490

Brain Research, 93 (1975) 490-496 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Delineation of dorsal lateral geniculate afferents from the cat brain stem as visualized by the horseradish peroxidase technique

L. LEGER, K. SAKAI, D. SALVERT, M. TOURET AND M. JOUVET Ddpartement de Mddeeine Expdrimentale, Facultd de Mddecine, Universitd Claude Bernard, 8 Av. Rockefeller, Lyon 69008 (France)

(Accepted April 25th, 1975)

Anatomical studies with the Golgi method and anterograde degeneration technique have revealed connections between the ponto-mesencephalic reticular formation and the dorsal lateral geniculate nucleus (LGNd) in the mouse 19 and in the cat 3. Further, several histochemical studies in the rat have demonstrated acetylcholinesterase (AChE)-containing 13, catecholaminergic (CA) 14, 20, and serotonergic (5-HT) 7 fibers ascending from the brain stem to the LGNd. The presence of CA fibers in the L G N d has been also observed in the cat 16. In addition, a recent autoradiographic study z has pointed to a direct connection between the nucleus raphe dorsalis (NRD) and the LGNd. Physiological investigations in several species of animals 4-6,11,1s have also shown that impulses originating in the brain stem reach the LGNd. In the present study, using a recently developed retrograde tracer technique with horseradish peroxidase (HRP)S-10,12, ~7, we have attempted to identify the afferent connections to the L G N d from the brain stem reticular formation. Using 21 cats weighing between 1.5 and 3.8 kg, H R P (Sigma, type VI) was injected hydraulically (0.05, 0.2, 0.5 and 1.0 #l of a 33 ~ solution) into the L G N d with a Hamilton 5 #1 syringe. In 5 animals (later sacrificed at 13 or 16 h), 0.05-0.1 /zl of the H R P was injected into the ventral lateral geniculate nucleus (LGNv), pulvinar, or reticular nucleus of the thalamus in the vicinity of the LGNd. At either 9, 13, 16, 20, 24, 30, or 72 h, the animals were sacrificed by cardiac perfusion with a solution containing 3 ~ paraformaldehyde-1 ~ glutaraldehyde in 0.1 M phosphate buffer (pH 7.2-7.4). The brain was removed, left in the same fixative for about 4 h and then transferred to 0.1 M phosphate buffer overnight. Frontal sections (60 #m thick) were cut on a freezing microtome from Horsley-Clarke plane A13 to P10. After rewashing in Tris.HC1 buffer (0.05 M, p H 7.6) for 1-2 h, the sections were soaked for 30 min in a medium containing 0.05 ~ 3,3'-diaminobenzidine (DAB), and then incubated for 30 rain in a 0.05 ~ DAB solution containing 0.01-0.02 HzO2. The sections were then washed and mounted. Half of these sections were counterstained with cresylecht violet. The limit of the spread of HRP at the injection site was determined according to Nauta et aL 17 and Graybiel and Hartwieg 9. Examples of'large' (1.0 #1) and 'small'

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Fig. 1. Location of HRP-positive neurons observed in the brain stem following injection (1.0 #I) of HRP into the LGNd (case H 48). Survival time: 16 h. Dots indicate HRP-positive neurons. Blackened areas and surrounding shaded areas indicate the central and peripheral areas of the injection site. Abbreviations for this and the following illustrations: BC, brachium conjunctivum; BCI, brachium of inferior colliculus; BP, brachium pontis; DBC, decussation of brachium conjunctivum; GC, griseum centralis; IC, inferior colliculus; ]P, interpeduncular nucleus; LC, locus coeruleus; LSC, locus subcoeruleus; LL, lateral lemniscus; LM, medial lemniscus; MLF, medial longitudinal fasciculus; mV, mesencephalic tract of trigeminus; NLL, nucleus of lateral lemniscus; NRC, nucleus raphe centralis; NRD, nucleus raphe dorsalis; NRL, nucleus raphe linearis; NRM, nucleus raphe magnus; NRP, nucleus raphe pontis; P, pyramidal tract; SC, superior colliculus; SOM, superior olive complex; TD, dorsal nucleus of Gudden; TV, ventral nucleus of Gudden; V, motor nucleus of trigeminus; III, oculomotor nucleus; IV, trochlear nucleus.

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Fig. 2. Location of HRP-positive neurons observed in the brain stem following injection (0.2/A) of HRP into the LGNd (case W 48). Survival time: 13 h. Symbols and abbreviations are described in the legend accompanying Fig. 1. (0.2/zl) injections are shown in Figs. 1 and 2, respectively. In each case, the injection site was centered in the L G N d , whereas diminished H R P activity was also found in the optic radiation, optic nerve, accessory optic nerve, reticular complex of the thalamus, L G N v and very slight activity in the pulvinar and the posterior and lateral posterior complex of the thalamus.

493 HRP-positive neurons were observed in the brain stem at 13-24 h with an H R P volume of 0.2-1.0 #I. Survival times of 13-16 h appeared optimal for demonstrating these neurons and their processes, while times of about 24 h seemed to be optimal for demonstrating the afferent connections with the cortex. An H R P volume smaller than 0.2 #1 failed to label brain stem neurons, while cortical neurons were labeled by injections as small as 0.05 #1. This latter observation is evidence of L G N d afferents from the cortex, and indeed H R P labeled neurons were observed scattered in the deepest laminae of the posterior splenial, lateral, suprasylvian, and ectosylvian gyri. The H R P reaction of these neurons was usually 2-3 times more intense than that of the brain stem neurons. When HRP-positive neurons were detected in the brain stem, the distribution of these labeled neurons was markedly similar for both large and small H R P injections (Figs. 1 and 2). At A2, labeled neurons were observed in the central linear nucleus (NRL) ventral to the oculomotor nucleus and dorsal to the decussation of the brachium conjunctivum (BC). More caudally, labeled neurons appeared in the dorsal raphe nucleus (NRD), mainly in its anterior part (Fig. 3B and C). At A1, numerous labeled cells were found bilaterally in the area which lies in and surrounds the BC, in particular its medial and lateral parts (Fig. 3G and H). From AP0 to P1, the labeled cells were mainly located in the area dorsal to the BC and ventral to the cuneiform nucleus, but also ventral to the periaqueductal gray and dorsomedial to the BC (Fig. 3F). This latter group of neurons appeared to extend caudally and join with a group of neurons in the nucleus locus coeruleus (LC) which were observed as far caudally as P4 (Fig. 3D and E). From P2-P3, a few labeled neurons were observed still closely related to the BC. Except for the raphe nuclei, HRP-positive neurons were about 2-3 times more numerous on the injection side than on the contralateral side (Figs. 1 and 2), and this was also the case with respect to the reaction intensity. Only a small percentage of neurons were labeled by H R P in each structure and no labeled neurons were detected in other parts of the mesencephalon and pons. In spite of the apparent spread of H R P from the injection site into the L G N v and pulvinar, no HRP-positive neurons appeared in the superior colliculus (SC). By contrast, even small (0.05 #1) injection of H R P directly into the L G N v or pulvinar caused the appearance of labeled cells in the SC, confirming the classical anatomical connections 1,s. The topography of labeled neurons in the case of large (0.5-1.0 #1) and small (0.2/~1) injections was remarkably similar, but the labeled cells were more numerous and their labeling was more intense in the former than in the latter case, confirming the findings of Nauta et al. ~7. Whether the situation described above is due to a difference in the number of HRP-capturing terminals located in the L G N d itself or indicates H R P uptake by synapses or fibers located just outside the L G N d is unclear, especially because no accumulation of H R P in the brain stem cell bodies after small (0.05 #1) injections was detectable. On the other hand, the lack of labeled cells in the SC after injection into the L G N d contrasts with the appearance of these cells after direct injection into the L G N v or pulvinar and leads us to conclude that

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495 peripheral zones of the injection site were not effective in eliciting the retrograde transport of HRP. The present study has supplied no evidence that could confirm the existence of L G N d afferents originating either in the magnocellular pontine reticular formation as described by Scheibel and Scheibe119 or in the region of the mesencephalic tegmentttm indicated by Bowsher 3 as the origin of such fibers. Nevertheless, the present experiment does not rule out these two afferent connections of the L G N d since, as previous workers have noticedg, 17, the H R P method may tail to label all afferents of a given nucleus. The present findings give further evidence of projections from the raphe nuclei to the LGNd, as well as between the locus coeruleus and the LGNd. As noted above, L G N d in the rat is known to contain CA fibers ascending from the LC. The origin of the CA fibers in the L G N d of the cat is thought to be the LC, especially its lateroventral part or locus subcoeruleus (LSC) 16. In the present study, HRP-positive neurons mainly appeared in the dorsomedial portion of the LC and only few cells were detected in the LSC area. This discrepancy suggests the existence of non-CA neuronal pools in LC efferents to the LGNd. We have here reported two additional sources of L G N d afferents: one of them is in the area just rostral to the LC, and the other in the area which lies in and surrounds the BC. The anatomical significance of these areas is unknown; however, the present results do give an anatomical basis to recent electrophysiological findings. Foote et al. "5 reported that stimulation of the lateral reticular formation as well as the dorsal nucleus of the raphe alters the spontaneous and evoked activity of single cells in the LGNd. The effect of the stimulation was reported to first produce an inhibition with a latency of 7-10 msec suggesting a monosynaptic connection between these structures. Similarly, stimulation of the region in and around the BC has been reported to elicit large macro-potentials (latency of 5-9 msec) in the L G N d as. In addition, bilateral electrolytic coagulations of the area completely abolish ' P G O waves' otherwise recorded in the L G N d TM. Whether H R P labeled neurons observed in the raphe nuclei and LC correspond respectively to 5-HT-containing and CA-containing neurons is unknown. The precise neurochemical structure and functions of these and other H R P labeled neurons should be determined. As demonstrated and suggested by I_jungdahl et al. 15, the H R P method combined with transmitter histochemistry may well be able to resolve these problems.

Fig. 3. A: low-power photomicrograph illustrating injection site in case W 48. B: dark-field photomicrograph of HRP-positive neurons in the NRD (case H 48). × 140. C: higher magnification of part of B. × 1200. D: dark-field photomicrograph of HRP-positive neurons in the dorsomedial part of the LC coutralateral to the injection site (case H 48). Arrows indicate the lecation of HRPpositive neurons. × 140. E: higher magnification of a part of D. × 1200. F: dark-field photomicrograph of HRP-positive neurons in the area just ventrolateral to the GC on the injection side (case H 48). × 200. G : dark-field photomicrograph of HRP-positive neurons in the area just dorsolateral to the BC at AI on the injection side (case T 48). × 200. H : bright-field photomicrograph of some of the HRP-positive neurons seen in G. × 580.

496 T h i s w o r k was s u p p o r t e d b y I N S E R M

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