Brain Research, 95 (1975) 211-220 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
211
D I E N C E P H A L I C PROJECTIONS F R O M T H E M I D B R A I N R E T I C U L A R FORMATION
DAVID BOWSHER Neurobiology Laboratory, Department of Anatomy, University of Liverpool, Liverpool L69 3BX (Great Britain)
SUMMARY (1) Physiologically guided stereotaxic coagulation was placed so as to avoid major through pathways in the midbrain reticular formation of 7 cats. Diencephalic degeneration resulting from this was traced by the Nauta method. (2) Preterminal degeneration was found in: the intralaminar nuclei and the posterior group (PO); the ventral group of thalamic nuclei; the ventral thalamus, including the zona incerta, subthalamus and fields of Forel; and the lateral hypothalamus. (3) The results are discussed in relation to somatomotor reactions, reticular influences on the electroencephalogram and telencephalic representation of pain.
I NTRODUCTION Perhaps because the properties of the reticular activating system 26 are so obviously similar to those previously described in the thalamic intralaminar* nuclei zS, much research, and theories based upon the results of that research, has been very largely concerned with reticulointralaminar relationships. However, the existence of reticular projections to diencephalic structures other than the intralaminar nuclei were early demonstrated by experimental methods~S; and the presence of reticular afferents to cortically projecting thalamic nuclei, as seen in Golgi material, has also been stressed 33. Reticular influences on the lateral geniculate nucleus 12 and anterolateral spinal influences on the ventrolateral thalamic nucleus14,21, z2 have been described, as have the extralemniscal properties of ventrobasal thalamic units in cats with interruption of the medial lemniscus 8. * Intralaminar thalamic nuclei, in accordance with widespread physiological practice, are here taken to include the centromedian-parafascicular complex, even though such definition may not be strictly anatomically accurate.
212 The following account, based on a description of projections from the midbrain reticular formation to the diencephalon, will attempt to underline the functional significance of all these connections and to redress the imbalance produced by undue concentration on the intralaminar nuclei of the thalamus. MATERIALS AND METHODS
In each of 7 cats under chloralose anaesthesia (70 mg/kg, intraperitoneal) a bipolar electrode was lowered stereotaxically into the midbrain through a small hole made in the cranium with a dental burr. Loci were sought in the reticular formation of the dorsal mesencephalic tegmentum in which evoked potentials could be elicited by stimulation of each of the 4 limbs. When such a site was satisfactorily identified, a small coagulation was made, using the central pole of the electrode as the cathode, while an 'indifferent' anode was clipped to the scalp incision. The lesions extended from stereotaxic plane A I to plane A5, at 2.5 or 3.0 m m from the midline. The dorsal reticular region chosen for recording and coagulation was designed to avoid damage to fibres of passage in specific thalamopetal bundles such as the brachium conjunctivum ~ and medial lemniscus; and was in the general area whose physiological properties we had previously analysed in a single unit study 10. The animals were killed by intracarotid perfusion of 10 % neutral formalin under anaesthetic 4 days after operation. The brains were removed into fixative and stored under refrigeration until they were processed. The brains were cut in serial frozen sections at 30 # m and assembled t0 to a pot in 5 % formalin. One section from each pot was stained by the Nissl method and one was impregnated by the technique of Nauta 27. Thus, sections at intervals of approximately 300 #m were available for examination. Each section was projected at a magnification of 10 times and all recognisable structures traced. The Nauta impregnated sections were then examined in the microscope and degenerating fibres and preterminals filled in on the outline drawings. Four brains were cut in coronal section and 3 in horizontal section. in two further animals, the active region of the midbrain reticular formation was again found with a bipolar electrode, the central pole of which was then removed, leaving a cannula. Down this, 0.2 #1 of [3H]leucine was then injected over 20 rain. Under the same anaesthetic (chloralose), repeated if necessary, the animals were perfused 24 h later. The brains were embedded in paraffin and cut at 8 #m in serial coronal sections. Autoradiographs were prepared 13 using llford G4 emulsion; the material awaits analysis. RESULTS
A typical case in the transverse series is illustrated in Fig. 1. Midbrain. Degenerating fibres could be seen coursing over the dorsal aspect of the aqueduct in the ventral part of the posterior commissure; dense preterminal degeneration was present in the midbrain reticular formation on the side contralateral to the lesion. On both sides, preterminal degeneration was observed in the ventral
213 tegmental area of Tsai and in the substantia nigra. At the mesodiencephalic junction degeneration was present in the periaqueductal grey matter. Diencephalon. In general, degeneration was denser on the side of the lesion; this was particularly noticeable in the case of very small lesions. The total amount of diencephalic degeneration attributable to destruction of the midbrain reticular formation (see Discussion) appeared to vary directly with the size of the lesion. Some degeneration could be attributed to incidental destruction of non-reticular structures, the most frequently involved of which were the deep layers of the superior colliculus and fastigiothalamic fibres running dorsolateral to the red nucleus 4. Dorsal thalamus. The intralaminar nuclei were in all cases filled with preterminal degeneration, the heaviest being in the nuclei centrum medianum and centralis lateralis. There appears to be a tendency for degeneration to be heavier laterally than medially within the group. The posterior group (PO), as defined by Rinvik 29, was always filled with preterminal degeneration, right up to its anterior limit as represented by the rostral border of the lateral geniculate nucleus. In some cases, preterminal degeneration in the lateral part (PO1) was almost absent, and in all cases was sparser than in the medial division (POm). Preterminal degeneration was observed in the part of nucleus lateralis posterior (LP) superjacent to PO. This was heavier when there was involvement of the deeper layers of the superior colliculus but was present to some extent in all cases. Fairly heavy degeneration of fibres of passage could always be observed in the radial bundles traversing PO and LP towards the internal capsule. In the ventral group of thalamic nuclei, scattered preterminal degeneration was always seen in the medial magnocellular part of the medial geniculate nucleus, in the (dorsal) lateral geniculate nucleus 9 (and in one instance in the ventral lateral geniculate nucleus) and in the ventrobasal complex, more heavily in its medial part and in the ventrolateral complex, including nucleus ventralis anterior. As in more dorsally placed nuclei, the radial bundles contain degenerating fibres of passage. In 3 cases there were degenerating fibres in the habenulo-interpeduncular tract. In two of these cases it was very sparse, while in the third it was extremely heavy on the side opposite the lesion (Fig. 1). Despite this, there was no degeneration in the interpeduncular nucleus. In the region of the habenular nuclei, there were only a very few degenerated preterminals in the lateral habenular nucleus; degenerating fibres of passage appeared rather to encapsulate the habenular nuclei, also running between the medial and lateral nuclei. Further degenerating fibres of passage were to be seen along the whole perithalamic course of the stria medullaris. Ventral thalamus. In all cases, plentiful preterminal degeneration was found in the zona incerta; less dense degeneration was seen in the fields of Forel (HI and H2), and in the subthalamic nucleus (corpus Luysii). Some preterminal degeneration was also observed in the rostral extension of the substantia nigra, pars reticulata. Hypothalamus. Degenerated fibres occurred in the body and descending columns of the fornix, more heavily on the side of the lesion than contralaterally; they apparently gained the contralateral side in the hippocampal commissure. Given the dorsal position of the hippocampus in the cat, it is possible that this structure has
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Fig. 1. a-e: projection drawings from a transverse series (4153). Stereotaxic planes refer to the thalamic part of each section; the ventral part is more rostral than the dorsal. The arrow is on the side of the lesion. Dots represent preterminal degeneration, lines degenerating fibres of passage. a: shows the greatest extent of the lesion, b ~ : distribution of resulting degeneration. Abbreviations: CL, n. centratis lateralis; CM, n. centrum medianum; C.Me, n. centralis medialis; Forel, fields of Forel; Fx, fornix; GL, n. geniculatus lateralis; G M , n. geniculatus medialis; Int, n. interpeduncularis; Lat. hp, lateral hypothalamus; Mm. lat, n. mammillaris lateralis; NR, n. ruber; Pf, n. parafascicularis; PO, posterior group; POre, posterior group, pars medialis; SN, substantia nigra: STh, n. subthalamicus, VP, n. ventralis posterior; ZI, zona incerta.
216 been mechanically damaged by the penetrating electrode; but it is hard to explain all the degeneration seen by this minimal trauma. As the descending columns of the fornix pass through the hypothalamus, very small amounts of preterminal degeneration were seen in association with it in the anterior and dorsal hypothalamus. Much heavier preterminal degeneration was seen in association with the terminal part of the fornix in the lateral hypothalamus and in the lateral mammillary nucleus; this latter degeneration, as well as that observed in the posterior hypothalamus, was also continuous with that seen in the ventral tegmental area of Tsai in the midbrain. DISCUSSION
It should be emphasised that the distribution of preterminal degeneration in the telencephalon has not been studied in the present investigation, which has been deliberately restricted to the diencephalon. Comparison with other studies. The distribution of preterminal degeneration found in this investigation is remarkably similar to that reported by Nauta and Kuypers 2s. Taking account of the revision of thalamic topography by Rinvik 29, it can be seen that PO is also implicated by the midbrain lesion of Nauta and Kuypers (see their Figs. 24-33, 34-36 and 37-42). The very extensive diencephalic projections of the reticular formation have also been described in normal material by the Scheibels 33. The evoked potential and microelectrode studies of Rudomin et al. 31,3z in the medial diencephalon (chiefly the hypothalamus) show a distribution similar to that of the degeneration in the present investigation, except for the finding of evoked potentials in nucleus lateralis dorsalis, where no degeneration was found by either Nauta and Kuypers 2s or ourselves. Rudomin et al. 3~,32 stimulated the sciatic nerve or its branches; Robertson et al. ~o stimulated the mesencephalic tegmentum directly and described a much more restricted distribution of evoked potentials and unit responses in the dorsal and ventral thalamus. McClure and Clark 2° made lesions in the pontine reticular formation and reported heavy degeneration in the posterolateral hypothalamus. Massopust and Thompson 23 described a pathway from the interpeduncular nucleus ascending in the habenulointerpeduncular tract (fasciculus retroflexus) which terminates mainly in the lateral habenular nucleus with some fibres continuing rostrally in the stria medullaris. It has already been mentioned that some dorsal fastigiothalamic fibres of passage may have been involved in some lesions. These fibres are known to be distributed principally to the ventrolateral nucleus (VL), with less dense projections to the ventromedial and ventroanterior nuclei; the VL projection is bilateral, the fibres crossing through the midline thalamus and leaving a few degenerating preterminals in the nuclei centralis medialis and reuniens 4. However, electrophysiological investigations have shown that there is an input to VL which travels in the anterolateral columns of the spinal cord and does not relay through the cerebellum 14,21,22. The superior colliculus, the deeper layers of which have been injured in some
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Fig. 2. Schematic representation of degeneration resulting from a mesencephalic tegmental lesion (hatched) projected onto parasagittal sections at Lz and L6. Abbreviations as in Fig. 1 and: D M , n. dorsalis medialis; LP, n. lateralis posterior; Pul, pulvinar; VL, n. ventralis lateralis.
218 of the present cases, is known to project to the nucleus lateralis posterior. Again, however, short latency responses have been found in this nucleus when stimulating the midbrain tegmentum well caudal and ventral to the superior colliculus a0. Finally, account must be taken of the projections from the rhombencephalic reticular formation to the thalamus, as fibres of passage in the central tegmental tract have been damaged in many of the present cases. Following lesions of the gigantocellular region, distribution of preterminal degeneration was found to be restricted to medial intralaminar regions and the magnocellular portion of the dorsomedial nucleus 7, although Nauta and Kuypers za (their Figs. 12-19) also reported degeneration in nucleus centralis lateralis and the zona incerta consequent upon a tegmental lesion at the level of the superior olive. Functional implications. Particular functions may be considered in relation to the following groups of terminal areas (Fig. 2): (a) the intralaminar nuclei and PO; (b) the ventral, extrinsic, or lemniscal group of thalamic nuclei; (c) the ventral thalamus; and (d) the hypothalamus. (1) Somatic motor reactions. It is now well established that the principal output of the intralaminar nuclei is to the basal ganglia 1~, and Graybie116 has demonstrated projections from PO to the neostriatum, while the zona incerta, fields of Forel, and subthalamic nucleus are themselves involved as a part of non-pyramidal upper motor circuitry. These telediencephalic structures would represent an even higher level of integrated motor reaction to somatic, visual and auditory stimulation travelling through the central reticular core than that involved in spino-bulbo-spinal circuits 11. (2) The electroencephalogram. Projections to the neocortex from the anterior intralaminar nuclei have been demonstrated by retrograde degeneration 1 and by antidromic invasionL Following lesions of the posterior intralaminar nuclei in cats, degenerating fibres of passage were traced to widespread areas of the subcortical white matter 6, but no preterminal degeneration was seen with certainty in the cortex itself; a number of investigators (including the present author) have been unable to invade cells of the posterior intralaminar nuclei by cortical stimulation. However, retrograde transport of horseradish peroxidase to all intralaminar nuclei from the cortex has recently been demonstrated is, and we have seen, using [aH]leucine, a widespread projection from CM to the deep layers of the cortex (Bower and Bowsher; unpublished). GraybieP 6,~7 has recently shown that PO projects to the sulcal cortex bordering the auditory and second somatic areas and less densely to the second somatosensory area itself. The cortical projection of the extrinsic ventral thalamic nuclei is too well known to need recapitulation. It may be seen, therefore, that in toto very widespread areas of neocortex are reached by those thalamic regions to which the upper brain stem reticular formation projects. While the thalamocortical projection from nuclei other than those of the ventral group are not somato-, retino-, or tonotopically organised, they are, nevertheless, orderly. There is no need to invoke a double thalamocortical projection to explain the widespread asynchronous rapid low-voltage electrocortical or electroencephalographic activity seen following tegmental stimulation at suitable parameters.
219 That such activity cart also be observed as a result of thalamic centromedian stimulation may not be unrelated to the fact that transsynaptic activity can be seen in the midbrain reticular formation (and in VPL) following single shock stimulation of the centrum medianum (Bowsher and Molony, unpublished). Further analysis suggests that reticular afferents reaching the classical cortically projecting nuclei are collaterals of axons terminating in the intralaminar nuclei. (3) Pain. Since the demonstration that the principal end station of fibres ascending in the anterolateral quadrant of the spinal cord is the reticular formation of the brain stem 5, the latter has been implicated in the upward conduction of impulses generated by noxious stimulation. The reaction to such stimulation is very complex, involving activity at almost every level of the central nervous system. If it be accepted that the cortex is the seat of conscious sensation, then the pathways described above in connection with arousal may be invoked. Similarly, the motor reactions to pain may use the circuitry described above. Another diencephalic region implicated in pain is the lateral hypothalamus, since it was shown that stimulation of this area induces the same behavioural patterns as does noxious stimulation of the periphery. In this context, it is of great interest that transphenoidal injection of alcohol into the pituitary fossa has been shown to produce very rapid relief of pain 24, even in cases of non hormone-dependent cancer; for Lipton 19 has shown radiographically that the injected material passes up around and through the pituitary stalk to those posterior hypothalamic regions where the ascending input to the lateral hypothalamus could be interrupted. It seems, then, that all the diencephalic regions to which the midbrain reticular formation can be shown to project are in some way involved in various reactions to noxious stimulation. Indeed, this brief review shows that reticulodiencephalic projections may mediate a number of basic phenomena which cannot be explained by the restricted connections of more recently evolved lemniscal systems. ACKNOWLEDGEMENT This investigation was supported by a grant to the author from the Medical Research Council. REFERENCES 1 ADRIANOV,O. S., Sur les liaisons et les fonctions des noyaux thalamiques du syst6me 'nonsp~ifique', Acta neurol, belg., 60 (1960) 704-722. 2 ALnE-FESSARD,D., LEvAr,rrE,A., AND ROKY'rA,R., Cortical projections of cat medial thalamic cells, Int. J. Neurosci., 1 (1971) 327-338. 3 ANGAUT,P., Etude anatomique expdrimentale des effdrences cdrdbelleuses ascendantes. Analyse dlectro-anatomique des projections cdrdbelleuses sur le noyau ventral latdral du thalamus, Thesis, Paris, 1969. 4 ANGAUT,P., AND BOWSHER,D., Ascending projections of medial cerebellar (fastigial) nucleus: an experimental study in the cat, Brain Research, 24 (1970) 49-68. 5 BOWSHER,D., Termination of t"e central pain pathway in man: the conscious appreciation of pain, Brain, 80 (1957) 606-622. 6 BOWSHER,D., Some afferent and efferent connections of the parafascicular-center median complex.
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In D. P. PURPURA AND M. D. YAHR (Eds.), The Thalamus, Columbia University Press, New York, 1966, pp. 99-108. BOWSHER, D., Etude compar6e des projections thalamiques de deux zones localis6es des formations r6ticul6e hulbaires et m6senc6phaliques, C.R. Acad. Sci. (Paris}, 265D (1967) 340-342. BOWSHER,D., Reticular projections to lateral geniculate in cat, Brain Research, 23 (1970) 247-249. BOWSHER,D., Properties of ventrobasal thalamic neurones in cat following interruption of specific afferent pathways, Arch. ital. Biol., 109 (1971) 59-74. BOWSHER, D., AND PETIT, D., Place and modality analysis in nucleus of posterior commissure, J. Physiol. (Lond.J, 206 (1970) 663-675. BRODAL, A., The Reticular Formation of the Brainstem, Oliver and Boyd, Edinburgh, 1957. BUSER, P., ET SEGUNDO, J., Influences r6ticulaires somesth6siques et corticales au niveau du corps genouill6 lat6ral du thalamus chez le chat, C.R. Acad. Sci. (Paris}, 249 (1959) 571-573. COWAN, W. M., GOTTLIEB, D. l., HENDRICKSON, A. E., PRICE, J. L., AND WOOLSEY, Z. A., The autoradiographic demonstration of axonal connections in the central nervous system, Brain Research, 37 (1972) 21-51. DORMONT, J. F., ET MASS1ON, J., D6termination des voies spinales afferentes au noyau ventrolat6ral chez le chat, J. Physiol. (Paris'), 57 (1965) 240-241. DROOGLEVER-FORTUYN, J., AND STEFENS, R., On the anatomical relation of the intralaminar and midline cells of the thalamus, Electroenceph. clin. Neurophysiol., 3 (1951) 393-400. GRAYBIEL, A. M., The thalamo-cortical projection of the so-called posterior nuclear group: a study with anterograde degeneration methods in the cat, Brain Research, 49 (1973) 229-244. GRAYBIEL, A. M., Studies on the anatomical organization of posterior association cortex. In F. O. SCHMITT AND F. G. WORDEN (Eds.), The Neurosciences. Third Study Program, The MIT Press, Cambridge, Mass., 1974, pp. 205-214. JONES, E. G., AND LEAVITT, R. Y., Retrograde axonal transport and the demonstration of nonspecific projections to the cerebral cortex and striatum from thalamic intralaminar nuclei in the rat, cat and monkey, J. comp. Neurol., 154 (1974) 349-378. LIPTON, S., Personal communication (1975). McCLURE, T. D., AND CLARK, G., An ascending fiber projection from the pontine reticular formation to the hypothalamus in the cat, Exp. Neurol., 37 (1972) 510-514. MASSION, J., ANGAUT, P., ET ALBE,FESSARD, D., Activit6s 6voqu6es chez le chat dans la r6gion du nucleus ventralis lateralis par diverses stimulations sensorielles. I. Etude macrophysioiogique, Electroenceph. clin. Neurophysiol., 19 (1965) 433-451. MASSION, J., ANGAUT, P., ET ALBE-FESSARD, D., Activit6s 6voqu6es chez le chat dans la r6gion du nucleus ventralis lateralis par diverses stimulations sensorielles. II. Etude microphysiologique, Electroenceph. clin. Neurophysiol., 19 (1965) 452-469. MASSOPUST, L. C., AND THOMPSON, R., A new interpedunculo-diencephalic pathway in rats and cats, J. comp. Neurok, 118 (1962) 97-105. MORICCA, G., Chemical hypophysectomy for cancer pain, Advanc. Neurol., 4 (1974) 707-714. MORISON, R. S., AND DEMPSEY, E. W., A study of thalamo-cortical relations, Amer. J. Physio/., 135 (1942) 281-292. MoRuzzI, G., AND MAGOUN, H. W., Brain stem reticular formation and activation of the LEG, Electroenceph. clin. Neurophysiol., 1 (1949) 455473. NAUTA, W. J. I"l., Silver impregnation of degenerating axons. In W. F. WINDLE (Ed.), New Research Techniques ofNeuroanatomy, C. C. Thomas, Springfield, 111., 1957, pp. 17-26. NAUTA, W. J H , AND KUYPERS, H. G. J. M., Some ascending pathways in the brain stem reticular formation. In H. H. JASPER, L. O. PROCTOR, R. S. KNIGHTON, W. C. NOSHAYAND R. T. COSTELLO (Eds.), Reticular Formation of the Brain, Little, Brown and Co., Boston, Mass., 1958, pp. 3-30. RINVIK, E., A re-evaluation of the cytoarchitecture of the ventral nuclear complex of the cat's thalamus on the basis of cortico-thalamic connections, Brain Research, 8 (1968) 237-254. ROaERTSON, R. T., LYNCH, G. S., AND THOMPSON, R. F., Diencephalic distributions of ascending reticular systems, Brain Research, 55 (1973) 309-322. RUDOMfN, P., MALLIANI, A., BORLONE, M., AND ZANCHETTI, A., Distribution of electrical responses to somatic stimuli in the diencephalon of the cat, with special reference to the hypothalamus, Arch. ital. Biok, 103 (1965) 60-89. RUDOMfN, P., MALLIANI, A., AND ZANCHETTI, A., Microelectrode recording of slow wave and unit responses to afferent stimuli in the bypothalamus of the cat, Arch. ital. Biok, 103 (1965) 90-118. SCHEIREL, M. E., AND SCHEIBEL, A. B., Patterns of organization in specific and nonspecific thalamic fields. In D. P. PURPURA AND M. D. YAHR (Eds.), The Thalamus, Columbia University Press, New York, 1966, pp. 13-46.
211
D I E N C E P H A L I C PROJECTIONS F R O M T H E M I D B R A I N R E T I C U L A R FORMATION
DAVID BOWSHER Neurobiology Laboratory, Department of Anatomy, University of Liverpool, Liverpool L69 3BX (Great Britain)
SUMMARY (1) Physiologically guided stereotaxic coagulation was placed so as to avoid major through pathways in the midbrain reticular formation of 7 cats. Diencephalic degeneration resulting from this was traced by the Nauta method. (2) Preterminal degeneration was found in: the intralaminar nuclei and the posterior group (PO); the ventral group of thalamic nuclei; the ventral thalamus, including the zona incerta, subthalamus and fields of Forel; and the lateral hypothalamus. (3) The results are discussed in relation to somatomotor reactions, reticular influences on the electroencephalogram and telencephalic representation of pain.
I NTRODUCTION Perhaps because the properties of the reticular activating system 26 are so obviously similar to those previously described in the thalamic intralaminar* nuclei zS, much research, and theories based upon the results of that research, has been very largely concerned with reticulointralaminar relationships. However, the existence of reticular projections to diencephalic structures other than the intralaminar nuclei were early demonstrated by experimental methods~S; and the presence of reticular afferents to cortically projecting thalamic nuclei, as seen in Golgi material, has also been stressed 33. Reticular influences on the lateral geniculate nucleus 12 and anterolateral spinal influences on the ventrolateral thalamic nucleus14,21, z2 have been described, as have the extralemniscal properties of ventrobasal thalamic units in cats with interruption of the medial lemniscus 8. * Intralaminar thalamic nuclei, in accordance with widespread physiological practice, are here taken to include the centromedian-parafascicular complex, even though such definition may not be strictly anatomically accurate.
212 The following account, based on a description of projections from the midbrain reticular formation to the diencephalon, will attempt to underline the functional significance of all these connections and to redress the imbalance produced by undue concentration on the intralaminar nuclei of the thalamus. MATERIALS AND METHODS
In each of 7 cats under chloralose anaesthesia (70 mg/kg, intraperitoneal) a bipolar electrode was lowered stereotaxically into the midbrain through a small hole made in the cranium with a dental burr. Loci were sought in the reticular formation of the dorsal mesencephalic tegmentum in which evoked potentials could be elicited by stimulation of each of the 4 limbs. When such a site was satisfactorily identified, a small coagulation was made, using the central pole of the electrode as the cathode, while an 'indifferent' anode was clipped to the scalp incision. The lesions extended from stereotaxic plane A I to plane A5, at 2.5 or 3.0 m m from the midline. The dorsal reticular region chosen for recording and coagulation was designed to avoid damage to fibres of passage in specific thalamopetal bundles such as the brachium conjunctivum ~ and medial lemniscus; and was in the general area whose physiological properties we had previously analysed in a single unit study 10. The animals were killed by intracarotid perfusion of 10 % neutral formalin under anaesthetic 4 days after operation. The brains were removed into fixative and stored under refrigeration until they were processed. The brains were cut in serial frozen sections at 30 # m and assembled t0 to a pot in 5 % formalin. One section from each pot was stained by the Nissl method and one was impregnated by the technique of Nauta 27. Thus, sections at intervals of approximately 300 #m were available for examination. Each section was projected at a magnification of 10 times and all recognisable structures traced. The Nauta impregnated sections were then examined in the microscope and degenerating fibres and preterminals filled in on the outline drawings. Four brains were cut in coronal section and 3 in horizontal section. in two further animals, the active region of the midbrain reticular formation was again found with a bipolar electrode, the central pole of which was then removed, leaving a cannula. Down this, 0.2 #1 of [3H]leucine was then injected over 20 rain. Under the same anaesthetic (chloralose), repeated if necessary, the animals were perfused 24 h later. The brains were embedded in paraffin and cut at 8 #m in serial coronal sections. Autoradiographs were prepared 13 using llford G4 emulsion; the material awaits analysis. RESULTS
A typical case in the transverse series is illustrated in Fig. 1. Midbrain. Degenerating fibres could be seen coursing over the dorsal aspect of the aqueduct in the ventral part of the posterior commissure; dense preterminal degeneration was present in the midbrain reticular formation on the side contralateral to the lesion. On both sides, preterminal degeneration was observed in the ventral
213 tegmental area of Tsai and in the substantia nigra. At the mesodiencephalic junction degeneration was present in the periaqueductal grey matter. Diencephalon. In general, degeneration was denser on the side of the lesion; this was particularly noticeable in the case of very small lesions. The total amount of diencephalic degeneration attributable to destruction of the midbrain reticular formation (see Discussion) appeared to vary directly with the size of the lesion. Some degeneration could be attributed to incidental destruction of non-reticular structures, the most frequently involved of which were the deep layers of the superior colliculus and fastigiothalamic fibres running dorsolateral to the red nucleus 4. Dorsal thalamus. The intralaminar nuclei were in all cases filled with preterminal degeneration, the heaviest being in the nuclei centrum medianum and centralis lateralis. There appears to be a tendency for degeneration to be heavier laterally than medially within the group. The posterior group (PO), as defined by Rinvik 29, was always filled with preterminal degeneration, right up to its anterior limit as represented by the rostral border of the lateral geniculate nucleus. In some cases, preterminal degeneration in the lateral part (PO1) was almost absent, and in all cases was sparser than in the medial division (POm). Preterminal degeneration was observed in the part of nucleus lateralis posterior (LP) superjacent to PO. This was heavier when there was involvement of the deeper layers of the superior colliculus but was present to some extent in all cases. Fairly heavy degeneration of fibres of passage could always be observed in the radial bundles traversing PO and LP towards the internal capsule. In the ventral group of thalamic nuclei, scattered preterminal degeneration was always seen in the medial magnocellular part of the medial geniculate nucleus, in the (dorsal) lateral geniculate nucleus 9 (and in one instance in the ventral lateral geniculate nucleus) and in the ventrobasal complex, more heavily in its medial part and in the ventrolateral complex, including nucleus ventralis anterior. As in more dorsally placed nuclei, the radial bundles contain degenerating fibres of passage. In 3 cases there were degenerating fibres in the habenulo-interpeduncular tract. In two of these cases it was very sparse, while in the third it was extremely heavy on the side opposite the lesion (Fig. 1). Despite this, there was no degeneration in the interpeduncular nucleus. In the region of the habenular nuclei, there were only a very few degenerated preterminals in the lateral habenular nucleus; degenerating fibres of passage appeared rather to encapsulate the habenular nuclei, also running between the medial and lateral nuclei. Further degenerating fibres of passage were to be seen along the whole perithalamic course of the stria medullaris. Ventral thalamus. In all cases, plentiful preterminal degeneration was found in the zona incerta; less dense degeneration was seen in the fields of Forel (HI and H2), and in the subthalamic nucleus (corpus Luysii). Some preterminal degeneration was also observed in the rostral extension of the substantia nigra, pars reticulata. Hypothalamus. Degenerated fibres occurred in the body and descending columns of the fornix, more heavily on the side of the lesion than contralaterally; they apparently gained the contralateral side in the hippocampal commissure. Given the dorsal position of the hippocampus in the cat, it is possible that this structure has
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Fig. 1. a-e: projection drawings from a transverse series (4153). Stereotaxic planes refer to the thalamic part of each section; the ventral part is more rostral than the dorsal. The arrow is on the side of the lesion. Dots represent preterminal degeneration, lines degenerating fibres of passage. a: shows the greatest extent of the lesion, b ~ : distribution of resulting degeneration. Abbreviations: CL, n. centratis lateralis; CM, n. centrum medianum; C.Me, n. centralis medialis; Forel, fields of Forel; Fx, fornix; GL, n. geniculatus lateralis; G M , n. geniculatus medialis; Int, n. interpeduncularis; Lat. hp, lateral hypothalamus; Mm. lat, n. mammillaris lateralis; NR, n. ruber; Pf, n. parafascicularis; PO, posterior group; POre, posterior group, pars medialis; SN, substantia nigra: STh, n. subthalamicus, VP, n. ventralis posterior; ZI, zona incerta.
216 been mechanically damaged by the penetrating electrode; but it is hard to explain all the degeneration seen by this minimal trauma. As the descending columns of the fornix pass through the hypothalamus, very small amounts of preterminal degeneration were seen in association with it in the anterior and dorsal hypothalamus. Much heavier preterminal degeneration was seen in association with the terminal part of the fornix in the lateral hypothalamus and in the lateral mammillary nucleus; this latter degeneration, as well as that observed in the posterior hypothalamus, was also continuous with that seen in the ventral tegmental area of Tsai in the midbrain. DISCUSSION
It should be emphasised that the distribution of preterminal degeneration in the telencephalon has not been studied in the present investigation, which has been deliberately restricted to the diencephalon. Comparison with other studies. The distribution of preterminal degeneration found in this investigation is remarkably similar to that reported by Nauta and Kuypers 2s. Taking account of the revision of thalamic topography by Rinvik 29, it can be seen that PO is also implicated by the midbrain lesion of Nauta and Kuypers (see their Figs. 24-33, 34-36 and 37-42). The very extensive diencephalic projections of the reticular formation have also been described in normal material by the Scheibels 33. The evoked potential and microelectrode studies of Rudomin et al. 31,3z in the medial diencephalon (chiefly the hypothalamus) show a distribution similar to that of the degeneration in the present investigation, except for the finding of evoked potentials in nucleus lateralis dorsalis, where no degeneration was found by either Nauta and Kuypers 2s or ourselves. Rudomin et al. 3~,32 stimulated the sciatic nerve or its branches; Robertson et al. ~o stimulated the mesencephalic tegmentum directly and described a much more restricted distribution of evoked potentials and unit responses in the dorsal and ventral thalamus. McClure and Clark 2° made lesions in the pontine reticular formation and reported heavy degeneration in the posterolateral hypothalamus. Massopust and Thompson 23 described a pathway from the interpeduncular nucleus ascending in the habenulointerpeduncular tract (fasciculus retroflexus) which terminates mainly in the lateral habenular nucleus with some fibres continuing rostrally in the stria medullaris. It has already been mentioned that some dorsal fastigiothalamic fibres of passage may have been involved in some lesions. These fibres are known to be distributed principally to the ventrolateral nucleus (VL), with less dense projections to the ventromedial and ventroanterior nuclei; the VL projection is bilateral, the fibres crossing through the midline thalamus and leaving a few degenerating preterminals in the nuclei centralis medialis and reuniens 4. However, electrophysiological investigations have shown that there is an input to VL which travels in the anterolateral columns of the spinal cord and does not relay through the cerebellum 14,21,22. The superior colliculus, the deeper layers of which have been injured in some
217 L2 121
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Fig. 2. Schematic representation of degeneration resulting from a mesencephalic tegmental lesion (hatched) projected onto parasagittal sections at Lz and L6. Abbreviations as in Fig. 1 and: D M , n. dorsalis medialis; LP, n. lateralis posterior; Pul, pulvinar; VL, n. ventralis lateralis.
218 of the present cases, is known to project to the nucleus lateralis posterior. Again, however, short latency responses have been found in this nucleus when stimulating the midbrain tegmentum well caudal and ventral to the superior colliculus a0. Finally, account must be taken of the projections from the rhombencephalic reticular formation to the thalamus, as fibres of passage in the central tegmental tract have been damaged in many of the present cases. Following lesions of the gigantocellular region, distribution of preterminal degeneration was found to be restricted to medial intralaminar regions and the magnocellular portion of the dorsomedial nucleus 7, although Nauta and Kuypers za (their Figs. 12-19) also reported degeneration in nucleus centralis lateralis and the zona incerta consequent upon a tegmental lesion at the level of the superior olive. Functional implications. Particular functions may be considered in relation to the following groups of terminal areas (Fig. 2): (a) the intralaminar nuclei and PO; (b) the ventral, extrinsic, or lemniscal group of thalamic nuclei; (c) the ventral thalamus; and (d) the hypothalamus. (1) Somatic motor reactions. It is now well established that the principal output of the intralaminar nuclei is to the basal ganglia 1~, and Graybie116 has demonstrated projections from PO to the neostriatum, while the zona incerta, fields of Forel, and subthalamic nucleus are themselves involved as a part of non-pyramidal upper motor circuitry. These telediencephalic structures would represent an even higher level of integrated motor reaction to somatic, visual and auditory stimulation travelling through the central reticular core than that involved in spino-bulbo-spinal circuits 11. (2) The electroencephalogram. Projections to the neocortex from the anterior intralaminar nuclei have been demonstrated by retrograde degeneration 1 and by antidromic invasionL Following lesions of the posterior intralaminar nuclei in cats, degenerating fibres of passage were traced to widespread areas of the subcortical white matter 6, but no preterminal degeneration was seen with certainty in the cortex itself; a number of investigators (including the present author) have been unable to invade cells of the posterior intralaminar nuclei by cortical stimulation. However, retrograde transport of horseradish peroxidase to all intralaminar nuclei from the cortex has recently been demonstrated is, and we have seen, using [aH]leucine, a widespread projection from CM to the deep layers of the cortex (Bower and Bowsher; unpublished). GraybieP 6,~7 has recently shown that PO projects to the sulcal cortex bordering the auditory and second somatic areas and less densely to the second somatosensory area itself. The cortical projection of the extrinsic ventral thalamic nuclei is too well known to need recapitulation. It may be seen, therefore, that in toto very widespread areas of neocortex are reached by those thalamic regions to which the upper brain stem reticular formation projects. While the thalamocortical projection from nuclei other than those of the ventral group are not somato-, retino-, or tonotopically organised, they are, nevertheless, orderly. There is no need to invoke a double thalamocortical projection to explain the widespread asynchronous rapid low-voltage electrocortical or electroencephalographic activity seen following tegmental stimulation at suitable parameters.
219 That such activity cart also be observed as a result of thalamic centromedian stimulation may not be unrelated to the fact that transsynaptic activity can be seen in the midbrain reticular formation (and in VPL) following single shock stimulation of the centrum medianum (Bowsher and Molony, unpublished). Further analysis suggests that reticular afferents reaching the classical cortically projecting nuclei are collaterals of axons terminating in the intralaminar nuclei. (3) Pain. Since the demonstration that the principal end station of fibres ascending in the anterolateral quadrant of the spinal cord is the reticular formation of the brain stem 5, the latter has been implicated in the upward conduction of impulses generated by noxious stimulation. The reaction to such stimulation is very complex, involving activity at almost every level of the central nervous system. If it be accepted that the cortex is the seat of conscious sensation, then the pathways described above in connection with arousal may be invoked. Similarly, the motor reactions to pain may use the circuitry described above. Another diencephalic region implicated in pain is the lateral hypothalamus, since it was shown that stimulation of this area induces the same behavioural patterns as does noxious stimulation of the periphery. In this context, it is of great interest that transphenoidal injection of alcohol into the pituitary fossa has been shown to produce very rapid relief of pain 24, even in cases of non hormone-dependent cancer; for Lipton 19 has shown radiographically that the injected material passes up around and through the pituitary stalk to those posterior hypothalamic regions where the ascending input to the lateral hypothalamus could be interrupted. It seems, then, that all the diencephalic regions to which the midbrain reticular formation can be shown to project are in some way involved in various reactions to noxious stimulation. Indeed, this brief review shows that reticulodiencephalic projections may mediate a number of basic phenomena which cannot be explained by the restricted connections of more recently evolved lemniscal systems. ACKNOWLEDGEMENT This investigation was supported by a grant to the author from the Medical Research Council. REFERENCES 1 ADRIANOV,O. S., Sur les liaisons et les fonctions des noyaux thalamiques du syst6me 'nonsp~ifique', Acta neurol, belg., 60 (1960) 704-722. 2 ALnE-FESSARD,D., LEvAr,rrE,A., AND ROKY'rA,R., Cortical projections of cat medial thalamic cells, Int. J. Neurosci., 1 (1971) 327-338. 3 ANGAUT,P., Etude anatomique expdrimentale des effdrences cdrdbelleuses ascendantes. Analyse dlectro-anatomique des projections cdrdbelleuses sur le noyau ventral latdral du thalamus, Thesis, Paris, 1969. 4 ANGAUT,P., AND BOWSHER,D., Ascending projections of medial cerebellar (fastigial) nucleus: an experimental study in the cat, Brain Research, 24 (1970) 49-68. 5 BOWSHER,D., Termination of t"e central pain pathway in man: the conscious appreciation of pain, Brain, 80 (1957) 606-622. 6 BOWSHER,D., Some afferent and efferent connections of the parafascicular-center median complex.
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