamina II in representative spinal segments L3, L5, and L6 and in the nucleus dorsalis (ND) at L3 segments after electro-acupuncture (EA) in cats subjected to removal of L1-L5 and L7-S2 DRG. Following dorsal root ganglionectomy, there was a significant increase in the density of c-jun immunoreactivity in the neurons and glia in spinal lamina II and in the ND; there was also marked elevation in the expression of c-fos in ND. In both cases there was no change in the c-jun and c-fos immunoreactivity in the DRG. After EA in the operated animals, there was an up-regulation in the expression of c-jun in the L6 DRG and the associated spinal lamina II; however, increased c-fos expression was detected only in the L6 DRG. Western blot and RT-PCR were also performed to quantitatively explore the mRNA and protein expression changes in the spinal dorsal horn and associated DRG. Following partial deafferentation, there was a significant increase in the protein level of both c-jun and c-fos in the dorsal horn, while, in both cases there was no change in c-jun and c-fos protein and mRNA in the DRG. After EA in the operated animals, both c-jun protein and its mRNA in the L6 DRG as well as the associated dorsal horn of L6 spinal segment were upregulated, but increased c-fos protein and its mRNA was observed only in the L6 DRG. These findings suggested that c-jun and c-fos might be related to the acupuncture promoted spinal cord plasticity as reported previously.

A role for substance P in arthritis?

Substance P is a neuropeptide that is released from sensory nerves and which has a number of pro-inflammatory effects. In this article, we review the evidence for a role of substance P in arthritis, both in experimental animal models and rheumatoid arthritis patients. Substance P expression is altered in the joint and dorsal horn of arthritic animals, exogenous substance P and neurokinin 1 (NK(1)) receptor antagonists modulate responses in the joint, and there is some evidence for a role of substance P in human joint disease. However, the therapeutic potential of NK(1) receptor antagonists in the treatment of rheumatoid arthritis remains controversial.

Differentiation of lamina I spinomedullary and spinothalamic neurons in the cat.

We characterized spinomedullary neurons that project to the ventrolateral portion of the medulla that receives lamina I terminations in two sets of experiments in the cat. First, their distribution was examined using single unilateral iontophoretic injections of cholera toxin subunit B. The injection sites were characterized by microelectrode recordings from nociceptive- and thermoreceptive-specific units, indicative of lamina I input. The spinomedullary neurons were symmetrically distributed bilaterally, predominantly (63-69%) in lamina I but also in laminae V-VIII and the thoracic lateral horn (intermediolateral cell column). In horizontal sections, spinomedullary lamina I neurons included all three main morphological types described earlier. Second, spinomedullary and spinothalamic neurons were compared in retrograde double-labeling experiments. Different combinations of tracers were injected in the right thalamus and the left or right ventrolateral medulla (guided by recordings). The numbers of spinomedullary and spinothalamic neurons on the left side were comparable, and the segmental and laminar distributions were similar, except that a greater proportion of spinomedullary neurons originated from thoracic segments. However, the proportion of double-labeled neurons was consistently approximately 1%, indicating that spinomedullary and spinothalamic pathways arise from separate subpopulations. Spinomedullary neurons were more ventrally located within lamina I than spinothalamic neurons. A significantly greater proportion of spinomedullary neurons had fusiform somata (49% vs. 36%). These observations indicate that lamina I is the major source of spinal input to this portion of the ventrolateral medulla, that the projection includes several morphological types of inputs, and that this projection is distinct from the spinothalamic projection. These findings are consistent with the concept that lamina I projections constitute an ascending homeostatic afferent pathway relating the physiological condition of the body.

Separate, parallel sensory and hedonic pathways in the mammalian somatosensory system.

We propose that separate sensory and hedonic representations exist in each of the primary structures of the somatosensory system, including brainstem, thalamic and cortical components. In the dorsal horn of the spinal cord, the hedonic representation, which consists primarily of nociceptive-specific, wide dynamic range, and thermoreceptive neurons, is located in laminae I and II, while the sensory representation, composed primarily by low-threshold and wide dynamic range neurons, is found in laminae III through V. A similar arrangement is found in the caudal spinal trigeminal nucleus. Based on the available anatomical and electrophysiological data, we then determine the corresponding hedonic and sensory representations in the area of the dorsal column nuclei, ventrobasal and posterior thalamic complex, and cortex. In rodent primary somatosensory cortex, a hedonic representation can be found in laminae Vb and VI. In carnivore and primate primary and secondary somatosensory cortical areas no hedonic representation exists, and the activities of neurons in both areas represent the sensory aspect exclusively. However, there is a hedonic representation in the posterior part of insular cortex, bordering on retroinsular cortex, that receives projections from two thalamic areas in which hedonics are represented. The functions of the segregated components of the system are discussed, especially in relation to the subjective awareness of pain.

Stimulation of the greater occipital nerve induces increased central excitability of dural afferent input.

Patients with primary headaches often report pain that involves not only the front of the head, innervated by the first (ophthalmic) division of the trigeminal nerve, but also the back of the head, innervated by the greater occipital nerve (GON) that is a branch of the C(2) spinal root. The aim of this work was to study the physiology of trigeminocervical input in a model of cranial nociception by describing a population of nociceptive neurones that receive convergent input from the supratentorial dura and the GON. Rats were anaesthetized with pentobarbital, paralysed and ventilated. The parietal dura above the middle meningeal artery was stimulated through a closed cranial window. The GON was exposed in the neck and stimulated. Recordings were made from 67 neurones (52 wide dynamic range neurones/15 nociceptive-specific-neurones) in the C(2) spinal dorsal horn, which responded to stimulation of the dura and the GON. Most neurones showed receptive fields corresponding to the first trigeminal division as well as input from C(2) skin and muscle. Neurones were recorded in superficial and deep layers of the dorsal horn. All neurones tested received input from the ipsilateral and contralateral GON (n = 5). The responses to dural stimulation were analysed before and after stimulation of the GON. Supramaximal electrical stimulation of the GON (20 s to 5 min) enhanced afferent dural input in 8/20 neurones. Application of the C-fibre activator mustard oil (MO) to the cutaneous receptive field or suboccipital muscles innervated by the GON induced an increased excitability of dural responses in 8/12 and 9/10 neurones, respectively. Stimulation of muscle afferents had a significant longer facilitatory effect than cutaneous stimulation (P < 0.05). These results show that a considerable population of neurones show convergent input from both dura as well as cervical cutaneous and muscle territories, which supports the view of a functional continuum between the caudal trigeminal nucleus and upper cervical segments involved in cranial nociception. The facilitatory effect of GON stimulation on dural stimulation suggests a central mechanism at the second order neurone level. This mechanism may be important in pain referral from cervical structures to the head and therefore have implications for most forms of primary headache.

Reduction in inflammation-induced sensitization of dorsal horn neurons by transcutaneous electrical nerve stimulation in anesthetized rats.

Transcutaneous electrical nerve stimulation (TENS) is utilized to treat a variety of painful conditions. Inflamed animals present with an increased response to noxious stimuli, i.e., hyperalgesia, at the site of injury (primary hyperalgesia) and outside the site of injury (secondary hyperalgesia). Further, following acute inflammation, dorsal horn neurons show an increased responsiveness to peripherally applied stimuli, which has been termed sensitization. Previous studies demonstrate a reduction in dorsal horn neuron activity following TENS treatment in normal animals and a reduction in primary and secondary hyperalgesia in acutely inflamed animals. The purpose of this study was to examine the effects of TENS on dorsal horn neurons sensitized by acute inflammation. Extracellular recordings from wide dynamic range (WDR), high threshold (HT) and low threshold (LT) dorsal horn neurons in anesthetized rats were assessed for spontaneous activity, responses to innocuous and noxious mechanical stimulation and receptive field size. Responses were measured before and 3 h after induction of inflammation, and immediately and 1 h after application of either high (100 Hz) or low (4 Hz) frequency TENS (motor intensity, pulse duration = 100 microseconds). TENS was applied to the inflamed paw to encompass the receptive field of the neuron for 20 min. WDR and HT dorsal horn neurons sensitized to mechanical stimulation after induction of inflammation. Application of either high or low frequency TENS to the inflamed paw reduced both innocuous and noxious evoked responses of WDR and HT dorsal horn neurons immediately and 1 h after treatment with TENS. Comparison of responses after TENS with baseline responses showed that the evoked responses in the majority of WDR and HT cells returned to or fell below baseline responses. TENS had no effect on responses of LT neurons. In summary, central neuron sensitization is reduced by TENS and may underlie the reduction in hyperalgesia observed after treatment with TENS.

Non-opioid actions of lamotrigine within the rat dorsal horn after inflammation and neuropathic nerve damage.

Some opioid-resistant pain conditions can be alleviated by voltage-dependent Na(+) channel blockers such as lamotrigine. The mu-opioid-receptor agonist morphine can modulate cation entry into cells to affect overall cellular excitability, an effect which can in turn be endogenously antagonised by the neuropeptide cholecystokinin (CCK). However, lamotrigine may also modulate cellular excitability by non-specifically blocking voltage-dependent ion channels. We have looked for interactions of lamotrigine with the opioid/CCK pathway within the spinal dorsal horn, to rule out the possibility that lamotrigine may attenuate nociceptive responses via actions on this pathway. Both lamotrigine and the mu-opioid agonist DAMGO inhibited mustard oil-evoked cell firing by approximately 50% compared with control levels. Co-application of CCK8S reversed DAMGO-, but not lamotrigine-induced inhibition of cell firing and this reversal was prevented with the selective CCK(B) receptor antagonist PD 135158. Although lamotrigine inhibited both brush- and cold-evoked cell firing in neuropathic animals, lamotrigine inhibition of mustard oil-evoked cell firing in the same animals was not significantly greater than that observed in controls. These results suggest that the antinociceptive properties of lamotrigine within the spinal dorsal horn are unlikely to be mediated via interactions with the opioid/CCK pathway.

Lamination of spinal cells projecting to the zona incerta of rats.

In this study, the lamination patterns of spinal cells projecting to the zona incerta (ZI), intralaminar nuclei and ventral posterior nucleus of the thalamus have been explored. Injections of cholera toxin subunit B or latex beads were made into the ZI, intralaminar and ventral posterior nuclei of Sprague Dawley rats. The brain and spinal cord were then aldehyde fixed and processed using standard methods. Our results show two major findings. First, after injections into the ZI, there is a distinct pattern of lamination of labelled cells in the spinal cord, a pattern that changes across the different levels. At cervical levels, labelled cells are located within the medial region of the deep dorsal horn, while at lumbar and sacral levels, they are found in the intermediate grey matter. These results are similar to those seen after injections into the intralaminar or ventral posterior nuclei, except that in the latter cases, more labelled cells are located in the superficial laminae of the dorsal horn, particularly from the ventral posterior nucleus. Second, the ZI is not associated uniformly with all spinal levels; labelling is heaviest at cervical and lightest at thoracic levels. From each thalamic injection site, labelling is noted on both sides of the spinal cord, with a clear contralateral predominance. In conclusion, the results indicate that the ZI receives a distinct set of spinal projections principally from the cervical level. The particular pattern of lamination of spinal cells projecting to the ZI suggests that the type of information relayed is from deep somatic and/or visceral structures, and probably nociceptive in nature.

Enhanced phosphorylation of NMDA receptor 1 subunits in spinal cord dorsal horn and spinothalamic tract neurons after intradermal injection of capsaicin in rats.

The functional enhancement of NMDA receptors after peripheral tissue injury is proposed to contribute to the sensitization of spinothalamic tract (STT) cells and hyperalgesia. Protein phosphorylation is a major mechanism for the regulation of NMDA receptor function. In this study, Western blots, immunofluorescence double labeling, and the retrograde tracing method were used to examine whether phosphorylation of NMDA receptor 1 (NR1) subunits increases in spinal cord tissue and spinal dorsal horn neurons, especially in STT cells, after injection of capsaicin (CAP) into the glabrous skin of one hindpaw of anesthetized rats. Western blots showed that phosphorylated NR1 protein in spinal cord tissue was increased 30 min after CAP injection. Immunofluorescence double-labeling staining showed no significant difference in the number of the NR1-like immunoreactive neurons in laminae I-VII in the lumbosacral segments (L(4)-S(1)) on the ipsilateral and the contralateral sides 30 min after CAP or vehicle injection. However, the numbers of phospho-NR1-like immunoreactive neurons were significantly increased on the ipsilateral side compared with the vehicle injection group. STT cells were labeled by bilateral microinjections of the retrograde tracer fluorogold into the lateral thalamus, including the ventral-posterior lateral nucleus. Immunofluorescence staining was performed at 30, 60, and 120 min after CAP injection or at 30 min after vehicle injection. There was a significant increase in the proportion of STT cells with phosphorylated NR1 subunits compared either with the contralateral side 30 and 60 min after CAP injection or either side of animals after intradermal injection of vehicle. These results provide direct evidence that NMDA receptors in STT cells are phosphorylated after CAP injection.

Modulation of GABAergic synaptic transmission by the non-benzodiazepine anxiolytic etifoxine.

We have investigated the effects of 2-ethylamino-6-chloro-4-methyl-4-phenyl-4H-3,1-benzoxazine hydrochloride (etifoxine) on GABA(A) receptor function. Etifoxine displaced [(35)S]TBPS (t-butylbicyclophosphorothionate) from GABA(A) receptors of rat cortical membranes with an IC(50) of 6.7+/-0.8 microM and [(3)H]PK11195 from peripheral (mitochondrial)-type benzodiazepine receptors (PBRs) of rat heart homogenates with an IC(50) of 27.3+/-1.0 microM. Etifoxine displayed anxiolytic properties in an anticonflict test in rats, and potentiated GABA(A) receptor-mediated membrane currents elicited by submaximal (5-10 microM) but not saturating (0.5 mM) concentrations of GABA in cultured rat hypothalamic and spinal cord dorsal horn neurones. In hypothalamic cultures, etifoxine induced a dose-dependent inward current for concentrations >1 microM which reflected the post-synaptic potentiation of a small ( approximately 20 pA) tonic and bicuculline-sensitive GABA(A) receptor-gated Cl(-) current. Etifoxine also increased the frequency of spontaneous and miniature GABAergic inhibitory post-synaptic currents without changing their amplitude and kinetic characteristics. Both effects of etifoxine were insensitive to flumazenil (10 microM), an antagonist of central-type benzodiazepine sites present at GABA(A) receptors, but were partly inhibited by PK11195 (10 microM) an antagonist of PBRs which control the synthesis of neurosteroids. Our results indicate that etifoxine potentiates GABA(A) receptor-function by a direct allosteric effect and by an indirect mechanism involving the activation of PBRs.

[Spinal segment distribution of neural innervation related to houhai acupoint and compared with zusanli and dazhui acupoints in domestic chicken].

OBJECTIVE: The aim of this study was to demonstrate the spinal segmental innervation of Houhai, Dazhui and Zusanli acupoints in domestic chicken. METHODS: The horseradish peroxidase conjugated cholera toxin B subunit (CB-HRP) was injected into the regions of the three acupoints namely, Houhai Dazhui and Zusanli respectively in separate groups of chicken. The frozen sections of spinal cord and dorsal ganglia were prepare for labelling using TMB as a chromogen. RESULTS: (1) In Houhai group the anterograde transganlionic labelling terminals were densely distributed in the dorsal horn and dorsal commissure nucleus. The moto-neurons in ventral horn labelled retrogradely appeared fine arborization of dendrites. There were labelled cell bodies in the dorsal region of the ventral horn (lamina VII) or dorsal commissure nucleus. (2) In Dazhui group, the bundles of labelled fibers presented from laminea I and II to IX in the caudal cervical enlargements and two upper thoracic segments. The labelled motoneurons were shown in medial and lateral groups. The most numourous of labelled ganglionic cells appeared in this group. (3) The labelling sensory ganglionic neurons and their terminals in spinal cord related Zusanli acupoint were overlaped with those related Houhai acupoint. The largest numbers of the labelled ventral motoneurons occured in the Zusanli group. CONCLUSIONS: The results demontrated the patterns of segmental innervation corresponding respectively to Houhai, Dazhui, and Zusanli acupoints. The anterograde afferent labelling terminals and interrelationship of the dendritic arborization of retrograde labelling motoneurons (somatic or visceral) provided neuroanatomical evidences for the innervation of the acupoints.