Mesencephalic projections to the thalamic centralis lateralis and medial prefrontal cortex: a WGA-HRP study

Brain Research, 509 (1990) 321-324

321

Elsevier BRES 23943

Short Communications

Mesencephalic projections to the thalamic centralis lateralis and medial prefrontal cortex: a WGA-HRP study Miguel Cond6s Lara 1, Martha Le6n Olea 2, Marcela S~,nchez AlvS.rez 2 and Imelda Omafia Z a p a t a 3 ~Departamento de Neurofisiologla and ZLaboratorio de Histologia, Divisidn de lnvestigaciones en Neurociencias, Instituto Mexicano de Psiquiatria, M~xico, D.F. (Mexico) and ~Coordinacidn de Estudios de Posgrado, lnvestigacidn y Desarrollo Acaddmico ENEP, UNAM, Zaragoza, MOxico, D.E (Mexico)

(Accepted 17 October 1989) Key words': Medial prefrontal cortex; Centralis lateralis nucleus; Dorsal raphe nucleus; Locus ceruleus; Ascending path; Rat

In order to provide anatomical information for a possible pathway involved in pain mechanisms, rats were injected with horseradish peroxidase wheat germ agglutinin (WGA-HRP) in the centralis lateralis nucleus of the thalamus (CI) or in the medial prefrontal cortex (PFCx) from which originated retrogradely labelled cells in the dorsal raphe nucleus (DR), locus ceruleus (LC) and surrounding structures. The locations of the C1 and the PFCx injections were previously determined by the presence of evoked single neuronal responses to noxious stimulations. The present study gives evidence for ascending pathways which originated in DR and LC and project to the CI and PFCx. LC and DR projections suggest a possible route to an ascending modulation pain system. In previous reports 7'8 we described that cells located in both the centralis lateralis nucleus (CI) of the intralaminar thalamus and the medial prefrontal cortex (PFCx) could exhibit responses to noxious stimulation and electrical stimulation of the dorsal raphe nucleus (DR). M o r e o v e r , D R cells could be antidromically activated by the C1 or PFCx electrical stimulation 9. R e c e n t works have claimed an ascending pain m o d u lation system, involving several thalamic nuclei, cortical regions and several mesencephalic structures such as D R 5'v'13"21. In an initial work carried out in h u m a n subjects with e x p e r i m e n t a l dental tooth pain stimulation, we o b s e r v e d a progressive diminution in the braine v o k e d potentials, galvanic skin responses, heart rate variations and verbal reports about pain sensations. This progressive diminution in pain responses has been correlated with somnolence and slow wave sleep 6. Besides, the i n t r a l a m i n a r thalamic nuclei, cerebral cortex and D R are also involved in sleep mechanisms 15. The f o r m e r evidence gave us the basis to search for an anatomical mesencephalic and pontine ascending modulation system. Thus, the aim of the present study is to d e t e r m i n e the projection of mesencephalic and pontine structures to C1 and PFCx in order to understand the pathways involved in a possible ascending pain modulation system, which could be involved, also, in sleep mechanisms.

Nine rats (Wistar) weighing 250-300 g were used. W h e a t - g e r m agglutinin horseradish peroxidase ( W G A H R P ) injections were a d m i n i s t e r e d under p e n t o b a r b i t a l anesthesia in 5 rats into the P F C x and in 4 rats in the CI nucleus using the stereotaxic coordinates of A l b e Fessard 3. The p l a c e m e n t of the injection sites was d e t e r m i n e d by the location of cells responding to noxious stimulation 7's. The injections were delivered using a syringe connected to a m i c r o p i p e t t e filled with a solution of 10% W G A - H R P in distilled water. The micropipettes used have an internal d i a m e t e r at the tip of about 10-15 Bm, controlled under the microscope. A volume of 0.03-0.01 ~ll was injected. A f t e r allowing a survival time of 48 h the animals were anesthetized and perfused intracardially with Ringer solution followed by a solution of 4% p a r a f o r m a l d e h y d e , 2cA g l u t a r a l d e h y d e in 0.1 M saline p h o s p h a t e buffer. The brains were removed immediately, left in the perfusion solution overnight and transferred to a p h o s p h a t e buffer with a 10% sucrose solution for 48 h. The brains were then sectioned on a freezing m i c r o t o m e at 40 Bm and the sections treated with T M B reaction 19. The sections at mesencephalic and pontine level were m o u n t e d , counterstained with Safranine, and examined with a light microscope using the coordinates of Paxinos and Watson 2°. Also, the brain regions with the injection

M. Cond6s Lara, Departamento de Neurofisiologia, Divisi6n de lnvestigaciones en Neurociencias, lnstituto Mexicano de Psiquiatr~a, Calzada M6xico-Xochimilco No. 101, M~xico. D.F. 1437[) Mexico. Correspondence:

0006-8993/90/$03.50 © 199(I Elsevier Science Publishers B.V. (Biomedical Division)

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Fig. 1. Microphotographshowing a WGA-HRP injection located in the CI nucleus. FD, fascia dentata; Hb, habenularis nucleus: L, lateralis nucleus; lme, lamina medullaris externa; MD, medialis dorsalis nucleus; MV, medialis ventralis; NCM, centralis medianus nucleus; PV, paraventricularis nucleus; Sm, stria medularis: trot, tractus mamillo-thalamicus; VL, ventralis lateralis nucleus; VP, ventralis posterior; ZI, zona incerta.

sites were cut in order to control the size and location of the injections (Fig. 1). C1 W G A - H R P injections give rise to retrograde transport to mesencephalic structures in various regions. Fig. 2 shows labelled cells in DR, iaterodorsal tegmental nucleus (LDTg), median raphe nucleus (MnR), and a well-localized group of labelled cells in the locus ceruleus (LC). C1 injections also provide anterograde W G A - H R P transport in DR, MnR, and the nucleus of the central gray at the ports level (CGPn). We also found retrogradely labelled cells at mesencephalic levels after PFCx W G A - H R P injections. Labelled cells were found in the DR, MnR, caudal linear raphe nucleus (CLi) and in the surrounding reticular formation. Fig. 3 shows retrogradely labelled cells in DR as a result of a PFCx injection. Another region strongly labelled by

PFCx injections was LC. The LC labelled cells were very obvious and, as in the case of CI W G A - H R P injections, the location was unmistakable. The LC and the tk)rmer structures only show retrograde transport after PFCx W G A - H R P injections. In addition, after PFCx injections the contralateral cortex was strongly marked with retrograde and anterograde transport of WGA-HRP. The DR and the LC projections to the CI and to the PFCx were found to be mainly ipsilateral, but there were also some contralateral labelled cells. The LC labelled cells were identified in the posterior pole of the description by Cintra et al. ~2. The W G A - H R P injections in C1 or in PFCx regions shows and confirms 2'14 the reciprocal connections of both structures. Our observations can be summarized as follows: CI W G A - H R P injections generate labelld cells at DR, MnR, LDTg and LC. PFCx W G A - H R P injections cause labelled cells at DR, LC, MnR, CLi and contralateral PFCx. The main point of these results is that the efferent projection of D R and LC have the same or similar target cells at PFCx and CI levels. We also want to point out that the cells of the CI and the PFCx where the injections were made have a common characteristic; its responsiveness to noxious stimulation 7"8. This, and the fact that the CI and the PFCx receive the input from the DR and LC make them special structures involved in pain modulation. Serotoninergic and noradrenergic pathways originating in DR and LC nuclei respectively have been implicated in analgesic processes ~'17''z2. Both amine systems have distinct ascending projections through the medial forebrain bundle which innervate forebrain structures ~. Since an endogenous pain control mechanism in the brainstem was proposed 1~, considerable attention has focused on descending serotoninergic and noradrenergic pathways. Ascending pathways have received less attention until recently an ascending pain control system involving the DR and thalamic structures was suggested 5' 13,21. Moreover, corticothalamic relationships have been implicated in the ascending pain modulation system 7. Our results pointed out direct pathways from the DR and LC to the C1 and to the PFCx. Recently 9, in an electrophysiological study we described the DR antidromic activated cells by CI or PFCx stimulation, but we failed to show that the DR projecting fibers branche to both structures concluding that there are different DR populations involved. In the present study we do not use double retrograde transport techniques and we cannot conclude if the same DR or LC populations are involved in the pathways reaching the CI and the PFCx. But the location of the DR and LC projecting cells to the PFCx and C1 have a superimposed localization. The ascending projection of DR to the thalamus and

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Fig. 2. Labelled cells after CI W G A - H R P injection. A: the upper left side shows a drawing 2° with the structures displaying labelled cells. B: typical W G A - H R P - l a b e l l e d cells. C: localized labelled cells in LC.

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Fig. 3. PFCx W G A - H R P injection site and labelled cells. A: the upper part shows the site of W G A - H R P injection at cortical level, The lower part is a drawing 2° of the mesencephalic D R cells displayed in B. C: location of the LC labelled cells after PFCx W G A - H R P injection,

324 to the cortex were described using mainly immunohislochemical techniques. Cropper et al. m reported i m m u n o -

during repeated dental tooth electrical stimulation the diminution of verbal and evoked responses is accompa.-

reactive serotonin (5-HT) fibers on the thalamic distri-

nied by somnolence and sleep ~. It is possible that in the former case the spontaneous interaction of the involved

bution and completed earlier studies on the 5-HT i n n e r v a t i o n of the thalamus 4'11'23. Furthermore, they established that the most densely innervated regions in the dorsal thalamus were the intralaminar nuclei and periventricular nuclei. The existence of 5-HT in the cortex was also first demonstrated by histofluorescence j~ , Several investigations clearly established that the 5-HT projections from the M n R and the D R reach the cortex

structures (Cl, PFCx, D R and LC) gives rise Io the decrease of pain responses. It is interesting to point out that the described D R and LC projections have a widespread cortical projection and also project to several distinct thalamic nuclei. Nevertheless, this paper is only related to the D R and the LC

via the central serotoninergic bundle which is part of the medial forebrain bundle (mfb). Axons in the mfb enter

projection to the CI and PFCx, which has an impotant nociceptive input and could be related with a modulatory pain system 7'8.

the posterior and medial cortex and continue until they reach the frontal cortex 16.

Thus, the ascending projection of D R and LC to CL and PFCx could be a possible route for pain modulation.

The previous evidence is in agreement with our described ascending D R pathway. However, LC and D R are involved in functions other than analgesia, for example, D R nucleus seems to be involved in slow wave sleep and LC seems to participate in paradoxical sleep is. At this point it is interesting to note that in humans 1 Akil, H. and Mayer, D.J., Antagonism of stimulation-produced analgesia by p-CPA, a serotonin synthesis inhibitor, Brain Research, 44 (1972) 692-697. 2 Albe-Fessard, D., Cond6s-Lara, M., Sanderson, P. and Levante, A., Tentative explanation of the special role played by the areas of paleospinothalamic projection in patients with deafferentation pain syndromes. In L. Kruger and J.C. Liebeskind (Eds.), Advances in Pain Research and Therapy, Raven Press. New York, 1984, pp. 167-182. 3 Albe-Fessard, D., Stutinsky, F. and Libouban, S., Atlas St&Ootaxique du Diencdphale de Rat Blanc, Editions du Centre National de la Recherche Seientifique, Paris, 1966. 4 And6n, N.E., Dahlstrfm, A., Fuxe, K., Larsson, K., Olson, L. and Ungerstedt, U., Ascending monoamine neurons to the telencephalon and diencephalon, Acta Physiol. Scand., 67 (1966) 313-326. 5 Andersen, E., Periaqueductal gray and cerebral cortex modulate responses of medial thalamic neurons to noxious stimulation, Brain Research, 375 (1986) 30-36. 6 Cond6s-Lara, M., Calvo, J.M. and Fern~indez-Guardiola, A., Habituation to bearable experimental pain elicited by tooth pulp electrical stimulation, Pain, 11 (1981) 185-200. 7 Cond6s-Lara, M. and Omaha Zapata, I., Suppression of noxious thermal evoked responses in thalamic central lateral nucleus by cortical spreading depression, Pain, 35 (1988) 199-204. 8 Cond6s-Lara, M., Omaha Zapata, I., Le6n Olea, M. and S~inchez Alv~irez,M., Dorsal raphe and nociceptive stimulations evoke convergent responses on the thalamic centralis lateralis and medial prefrontal cortex neurons, Brain Research, 499 (1989) 145-152. 9 Cond6s-Lara, M., Omaha Zapata, 1., Le6n Olea, M. and S~inchez Alvarez, M., Dorsal raphe neuronal responses to thalamic centralis lateralis and medial prefrontal cortex electrical stimulation, Brain Research, 499 (1989) 141-144. 10 Cropper, E.C., Eisenman, J.S. and Azmitia, E.C., An immunocytochemical study of the serotonergic innervation of the thalamus of the rat, J. Comp. Neurol., 224 (1984) 38-50. 11 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamine in cell bodies of brain stem

This work was partially supported by a research grant from Consejo Nacional de Ciencia y Tecnologia CONACyT (PCEXCNA 040661 and P228CCOX880165). We thank Mr. Abel Ortega for technical assistance, Mr. Ratll Cardoso for the illustrations and Ratll Bernal for the photographs. neurons, Acta Physiol. Scand. Suppl., 232 (1964) 1-55. 12 Cintra, L., Dfaz-Cintra, S., Kemper, T. and Morgane, P.J., Nucleus locus coeruleus: a morphometric Golgi study in rats of 3 age groups, Brain Research, 247 (1982) 17-28. 13 Hardy, S.G.P. and Haigler, H.J., Prefrontal influences upon the midbrain: a possible route for pain modulation, Brain Research, 339 (1985) 285-293. 14 Jones, E.G., The Thalamus, Plenum, New York, 1985, 935 pp. 15 Jouvet, M., The role of monoamines and acetylcholine containing neurons in the regulation of the sleep waking cycle. In M. Jouvet and G. Moruzzi (Eds.), Neurophysiology and Neurochemistry of Sleep and Wakefulness, Rev. Physiol., VoL 64, Springer, Berlin, 1972, pp. 166-307. 16 Lamour, Y., Rivot, J.P., Pointis, D. and Ory-Lavollfe, L., Laminar distribution of serotonergic innervation in rat somatosensory cortex, as determined by in vivo electrochemical detection, Brain Research, 259 (1983) 163-166. 17 Mantz, J., Milla, C., Glowinski, J. and Thierry, A.M., Differential effects of ascending neurons containing dopamine and noradrenaline in the control of spontaneous activity and of evoked responses in the rat prefrontal cortex, Neuroscience, 27 (1988) 517-526. 18 Mayer, D.J. and Prince, D.D, Central nervous system mechanisms of analgesia, Pain, 2 (1976) 379-404. 19 Mesulam, M.M., Tracing Neuronal Connections with Horseradish Peroxidase, Wiley, New York, 1982, 251 pp. 20 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1982. 21 Quiao, J.T. and Dafny, N., Dorsal raphe stimulation modulates nociceptive responses in thalamic parafascicular neurons via an ascending pathway: further studies on ascending pain modulation pathways, Pain, 34 (1988) 65-74. 22 Sawynok, J. and Reid, A.. Lesions to ascending noradrenergic and serotoninergic pathways modify antinociception produced by intracerebroventricular administration of morphine, Neuropharmacology, 28 (1989) 141-147, 23 Ungerstedt, U., Stereotaxic mapping of the monoamine pathways in the rat brain, Acta Physiol. Scand. Suppl, 367 (1971) 1-48.