Deolinda Lima

The spinothalamic system of the rat: Structural types of retrogradely labelled neurons in the marginal zone (lamina I)

Retrogradely labelled lamina I neurons were studied after intrathalamic injections of free horseradish peroxidase mixed with dimethylsulphoxide, wheat germ agglutinin conjugated with horseradish peroxidase, and subunit B of cholera toxin. The first two tracers revealed only the perikaryal shape and the orientation of primary dendrites, while cholera toxin subunit B produced Golgi-like stainings. The morphological and morphometric analysis of the labelled marginal neurons in different planes showed them to belong to the pyramidal and the flattened types of our Golgi-based classification. These cells were located predominantly in the intermediate lateromedial portion of lamina I at all spinal levels, and it is suggested that their structural duality is matched by different functional properties. Distributions of the remaining spinothalamic cells labelled with the two horseradish peroxidase tracers were rather similar to those previously reported in the literature, including the almost exclusive occurrence of labelled cells, at lumbar levels, in the internal basilar column group. Cholera toxin subunit B labelled many more spinal cells and revealed considerable numbers of labelled cells in all cell groups at the lumbar enlargement.

The Paired Homeodomain Protein DRG11 Is Required for the Projection of Cutaneous Sensory Afferent Fibers to the Dorsal Spinal Cord

Cutaneous sensory neurons that detect noxious stimuli project to the dorsal horn of the spinal cord, while those innervating muscle stretch receptors project to the ventral horn. DRG11, a paired homeodomain transcription factor, is expressed in both the developing dorsal horn and in sensory neurons, but not in the ventral spinal cord. Mouse embryos deficient in DRG11 display abnormalities in the spatio-temporal patterning of cutaneous sensory afferent fiber projections to the dorsal, but not the ventral spinal cord, as well as defects in dorsal horn morphogenesis. These early developmental abnormalities lead, in adults, to significantly attenuated sensitivity to noxious stimuli. In contrast, locomotion and sensori-motor functions appear normal. Drg11 is thus required for the formation of spatio-temporally appropriate projections from nociceptive sensory neurons to their central targets in the dorsal horn of the spinal cord.

The medullary dorsal reticular nucleus as a pronociceptive centre of the pain control system.

The endogenous pain control system has long been considered as engaged in pain depression through the commitment of multiple descending actions that reduce the response capacity of spinal dorsal horn nociceptive neurones. Such a pure inhibitory antinociceptive nature was lately questioned by the observation of pronociceptive effects from areas classically regarded as antinociceptive. The thereby raised hypothesis of a more versatile functional arrangement that dynamically adjusts the pain modulatory effect to multiple conditions by balancing several excitatory and inhibitory actions found strong support on the recent discovery of a medullary area particularly dedicated to pain facilitation. Lesioning the medullary dorsal reticular nucleus (DRt) depresses nociceptive responses to acute and inflammatory pain, whereas stimulation produces the inverse effect. The decrease in formalin-induced pain behaviour following DRt lesioning is accompanied by a decrease of spinal noxious-evoked c-fos neuronal activation. DRt blocking by lidocaine results in a decrease of the nociceptive activity of spinal dorsal horn neurones, whereas stimulation by glutamate has the opposite effect. A reciprocal disynaptic putative excitatory circuit that links the DRt and the spinal dorsal horn and conveys nociceptive input through the ascending branch was described, indicating that the DRt pain facilitating action is mediated by a reverberating spino-DRt circuit that promotes the enhancement of the response capacity of spinal neurones to noxious stimulation.The demonstration of a primary pronociceptive centre in the endogenous pain control system brings new important data to the emerging concept of pain modulation as a dynamic and flexible process that integrates nociceptive processing by balancing multiple excitatory and inhibitory actions as the way of adapting to the various unsteady pain determinants.

The spino-latero-reticular system of the rat: Projections from the superficial dorsal horn and structural characterization of marginal neurons involved

The projections of the superficial dorsal horn to the lateral reticular nucleus of the medulla oblongata of the rat, and the morphological types of spinal cord lamina I neurons involved were studied after injecting the retrograde tracer cholera toxin subunit B in the caudal portion of the lateral reticular nucleus. Only injection sites located in the lateral part of the lateral reticular nucleus caused retrograde cell labelling in the superficial dorsal horn (laminae I-III). However, injection sites covering the lateral half of the lateral reticular nucleus and the region intermediate between its lateral border and the ventrocaudal tip of the trigeminal spinal nucleus also labelled cells in the neck of the dorsal horn. In contrast, injection sites confined to the intermediate region gave rise to an almost exclusive cell labelling in laminae I-III. Because the lateral part of the lateral reticular nucleus and the adjoining lateral region are rich in noradrenergic cells, it is suggested that these may be the specific targets of laminae I-III neurons. On the basis of the solid dendritic filling achieved, labelled lamina I cells were classified structurally. Most were fusiform cells (80%) and a minority pyramidal or flattened cells (10% each). Since fusiform cells also project selectively to the parabrachial nuclei, which together with the lateral reticular nucleus have been implicated in respiratory and cardiovascular reflexes, it is suggested that this cell type may convey nociceptive input originating autonomic responses. The pyramidal cells project also in large numbers to the mesencephalic periaqueductal gray which, like the lateral reticular nucleus, exerts descending inhibition on the dorsal horn nociceptive neurons. This suggests that this cell type may activate the spinal-midbrain pain modulatory loops centred on both nuclei.

Differential activation of c-fos in spinal neurones by distinct classes of noxious stimuli.

The laminar distribution of spinal cord neurones expressing immunoreactivity to the Fos protein was evaluated in the rat following chemical, thermal or mechanical noxious stimulation of the skin for 2 h. After stimulation by 20% or 5% formalin, Fos-immunoreactive neurones prevailed in lamina I where they accounted for 64% and 59%, respectively, of the entire population of Fos-immunoreactive spinal cells. Values in the remaining laminae were low (2-10%). Following thermal stimulation by radiant heat at 65 degrees C or 58 degrees C, Fos cells were concentrated in laminae I and IIo, amounting to 57% and 62%, respectively, in lamina I, and to 26% and 29% in lamina IIo. Values were lower than 10% in the remaining laminae. Following mechanical stimulation by pinching or needle prick, Fos-positive cells were regularly distributed throughout laminae I-V amounting to 25-26% in lamina I, and 10-20% in each of the remaining laminae. These findings suggest that the spinal neuronal groups upon by prolonged noxious stimulation differ according to the nature of the stimulus.

Neurons in the superficial dorsal horn of the rat spinal cord projecting to the medullary ventrolateral reticular formation express c-fos after noxious stimulation of the skin

The nociceptive nature of the neurons of the superficial dorsal horn (laminae I-III) which project to the medullary ventrolateral reticular formation is studied in the rat. Medullary injections of Fluoro-Gold showed exclusive retrograde labeling of laminae I-III cells when the tracer filled a zone intermediate between the lateral tip of the lateral reticular nucleus and the spinal trigeminal nucleus, pars caudalis. This zone is here called VLMlat. Following noxious mechanical or thermal stimulation of the skin, double-labeled neurons, which stained retrogradely and were Fos-immunoreactive, prevailed in laminae I and IIo. Double-labeled neurons were few in lamina IIi after thermal stimulation and entirely lacking in lamina III after the two kinds of stimulation. Findings in lamina I confirm previous electrophysiological data (see Menétrey et al., J. Neurophysiol., 52 (1984) 595-611) showing that lamina I cells projecting to the ventrolateral reticular medulla convey noxious messages. The occurrence of numerous double-labeled cells in lamina IIo suggests that this lamina is also involved in nociceptive transmission to the VLMlat.

A spinomedullary projection terminating in the dorsal reticular nucleus of the rat.

Spinal afferents to the medullary dorsal reticular nucleus were studied using the following retrograde tracers: horseradish peroxidase (diluted in dimethylsulfoxide), wheat germ agglutinin conjugated with horseradish peroxidase, and cholera toxin subunit B. Spinal cord cells projecting to that medullary region were located predominantly in medial lamina I and lamina X. Cell labelling was moderate in the medial part of laminae II-IV and sparse throughout laminae V-VII. Labelling was predominantly ipsilateral in the dorsal horn and bilateral in laminae VII and X. After mechanical lesions of the dorsal white matter which severed most of the ipsilateral cuneate fasciculus, the numbers of superficial dorsal horn cells that were labelled from the dorsal reticular nucleus were considerably decreased caudal to the lesion, which suggests that their axons utilize mostly the cuneate fasciculus. Since the medullary dorsal reticular nucleus of the rat has a predominant population of nociceptive specific neurons, it is suggested that this spino-dorsomedullary reticular pathway is involved in pain processing.

Orbitofrontal cortex lesions disrupt risk assessment in a novel serial decision-making task for rats

Neurobiological mechanisms of decision-making have been shown to be modulated by a number of frontal brain regions. Among those areas, the orbitofrontal cortex (OFC) is thought to play an important role in the decision of behavioral actions when faced with alternative options of ambiguous outcome. Here we present a novel neurobehavioral task to study affective decision-making in the rat, based on evaluation of consecutive choices between two levers associated with rewards of different value and probability. Two groups of animals were studied; a sham control group (n=6) and an OFC-lesioned group (n=7). In the first 30 trials both groups had similar preference patterns but at the end of the 90 trials of the task both groups developed specific preferences. The control group systematically preferred the lever associated with smaller but more reliable rewards (low risk lever) while the OFC lesion group preferred the high risk lever (index of preference of 0.21+/-0.21 vs. -0.45+/-0.10; t-test, P<0.05). Analysis of choice persistence (i.e. choosing the same lever in consecutive trials) suggests that the OFC-lesioned group became less sensitive to risk, seeking large rewards irrespective of their success probability.

Descending projections from the caudal medulla oblongata to the superficial or deep dorsal horn of the rat spinal cord

The location of neurons in the caudal medulla oblongata that project to the superficial or deep dorsal horn was studied in the rat, by means of retrograde labelling from confined spinal injection sites. The tracer cholera toxin subunit B was injected into laminae I–III (fuve rats) or I–V (three rats) at C4–7 spinal segments. Neurons projecting to the superficial dorsal horn were located in the dorsomedial part of the dorsal reticular nucleus ipsilaterally, the subnucleus commissuralis of the nucleus tractus solitarius bilaterally, and a region occupying the lateralmost part of the ventrolateral reticular formation between the lateral reticular nucleus and the caudal pole of the spinal trigeminal nucleus, pars caudalis, bilaterally. Neurons projecting to the deep dorsal horn, which were only labelled when laminae I–V were filled by the tracer, occurred in the dorsomedial and ventrolateral parts of the dorsal reticular nucleus and in the ventral reticular nucleus bilaterally. A few cells were located in the above described lateralmost portion of the ventrolateral reticular formation bilaterally and in the ventral portion of the ipsilateral cuneate nucleus. In the light of previous data demonstrating that dorsal horn neurons project to the dorsal reticular nucleus, the ventrolateral reticular formation, and the nucleus tractus solitarius, and that neurons in these three medullary regions are involved in pain inhibition at the spinal level, the descending projections demonstrated here suggest the occurrence of spino-medullary-spinal loops mediating the analgesic actions elicited in each nucleus upon the arrival of nociceptive input from the dorsal horn.

The medullary dorsal reticular nucleus facilitates acute nociception in the rat

The influence on pain processing caused by destruction or stimulation of the dorsal reticular nucleus (DRt) was studied using the tail-flick and the increasing temperature hot-plate tests. Lesions of the DRt were obtained by injecting quinolinic acid (180 nmol/microliters) unilaterally or bilaterally, and nociceptive responses were evaluated by both tests. Following unilateral lesions, the tail-flick latencies and the hot-plate response temperatures were increased, values differing statistically from controls in the latter test. Bilateral lesions resulted in statistically significant increases of both tail-flick latency and hot-plate response temperature. Stimulation of the DRt was performed by injecting glutamate (100 nmol/microliters) unilaterally, which was followed 1 min later by a significant decrease in the tail-flick latency compared to saline injected controls. These results suggest that the DRt is involved in the facilitation of nociception after acute thermal noxious stimulation. This effect may be mediated through a spino-DRt-spinal loop causing a rebound of excitation in lamina I cells receiving noxious input from their own receptive field.