Mengliang Zhang

Spinal Sensorimotor Transformation: Relation between Cutaneous Somatotopy and a Reflex Network

The projection of primary afferents onto spinal interneurons constitutes the first step in sensorimotor transformations performed by spinal reflex systems. Despite extensive studies on spinal somatotopy, uncertainties remain concerning the extent and significance of representational overlap and relation to spinal reflex circuits. To address these issues, the cutaneous projection from the hindpaw and its relation to the topography of lamina V neurons encoding withdrawal reflex strength (“reflex encoders”) was studied in rats. Thin and coarse primary afferent terminations in laminas II and III–IV, respectively, were mapped by wheat germ agglutinin-horseradish peroxidase and choleragenoid tracing. The functional weights of these projections were characterized by mapping nociceptive and tactile field potentials and compared with the topography of reflex encoders. Both anatomical and physiological data indicate that thin and coarse skin afferent input is spatially congruent in the horizontal plane. The representation of the hindpaw in the spinal cord was found to be intricate, with a high degree of convergence between the projections from different skin sites. “Somatotopic disruptions” such as the representation of central pads medial to that of the digits were common. The weight distribution of the cutaneous convergence patterns in laminas III–IV was similar to that of lamina V reflex encoders. This suggests that the cutaneous convergence and features such as somatotopic disruptions have specific relations to the sensorimotor transformations performed by reflex interneurons in the deep dorsal horn. Hence, the spinal somatotopic map may be better understood in light of the topography of such reflex systems.

The time course of serotonin 2A receptor expression after spinal transection of rats: an immunohistochemical study.

Hyperexcitability of motoneurons is one of the key mechanism that has been demonstrated to underlie the pathogenesis of spasticity after spinal injury. Serotonin (5-HT) denervation supersensitivity is one of the mechanisms underlying this increased motoneuron excitability. In this study, to examine whether the supersensitivity is caused by 5-HT receptor upregulation we investigated changes in levels of 5-HT2A receptor immunoreactivity (5-HT2AR-IR) following a spinal transection in the sacral spinal cord of rats at seven different time points post injury: 2, 8, 16 h, and 1, 2, 7 and 28 days, respectively. 5-HT2AR-IR density was analyzed in motoneurons (regions containing their somata and dendrites) in the spinal segments below the lesion. The results showed no significant changes in 5-HT2AR-IR in the motoneurons up to 16 h following the transection. After 1-day, however the levels of 5-HT2AR-IR increased in the motoneurons and their dendrites, with the density level being 3.4-fold higher in spinalized rats than in sham-operated rats. The upregulation increased progressively until a maximal level was reached at 28 days post-injury. We also investigated 5-HT and 5-HT transporter expressions at five different post injury time points: 1, 2, 7, 21 and 60 days and they showed concurrent down-regulation changes after 2 days. These results suggest that the upregulation of 5-HT2ARs may at least partly underlie the development of 5-HT denervation supersensitivity in spinal motoneurons following spinal injury and thereby implicates their involvement in the pathogenesis of the subsequent development of spasticity.

Intrinsic Properties of Mouse Lumbar Motoneurons Revealed by Intracellular Recording In Vivo

We have developed an in vivo model for intracellular recording in the adult anesthetized mouse using sharp microelectrode electrodes as a basis for investigations of motoneuron properties in transgenic mouse strains. We demonstrate that it is possible to record postsynaptic potentials underlying identified circuits in the spinal cord. Forty-one motoneurons with antidromic spike potentials (>50 mV) from the sciatic nerve were investigated. We recorded the intrinsic properties of the neurons, including input resistance (mean: 2.4 +/- 1.2 MOmega), rheobase (mean: 7.1 +/- 5.9 nA), and the duration of the afterhyperpolarization (AHP; mean: 55.3 +/- 14 ms). We also measured the minimum firing frequencies (F(min), mean 23.5 +/- 5.7 SD Hz), the maximum firing frequencies (F(max); >300 Hz) and the slope of the current-frequency relationship (f-I slope) with increasing amounts of current injected (mean: 13 +/- 5.7 Hz/nA). Signs of activation of persistent inward currents (PICs) were seen, such as accelerations of firing frequency or jumps in the membrane potential with increasing amounts of injected current. It is likely that the particular anesthetic regime with a mixture of Hypnorm and midazolam is essential for the possibility to evoke PICs. The data demonstrate that mouse spinal motoneurons share many of the same properties that have been demonstrated previously for cat, rat, and human motoneurons. The shorter AHP duration, steeper f-I slopes, and higher F(min) and F(max) than those in rats, cats, and humans are likely to be tailored to the characteristics of the mouse muscle contraction properties.

Robust upregulation of serotonin 2A receptors after chronic spinal transection of rats: an immunohistochemical study.

It is well known that spinal motoneurons below a spinal transection become supersensitive to a systemic administration of serotonin (5-HT) precursors, such as 5-hydroxytryptophan. This supersensitivity has been implicated in both the process of functional recovery following chronic lesions, and also in the development of symptoms such as hyperreflexia and spasticity. However, the mechanisms of this denervation supersensitivity are still largely unknown. In this study we have investigated the changes in 5-HT2A receptor immunoreactivity following chronic spinal transections at the level of the sacrocaudal spinal cord. The results show that in the spinalized rats the immunoreactivity of 5-HT2A receptors below the lesion is dramatically increased in the motoneuron soma and its proximal dendritic territory, most likely also in their distal dendritic territory, to a level 3-5-fold higher than that of sham-operated rats. We also found a small number of intraspinal 5-HT neurons and clusters of 5-HT fibers and their varicosities in the spinal cord caudal to the lesion, which may provide an intrinsic source of 5-HT to act upon the upregulated 5-HT2A receptors. These results indicate that the upregulation of 5-HT2A receptors at least partly underlies the 5-HT denervation supersensitivity of spinal motoneurons after a complete spinal transection.

Brainstem and Thalamic Projections from a Craniovascular Sensory Nervous Centre in the Rostral Cervical Spinal Dorsal Horn of Rats

To examine the ascending projections from the headache-related trigeminocer-vical complex in rats, biotinylated dextran amine (BDA) was injected into the ventrolateral dorsal horn of segments C1 and C2, a region previously demonstrated to receive input from sensory nerves in cranial blood vessels. Following injections into laminae I–II, BDA-labelled terminations were found bilaterally in several nuclei in the pons and the midbrain, including the pontine reticular nucleus, the parabrachial nuclei, the cuneiform nucleus and the periaqueductal grey. In the diencephalon, terminations were confined to the contralateral side and evident foremost in the posterior nuclear group, especially its triangular part, and in the ventral posteromedial nucleus. Following injections extending through laminae I-IV, anterograde labelling was more extensive. Some of the above regions are likely to be involved in the central processing of noxious signals of craniovascular origin and therefore putatively involved in mechanisms associated with primary headaches.

Central projections of sensory innervation of the rat superficial temporal artery

Elucidating the central sensory projection pathways of extra- and intracranial vessels appears to be of fundamental importance for understanding the pathogenetic mechanisms of primary headaches. In this paper, two kinds of tracers, choleragenoid (cholera toxin subunit b, CTb) and wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP), were used to transganglionically label the central sensory projections of the innervation of the superficial temporal artery (STA). Following either of the tracers applied on the adventitia of the STA, labelled terminations were found mainly in the ipsilateral C1-C3 spinal dorsal horns. Sparse labelling was also found in the interpolar and caudal parts of the spinal trigeminal nucleus. In the spinal cord, CTb labelled profiles were mainly located in laminae III and IV, whereas WGA-HRP labelled profiles were mainly located in laminae I and II. In the medulla, CTb but not WGA-HRP labelled terminals were found in a small dorsolateral extension of the cuneate nucleus. The present results indicate that the primary sensory nervous center of the STA is located in the rostral cervical spinal dorsal horn. The caudal parts of the spinal trigeminal nucleus, which has been demonstrated as a center of pain and temperature sensations of the head and face, transmits limited information from the STA to higher nervous centers.

Expression of calcium channel CaV1.3 in cat spinal cord: light and electron microscopic immunohistochemical study.

In spinal neurons, plateau potentials serve to amplify neuronal input signals. To a large extent, the underlying persistent inward current is mediated by a subtype of the L‐type calcium channel (CaV1.3). In the present investigation, we have studied its distribution and cellular localization in the cat spinal cord by light and electron microscopic immunohistochemistry. The results show that CaV1.3‐like immunoreactivity is widely distributed in all segments of the spinal cord but that the distribution in the different laminae of the spinal gray matter varies, with the highest density of labeled neurons in lamina IX and the lowest in lamina II. The labeling intensity was highest in neuronal somata, but a certain length of the proximal dendrite was also labeled. Some neuronal groups exhibited a particularly dense labeling; these include the lateral motoneuronal group in the cervical and the lumbar enlargements and the phrenic nucleus in cervical, Clarkes nucleus in lower thoracic and upper lumbar, and Onufs nucleus in upper sacral segments. At the ultrastructural level, CaV1.3‐immunoreactive products were found in neuronal somata and dendrites of different sizes. In the soma, they were predominantly associated with the rough endoplasmic reticulum but some also with the plasma membrane. In dendrites, they were associated with both intracellular organelles, including microtubules and microchondria, and the plasma membrane. These results indicate that significant proportions of the neurons in cat spinal cord, including projection neurons, interneurons, and motoneurons, are endowed with ion channels that subserve persistent inward currents and act to amplify synaptic input signals. J. Comp. Neurol. 507:1109–1127, 2008.

Spinal cord injury enables aromatic L-amino acid decarboxylase cells to synthesize monoamines.

Serotonin (5-HT), an important modulator of both sensory and motor functions in the mammalian spinal cord, originates mainly in the raphe nuclei of the brainstem. However, following complete transection of the spinal cord, small amounts of 5-HT remain detectable below the lesion. It has been suggested, but not proven, that this residual 5-HT is produced by intraspinal 5-HT neurons. Here, we show by immunohistochemical techniques that cells containing the enzyme aromatic l-amino acid decarboxylase (AADC) occur not only near the central canal, as reported by others, but also in the intermediate zone and dorsal horn of the spinal gray matter. We show that, following complete transection of the rat spinal cord at S2 level, AADC cells distal to the lesion acquire the ability to produce 5-HT from its immediate precursor, 5-hydroxytryptophan. Our results indicate that this phenotypic change in spinal AADC cells is initiated by the loss of descending 5-HT projections due to spinal cord injury (SCI). By in vivo and in vitro electrophysiology, we show that 5-HT produced by AADC cells increases the excitability of spinal motoneurons. The phenotypic change in AADC cells appears to result from a loss of inhibition by descending 5-HT neurons and to be mediated by 5-HT1B receptors expressed by AADC cells. These findings indicate that AADC cells are a potential source of 5-HT at spinal levels below an SCI. The production of 5-HT by AADC cells, together with an upregulation of 5-HT2 receptors, offers a partial explanation of hyperreflexia below a chronic SCI.

Localization of L-type calcium channel CaV1.3 in cat lumbar spinal cord - with emphasis on motoneurons

Voltage-dependent persistent inward currents (PICs) which underlie the plateau potentials are an important intrinsic property of spinal motoneurons. Electrophysiological experiments have indicated that a subtype of the low threshold L-type calcium channel, Ca(V)1.3, mediates this current. In mouse and turtle lumbar spinal cord it has been shown that these channel proteins are mainly found on motoneuron dendrites. In the present study we have used immunohistochemistry to locate these channels in lumbar spinal neurons, especially motoneurons, of the cat. The results indicate that Ca(V)1.3 immunoreactivity was unevenly distributed among the laminae of the spinal grey matter. The small neurons in superficial dorsal horn (laminae I-III) were sparsely and weakly labelled, while large neurons in ventral horn were frequently and densely labelled. Groups of motoneurons in lamina IX that were immunoreactive to choline acetyltransferase also co-expressed Ca(V)1.3. The immunoreactivity was mainly associated with neuronal somata and proximal dendrites. Double staining with antibodies against Ca(V)1.3 and MAP2 (a dendritic marker) showed that some fine fibres, which may include distal dendrites, were also labelled. These results in the cat spinal cord show some differences from studies in mouse and turtle motoneurons where the immunoreactivity against this channel was mainly localized to the dendrites.

Distribution of calcium channel CaV1.3 immunoreactivity in the rat spinal cord and brain stem

The function of local networks in the CNS depends upon both the connectivity between neurons and their intrinsic properties. An intrinsic property of spinal motoneurons is the presence of persistent inward currents (PICs), which are mediated by non-inactivating calcium (mainly Ca(V)1.3) and/or sodium channels and serve to amplify neuronal input signals. It is of fundamental importance for the prediction of network function to determine the distribution of neurons possessing the ion channels that produce PICs. Although the distribution pattern of Ca(V)1.3 immunoreactivity (Ca(V)1.3-IR) has been studied in some specific central nervous regions in some species, so far no systematic investigations have been performed in both the rat spinal cord and brain stem. In the present study this issue was investigated by immunohistochemistry. The results indicated that the Ca(V)1.3-IR neurons were widely distributed across different parts of the spinal cord and the brain stem although with variable labeling intensities. In the spinal gray matter large neurons in the ventral horn (presumably motoneurons) tended to display higher levels of immunoreactivity than smaller neurons in the dorsal horn. In the white matter, a subset of glial cells labeled by an oligodendrocyte marker was also Ca(V)1.3-positive. In the brain stem, neurons in the motor nuclei appeared to have higher levels of immunoreactivity than those in the sensory nuclei. Moreover, a number of nuclei containing monoaminergic cells, for example the locus coeruleus, were also strongly immunoreactive. Ca(V)1.3-IR was consistently detected in the neuronal perikarya regardless of the neuronal type. However, in the large neurons in the spinal ventral horn and the cranial motor nuclei the Ca(V)1.3-IR was clearly detectable in first and second order dendrites. These results indicate that in the rat spinal cord and brain stem Ca(V)1.3 is probably a common calcium channel used by many kinds of neurons to facilitate the neuronal information processing via certain intracellular mechanisms, for instance, PICs.