Are there separate central nervous system pathways for touch and pain?

Information about bodily events is conveyed by primary sensory fibres to higher brain centres through neurons in the dorsal column nuclei (DCN) and spinal dorsal horn. The DCN route is commonly considered a 'touch pathway', separate from the spinal pain pathway', in part because DCN neurons respond to gentle tactile stimulation of small skin areas. Here we report that DCN neurons can additionally respond to gentle and noxious stimulation of viscera and widespread skin regions. These and other experimental and clinical data suggest that the DCN and spinal routes cooperate, rather than operate separately, to produce the many perceptions of touch and pain, an ensemble view that encourages novel approaches to health care and research.

Vive la différence!

Hormonal effects are increasingly recognized as important influences on neuronal function and, ultimately, on animal behavior. Such 'higher' behavioral effects are well studied, particularly in relation to sexually dimorphic behaviors. Yet, somewhat surprisingly, a significant proportion of more basic neuroscience research papers fail to specify the sex of the subjects used. In this brief article Karen Berkley argues that knowledge of, and controlling for, the sex of research animals is important. In addition, if females are used, their reproductive-cycle status could provide a deliberate strategy to investigate the effects of gonadal steroid hormones on biological functions.

Estrous changes in responses of rat gracile nucleus neurons to stimulation of skin and pelvic viscera.

Multi- and single-unit recording was performed in the gracile nucleus in urethane-anesthetized rats to examine estrous variations in responses of its neurons to brushing the hindquarters and mechanical stimulation of the uterus, vaginal canal, cervix, and colon. Six rats each were studied in each of the four estrous stages: proestrus (P), estrus (E), metestrus (M), and diestrus (D). The magnitude of multi-unit responses to gentle brushing of the perineum, hip, and tail, but not the foot and leg, was significantly greater during proestrus than during other stages. Of 70 single units responsive to brush, 56 (80%) responded to stimulation of at least one viscus. Although this percentage did not change with estrous stage, the direction and latency of some responses did. Pressure on the cervix evoked significantly more inhibitory (vs excitatory) responses in P than in E and M, and the response latency was significantly longer in D and P than in E and M. The direction of response to vaginal distention did not change with estrous stage, but response latency was significantly longer in D than in P and E. Uterine distention evoked significantly more inhibitory responses in D than in P, with no estrous changes in latency. Responses to colon distention did not change. These variations in both magnitude of response to tactile stimulation and characteristics of response to stimulation of reproductive organs, but not the colon, correlate with changes in mating behaviors of the female rat, suggesting that the gracile nucleus is a component of neural systems that control reproductive behaviors.

Spatial relationships between the terminations of somatic sensory motor pathways in the rostral brainstem of cats and monkeys. II. Cerebellar projections compared with those of the ascending somatic sensory pathways in lateral diencephalon.

Previous studies have shown that ascending somatic sensory pathways arising from the dorsal column nuclei, lateral cervical nucleus and spinothalamic tract terminate in parts of the thalamus adjacent to those which receive cerebellar terminations. This termination pattern creates a border between the ventroposterolateral nucleus (VPL) and the ventrolateral nucleus (VL) in the cat and between the caudal and oral parts of VPL (VPLc and VPLo, respectively) in the monkey. Since it is not clear how sharp these borders are, a double orthograde labeling strategy was used in the present study to make direct comparisons of the projections to the thalamus from these sources of input. It was found that there was a change in the sources of afferent input to the different target areas that paralleled changes in cytoarchitecture. Moving caudally to rostrally, VPL in the cat and VPLc in the monkey received projections predominantly from the middle, dorsal (clusters) portion of the dorsal column nuclei. These projections were gradually replaced near the VPL-VL border in the cat and VPLc-VPLo border in the monkey first by input from the lateral cervical nucleus (cat only) and the rostral and ventral portions of the dorsal column nuclei and then by spinothalamic projections. Towards VL in the cat and the rostral parts of VPLo in the monkey (referred to as Vim by Hassler, '59 and Mehler, '71), these projections were in turn replaced by those from the cerebellum. This sequence resulted in a complex pattern (summarized in Fig. 10) where some thalamic territories received input predominantly from one source and others received converging input from several sources. The major region receiving converging ascending somatic sensory and cerebellar terminations was located at the border between VPL and VL in the cat and in the caudal parts of Olszewski's ('52) VPLo in the monkey (that is, between VPLc and Vim). In general, the results in the cat were similar to those in the monkey. One notable difference was that the domain containing terminals from the cerebellum and the rostral-ventral parts of the dorsal column nuclei was located medially between VPLc and Vim in the monkey, whereas it extended across the entire mediolateral border between VPL and VL in the cat. In both species, thalamic neurons received input predominantly from one afferent source and only minor input, if any, from other sources.(ABSTRACT TRUNCATED AT 400 WORDS)

Different targets of different neurons in nucleus gracilis of the cat.

Efferent projections of neurons in the cat nucleus gracilis to the ventrobasal complex (VB) of the thalamus and the dorsal accessory portion of the inferior olive (IOd) were studied using tracing techniques that exploit neuronal orthograde and retrograde intra-axonal transport processes. These projections were studied in order to determine if the heterogeneity of the morphology, physiology and afferent input of the nucleus gracilis is paralleled by a similar heterogeneity in its efferent projections. In an orthograde study, 3H-leucine and/or 3H-proline were injected into the nucleus gracilis of different subjects in an attempt to label different proportions of large (GREATER THAN 18 MU) AND SMALL CELLS. This procedure permitted study of the efferent targets of the two cell types. The amount of labeling in VB was a constant ratio of the number of large cells in the nucleus gracilis that had incorporated the isotope. This relationship was not observed for the small cells. The amount of labeling in IOd was dependent neither on the number of large nor the number of small cells that had incorporated the isotope. In a retrograde study following extensive injections of horseradish peroxidase (HRP) into the thalamus, only large cells (greater than 18 mu) in the dorsal column nuclei were filled with HRP reaction product. These cells were located predominantly in clusters in the middle zone of the nucleus gracilis as well as rostrally. After injections including (but not confined to) the inferior olive, small cells located in the rostral and middle zones were filled with reaction procduct. A few large cells located ventrally in the middle zone of the nucleus gracilis and rostrally were also filled. Some of these ventrally located large cells may project in a collateral fashion both to the thalamus and to the inferior alive. The results of both the orthograde and retrograde studies are consistent with other evidence on the heterogeneity of the nucleus gracilis. These data strongly support the conclusion that the population of cells in the nucleus gracilis that projects to the thalamus overlaps with but is not identical to the population of cells that projects to the inferior olive.

[Projections to the inferior olive of the cat. I. Comparisons of input from the dorsal column nuclei, the lateral cervical nucleus, the spino-olivary pathways, the cerebral cortex and the cerebellum].

The present experiments compare the projections from the dorsal column nuclei (DCN), the lateral cervical nucleus (LCN), the spino-olivary pathways (SO), the motor cortex (Ms1) and the cerebellum (CB) to the inferior olive of the cat. A differential labeling strategy was used for these comparisons. It was found that projections from the contralateral DCN and LCN and the ipsilateral SO overlap extensively with each other in the dorsal accessory division of the inferior olive and the caudal half of the medial accessory olive. Projections from the contralateral motor cortex partially overlap these somato-sensory projections but they also extend into the principal division and into the rostral half of the medial accessory olive where other authors have found input from other "motor" sources such as the red n., the globus pallidus and the caudate n. The contralateral cerebellum projects heavily to most of the inferior olive except for a few regions in the caudal portions of the two accessory nuclei. These results show that there is a partial segregation between somatosensory and motor inputs within the inferior olive and that the cerebellar feedback fibers appear to avoid at least parts of those regions where the somatosensory input is heavy. Some of this segregation, particularly within the medial accessory olive and the principal n., correlates with differences in both the olivo-cerebellar connectivity and the cytoarchitecture of these regions. Although the somatosensory and motor inputs are partially segregated, there are also several regions where these inputs overlap. These regions are: (1) an area in the medial half of the rostral portions of the dorsal accessory olive, and (2) a small oval zone in the middle of the caudal half of the medial accessory olive. This overlap provides an anatomical basis for electrophysiological evidence which demonstrates the existence of cells in some of these regions that respond to activation of both the cerebral cortex and the spinal cord (e.g., Crill and Kennedy, '67).

Projections to the inferior olive of the cat. II. Comparisons of input from the gracile, cuneate and the spinal trigeminal nuclei.

The present experiments compared the projections to the inferior olive of the cat from the gracile, cuneate and spinal trigeminal nuclei. A differential labeling strategy was used for these comparisons. It was found that all three somatic sensory nuclei project to portions of all three major divisions of the contralateral inferior olive. The spinal trigeminal n. may also project less densely to the ipsilateral medial accessory olive. Projections to the dorsal accessory nucleus (DAO) and the medially-adjacent ventral lamella of the principal nucleus are roughly somatotopically organized. Although there is considerable overlap between the projection zones, the gracile n. projects predominantly to lateral DAO, the cuneate n. projects predominantly to medial DAO, and the spinal trigeminal nucleus pars caudalis projects predominantly to the most medial portions of DAO and the ventral lamella of principal olive. Projections to the medial accessory olive, on the other hand, are not as highly organized. Instead, they overlap extensively within a small egg-shaped area in the middle of the caudal half of the nucleus. Whereas all portions of the gracile and cuneate nuclei project to the inferior olive, only the pars caudalis of the spinal trigeminal nucleus appears to do so. These results were compared with the three available olivocerebellar maps as well as with the available behavioral and electrophysiological evidence on cerebellar somatotopic organization. This comparison indicated that the inputs to the cerebellum from the three second-order somatosensory nuclei via the inferior olive appear to be generally consistent with cerebellar somatotopic organization. This consistency is apparent not only with respect to the longitudinally-organized, vermal and paravermal differences in the anterior lobe, but also with respect to the transversely-organized specific somatotopy of the intermediate zone of the anterior lobe and the paramedian lobule.

The location of cerebellar-projecting neurons within the lumbosacral spinal cord in the cat. An anatomical study with HRP and retrograde chromatolysis.

The locations of lumbosacral spinocerebellar neurons were examined by two anatomical methods in kittens. In one group of animals chromatolytic changes were provoked by cerebellar lesions. In another group horseradish peroxidase (HRP) was injected into the cerebellum. Projection laterality was investigated by making unilateral spinal lesions prior to the cerebellar HRP injections. Diaminobenzidine (DAB) or tetramethylbenzidine (TMB) was used as substrate fo HRP. The morphological characteristics of HRP-labeled neurons in the TMB-processed material were examined. Neurons marked by the two methods were located within the same regions. A greater number of cells were marked with the HRP method, however, than with the retrograde chromatolysis method. Spinocerebellar neurons were found in laminae IV-IX with large differences with regard to specific locations depending on segmental level. Numerous marked neurons were found in the following areas: laminae IV-Vi in L3-L7, the column of Clarke in L3-L4, the medial part of lamina VII in L6-L7, the lateral part of lamina VII in L3-L4, the dorsolateral nucleus of lamina IX in L3-L6, the ventrolateral nucleus of lamina IX in L4-L5, and the ventromedial nucleus of lamina IX in S3 (and Ca1). Dorsally located neurons were in general more likely to project ipsilaterally than ventrally located neurons. Marked structural differences were frequently observed between spinocerebellar neurons in different locations. These results provide additional information on the anatomical complexity of the spinocerebellar pathways from the lumbosacral region in the cat. Together with results from some other recent anatomical studies on spinocerebellar tracts, they also form a basis for further anatomical and physiological investigations which could contribute to a better understanding of the organization of the spinocerebellar tracts.

Pain threshold variations in somatic wall tissues as a function of menstrual cycle, segmental site and tissue depth in non-dysmenorrheic women, dysmenorrheic women and men.

Pain symptoms of many disorders are reported to vary with menstrual stage. This study investigated how pain thresholds to electrical stimulation of the skin, subcutis and muscle tissue varied with menstrual stage in normal women and compared these variations with those in women with dysmenorrhea and in healthy men at matched intervals. Thresholds of the three tissues were measured four times during the course of one menstrual cycle at four sites. Two of the sites were on the abdomen within the uterine viscerotome (abdomen-rectus abdominis, left and right) and two were outside it on the limbs (leg-quadriceps, arm-deltoid). Calculated from the beginning of menstruation (day 0), the menstrual phases studied were menstrual (days 2-6), periovulatory (days 12-16), luteal (days 17-22) and premenstrual (days 25-28). Spontaneous pain associated with menstruation was measured from diary estimates on a VAS scale. Whereas the highest thresholds always occurred in the luteal phase regardless of segmental site or stimulus depth, the lowest thresholds occurred in the periovulatory stage for skin, whereas those for muscle/subcutis occurred perimenstrually. Dysmenorrhea accentuated the impact of menstrual phase. For non-dysmenorrheic women menstrual trends were significant only in abdominal muscle and subcutis, but for dysmenorrheic women the trends were also significant in abdominal skin and in limb muscle and subcutis. Dysmenorrhea also lowered thresholds mainly in muscle and sometimes in subcutis, but never in skin, with the greatest hyperalgesic effects in left abdominis muscle. Abdominal sites were more vulnerable to menstrual influences than limb sites. Muscle thresholds, but not skin or subcutis thresholds, were significantly lower in abdomen than in limbs, particularly in dysmenorrheic women. The amount of abdominal muscle hyperalgesia correlated significantly with the amount of spontaneous menstrual pain. Only minor sex differences were observed for pain thresholds of the arm and leg, but there was a unanimous refusal by men, but not by women, to be tested at abdominal sites. These results indicate that menstrual phase, dysmenorrhea status, segmental site, tissue depth and sex all have unique interacting effects on pain thresholds, thus adding more items to the lengthy and still-growing list of biological factors that enter into an individual's judgment of whether or not a stimulus is painful.

Glial cells: possible determinants of neuronal uptake of tritiated proline.

When tritiated proline is injected into various sensory and integrative areas of the brain, it fails to be incorporated into the proteins of neuronal soma located within the injection site. In contrast, such incorporation does occur when [3H]proline is injected into dorsal root ganglia. The basis for this difference is unclear because brain and dorsal root ganglion tissue differ in configurational factors (e.g. synapses, dendrites) as well as in the embryological origin of their respective neuronal and non-neuronal cell populations. To determine if configurational factors might account for [3H]proline's incorporation into somal neuronal proteins in dorsal root ganglia, [3H]proline was injected into autonomic (pelvic and superior cervical) and sensory (dorsal root and nodose) ganglia in the rat. These ganglia differ in synaptic and cellular configurations, but have the same neural origin (neural crest). Virtually all neuronal soma were labeled in autoradiograms of all of these injection sites, suggesting that configurational factors do not account for the labeling of dorsal root ganglion neurons by [3H]proline. To address the issue of embryological origin, cellular labeling patterns after [3H]proline injection into the hypoglossal nucleus, dorsal motor nucleus of the vagus and the ventral horn of spinal cord were compared with those after [3H]proline injections into the adjacent solitary nucleus, gracile nucleus and central cervical nucleus of the spinal cord. The neurons in the former three nuclei (i.e. motoneurons) originate from the neural tube, but their axons are associated primarily with Schwann cells which originate from the neural crest. Although neurons in the latter three regions also originate from the neural tube, their axons are myelinated entirely by neural tube-derived glia (i.e. oligodendrocytes).(ABSTRACT TRUNCATED AT 250 WORDS)

A glial-neuronal-glial communication system in the mammalian central nervous system.

Previous studies have demonstrated that when tritiated proline [( 3H]Pro) is injected into the dorsal column nuclei (DCN) of cats, it labels macroglial cells, but fails to label neurons at the injection site. (Tritiated leucine [( 3H]Leu) in contrast, labels both neurons and some glial cells.) Despite the failure of [3H]Pro to label DCN neurons, labeling is still observed in DCN terminal targets. This result suggests that glial cells are involved in the translocation of [3H]Pro-labeled molecules from one part of the brain to another. The purpose of the present experiment was to use electron microscopic autoradiographic techniques to characterize the labeling produced in internal arcuate fiber tract axons arising from DCN neurons 24 h after injections of [3H]Pro (or [3H]Leu, for comparison) into DCN. It was reasoned that, if the translocation of [3H]Pro-labeled molecules from DCN to its targets is indeed carried out by glial cells, then only glial elements associated with the fibers should be labeled following [3H]Pro injections of DCN. If, on the other hand, the translocation involves an initial transfer of [3H]Pro-labeled molecules into neuronal perikarya followed by axonal transport, then only axoplasmic elements along the fiber pathway should be labeled. Injections of [3H]Pro into DCN labeled axoplasmic elements in samples of axons from the internal arcuate tract both 'near' (0.5-0.8 mm) and 'far' (2-4 mm) from the injection site at about an equal absolute density. However, glial elements associated with the axons were also labeled in both samples, but much more densely in the 'near' than in the 'far' axons. Injections of [3H]Leu labeled axoplasm more densely than did [3H]Pro (by a factor of 4 in the 'far' samples). Glial labeling by [3H]Leu near the injection site was much less than that of [3H]Pro, but, 'far' from the injection, the levels of [3H]Leu and [3H]Pro glial labeling were comparable. Taken together with the results of other studies, these data support the existence of a previously unrecognized system of communication between glial cells and neurons. In this putative system (Fig. 9), molecules containing both [3H]Leu and [3H]Pro are transferred from glial cells into adjacent neuronal soma and transported down the length of the axon where, all along the way, some of them are transferred from the axon into adjacent glial processes. The system is more readily apparent when [3H]Pro is used because of its avid and preferential uptake by glial cells. Potential functions of such a system are unknown, but could be trophic, protective and/or informative.

A re-examination of the spino-reticulo-diencephalic pathway in the cat.

One commonly accepted idea is that affective aspects of pain sensation are derived from a flow of information from the spinal cord through the reticular formation to the intralaminar thalamus and subthalamus. Little is known, however, about the extent to which spinoreticular terminations and reticulodiencephalic neuronal cell bodies overlap. This study used a combination of anterograde and retrograde tracing techniques to compare these distributions in the cat. Whereas spinoreticular terminations were concentrated caudally and laterally, neurons projecting to intralaminar thalamus and subthalamus were concentrated rostrally and medially. Thus, information conveyed from the spinal cord to the reticular formation appears to have direct access to intralaminar thalamus and subthalamus only by way of a few widely scattered neurons. When considered with the results of others, these results encourage less emphasis on a putative spino-reticulo-diencephalic pathway for pain. Rather, the reticular formation's role in pain is more likely to involve its full complement of interconnected descending and ascending connections.

Relays from the spinal cord and solitary nucleus through the parabrachial nucleus to the forebrain in the cat.

Projections from the spinal cord and solitary nucleus to the lateral parabrachial nucleus (PBN1) in the cat were directly compared using double anterograde tracing methods. The two inputs were found to overlap within a well-circumscribed zone in the rostral 2/3 of PBN1. This zone was flanked ventrally by a zone receiving only solitary nucleus input and dorsally by a zone receiving only spinal input. Other authors have shown that neurons within these three recipient zones (overlap area, solitary nucleus and spinal cord) project to different forebrain targets (hypothalamus, amygdala and thalamus, respectively). This orderly input-output organization is likely to provide part of the framework for PBN's complex involvement in the coordination of respiratory and cardiovascular activities and their association with pain, visceral sensation and emotion.

Spinal and vagal influences on the responses of rat solitary nucleus neurons to stimulation of uterus, cervix and vagina.

Neurons in the rat solitary nucleus (NTS) respond to mechanical stimulation of the uterine horn, cervix and vagina. The present study examined how these responses were affected by bilateral vagotomy and/or T10-T12 spinal transection for 12 single NTS neurons recorded in 12 rats anesthetized with halothane/nitrous oxide. Spinal transections all responses. Vagotomies eliminated responses only to uterine horn stimulation and either reduced the excitatory or enhanced the inhibitory responses to cervix and vaginal stimuli. These results suggest that NTS neuronal responses to cervix and vaginal stimulation depend upon input from the spinal dorsal horn and are facilitated by vagal input, whereas responses to uterine horn stimulation may require both spinal and vagal input.

Efferent projections of the gracile nucleus in the cat.

The efferent projections of different portions of the gracile nucleus in the cat were studied using both autoradiographic and degeneration tracing methods. The results suggest that there are two aspects to the functional organization of these projections. First, the somatotopic organization of the gracile n. (GR) is maintained, but inverted, by the topographic organization of its projections to VPL1. Fibers from the lateral portions of GR terminate medially in VPL1; fibers from the dorsal portions terminate ventrally. These fibers, especially those from the middle and caudal portions of GR, terminate in dense, precisely located groups of clusters. Dorsally located clusters in VPL1 (predominantly from middle-ventral portions of GR) are significantly smaller than ventrally located clusters (predominantly from middle-dorsal portions). The second aspect of this organization, involving the projections both to VPL1 and to other brain stem targets, is that some kind of functionally relevant sorting process appears to occur as fibers leave different portions of the gracile n. The afferent projections of the rostral (GRr) and middle-ventral portions (GRmv) of the gracile n. are different from those from the other portions of the nucleus. Projections to VPL1 from GRr are less dense, less likely to form clusters, less clearly topographically organized, and extend further rostrally and dorsally in VPL1 than those from the rest of GR. The clusters are small, like those from GRmv. Similarly, although all portions of GR project to several other brain stem regions, these projections appear to be derived preferentially from GRr and/or GRmv. These brain stem regions involve certain portions of the inferior olive, inferior and superior colliculi, red n., zona incerta, pretectum, thalamic posterior group and the H field of Forel. This dual organization of efferent connectivity is similar to that of the cuneate n.20, and is consistent with many of the differences in cytoarchitecture, afferent connectivity and response properties of cells within different portions of the dorsal column nuclei.

Responses of neurons in caudal solitary nucleus of female rats to stimulation of vagina, cervix, uterine horn and colon.

Neurons in the caudal part of the solitary nucleus (NTS) are known for their processing of information derived from many viscera, including cardiovascular, respiratory and alimentary tract organs. This study characterized responses of NTS neurons in female rats in estrus to mechanical stimulation of four pelvic visceral organs (i.e. the vaginal canal, cervix, uterine horn and colon) as well to gentle mechanical skin stimulation. Of the 90 neurons tested, 31% responded with excitation or inhibition to one (22%) or more (9%) visceral stimuli. Responses included 13% to vaginal distension, 12% to cervix stimulation, 10% to uterine distension and 4% to colon distension. None responded to gentle cutaneous stimuli. These results expand the domain of visceral functions of NTS neurons to include pelvic female reproductive organs. The failure of NTS neurons to respond to gentle cutaneous stimuli contrasts with convergent responses of neurons in the gracile nucleus to skin and pelvic visceral stimuli [13] indicating the two nuclei are involved in different aspects of visceral function.

Effects of colchicine on retrogradely-transported WGA-HRP.

The effects of colchicine treatment on retrogradely-transported WGA-HRP were examined in the cat. The ventroposterolateral nucleus of the thalamus was bilaterally injected with WGA-HRP followed 2 days later by a unilateral injection of colchicine into the dorsal column nuclei (DCN). The cats were sacrificed by perfusion 24 h later. Retrogradely-transported WGA-HRP within DCN neurons was visualized in coronal, vibratome-cut sections of the medulla using the procedure of Rye and collaborators (J. Histochem. Cytochem., 32 (1984) 1145-1153). On the uninjected side, as expected, the reaction product filled numerous neuronal perikarya and their dendrites. In contrast, on the colchicine-treated side, the reaction product was restricted to dendrites; little labeling was observed within perikarya. These findings appear to reflect colchicine's effects on the translocation of lysosomes from neuronal perikarya to their dendrites are important for the interpretation of data from experiments using colchicine to enhance perikaryal immunohistochemical staining.

Spinal input to the parabrachial nucleus in the cat.

The projections from the spinal cord to the parabrachial nucleus in the cat were investigated using both the degeneration method and the anterograde transport of wheat germ agglutinin-horseradish peroxidase conjugate. Both methods produced similar results. Spinal input to the parabrachial nucleus was bilateral, with a slight contralateral predominance. The termination area was localized predominantly in the dorsal part of the lateral parabrachial nucleus, with additional limited terminations in the Kölliker-Fuse subnucleus. Projections from different rostrocaudal levels of the spinal cord overlapped completely, suggesting that spinal input to the parabrachial nucleus is not topographically organized. Taking these results together with those of others indicating that spinal input to the parabrachial nucleus arises primarily from nociceptive-specific neurons in lamina I of the dorsal horn, it is concluded that the spinal projections to the parabrachial nucleus are likely to be involved in various generalized aspects of nociception.

Spino-diencephalic relays through the parabrachial nucleus in the cat.

Previous studies have shown that the spinal input to the parabrachial nucleus (PBN) in the cat is limited to certain portions of its lateral division 8,21,45. The purpose of the present study was to determine some of the output targets of PBN neurons located within this spinal terminal domain by means of single, double and triple light microscopic labeling strategies. Combinations of tracers included the retrograde transport of tritiated wheat germ agglutinin, wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) and Fluoro-Gold from the hypothalamus, amygdala or thalamus/zona incerta together with either the anterograde transport of WGA-HRP from the spinal cord or the degeneration of spinal terminals following spinal lesions. The results (summarized in Fig. 10) showed that the spinal terminal domain contains separable populations of neurons projecting to the thalamus/zona incerta and hypothalamus. Only a limited number of amygdala-projecting neurons was located in this domain. Evidence from several laboratories supports the conclusion that these potential spino-diencephalic relays are involved somehow in nociception. More information is needed, however, regarding differences in the response properties of these separable populations of spinal-recipient neurons before more specific hypotheses concerning the precise nature of their nociceptive functions can be formulated.