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Projection of brain stem neurons to the perigeniculate nucleus and the lateral geniculate nucleus in the cat
Brain Research, 238 (1982) 433438 Elsevier Biomedical Press
433
Projection of brain stem neurons to the perigeniculate nucleus and the lateral geniculate nucleus in the cat
GUNILLA AHLSI~N and FU-SUN LO* Department of Physiology, University of GSteborg, Box 33031, S-400 33 GSteborg (Sweden)
(Accepted December 31st, 1981) Key words: reticular formation - - retrograde horseradish peroxidase-labehng - - antidromic activation - - lateral geniculate nucleus - - perigeniculate nucleus
Small horseradish peroxidase injections in the perigeniculate nucleus (PGN) or the lateral geniculate nucleus (LGN) gave retrograde labeling of many cells in the pontomesencephalic reticular formation (RF), the nuclei raphe dorsalis and centralis linearis, locus coeruleus, nucleus of the optic tract and nucleus parabigeminalis. Antidromic stimulation was used to identify neurons in the RF projecting to the PGN-LGN complex. Threshold mapping through the PGN and the LGN shows separate projection from the reticular formation to the PGN and the LGN. As in other thalamic nuclei, transmission in the dorsal lateral geniculate nucleus ( L G N ) can be effectively facilitated by electrical stimulation in the reticular formation (RF) xs. It has been proposed that this effect is mainly due to disinhibition o f principal cells in the LGN14,17, as was first demonstrated for the ventrolateral thalamic nucleus (ref. 15). Principal cells in the L G N are inhibited both through a feed-forward pathway, via retinal ganglion cells and intrageniculate interneurons, and through a recurrent pathway including axon collaterals of principal cells and interneurons in the perigeniculate nucleus (PGN)4, al. Weak stimulation ( < 30 # A ) in the R F induces a long-lasting (about 100 ms) inhibition o f perigeniculate neurons with a latency o f 10-15 ms, as well as a corresponding reduction o f recurrent IPSPs in principal cells 2. A similar inhibition is evoked in intrageniculate interneurons assumed to mediate feed-forward inhibition (Ahls6n, Lindstr6m and Lo, unpublished observation), q-he latency o f the inhibition from the R F does not preclude a pathway with several relays, but might be due to slow conduction o f inhibitory reticular neurons projecting directly to P G N and L G N . It was therefore desirable to obtain information regarding the projection from the R F to the P G N and the L G N . Degeneration studies indicated the existence o f such a projection in the cat 20. More recently, the horseradish peroxidase (HRP) method revealed retrograde labeling o f cells in the pontomesencephalic R F after large H R P injections in the LGNS, 10. However, no labeled neurons were found in the brain stem after small H R P injections * Present address: Shanghai Brain Research Institute, 319 Yo-Yang Road, Shanghai, China. 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
434 (ref. 10). Since large injections give considerable diffusion outside P G N a n d LGN. It was desirable to reinvestigate the problem. It will be shown that small H RP injections in P G N or L G N m a y label a large n u m b e r of n e u r o n s in the RF. but o u r results also show that the H R P technique is n o t sufficiently discriminative to differentiate Izetween projections to P G N a n d L G N . A further analysis was therefore made using threshold m a p p i n g for a n t i d r o m i c s t i m u l a t i o n : since a previous study showed that strong s t i m u l a t i o n in the L G N gave a n t i d r o m i c activation of some brain stem cells t6. S
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Fig. 1. A-D: location of labeled cells in the brain stem after two 0.025 #1 injections of HRP (10 ~ in 1 M NaCI) m the PGN. Survival time 22 h. A: frontal sections through the centers of the two injection sites; DAB-procedure. Black areas represent densely HRP-stained central zones of the injection sites while dotted areas represent weaker stained peripheral areas (see ref. I). Note that the peripheral zone of the injection site covers not only the PGN but also a large part of the reticular nucleus of thalamus and extends into lamina A of the LGN. The correspondin~ contralaterat retinal labeling (cf. text) was not found in retinal regions projecting to more medial or lateral parts of the LGN, which makes it very unlikely that uptake and retrograde labeling has occurred from regions outside the dotted area. B-D: reconstructions of labeled cells in 3 consecutive 80 #m sections at A2 (B), APO ((3) arid PI (D). E and F: location of 49 cells antidromicaUy activated by stimulation in the PGN-LGN complex with thresholds below 200uA. Open circles represent cells with bilateral projection; E at P 0.3, F at P 1.0. Abbreviations: BC, brachium conjuuctivum; CNF, cuneiform nucleus; DR, dorsal raphe nuclei; [C. inferior colliculus; LGN, lateral geniculate nucleus; MLB, medial longitudinal bundle; PAG, periaqueductal gray; PGN, perigeniculate nucleus; RNT, reticular nucleus of thalamus: SC. superior colliculus; TV, ventral tegmental nucleus; lII, oculomotor nucleus.
435 The experiments were made on cats anesthetized with pentobarbital sodium. Small volumes (0.02-0.15/A usually divided in 2-3 injections) of 10 ~ H RP solution (Sigma type VI or Boehringer grade I, in 1 M NaC1) were pressure-injected from a glass micropipette into the PGN or the LGN. After 16-24 h survival time the brains were fixed 1 and relevant regions removed. Frontal sections, 80 /~m, were cut on a freeze microtome. The brain stem sections were reacted for H R P using the tetramethylbenzidine (TMB) method a3. The P G N - L G N sections and the retinae were processed with diaminobenzidine (DAB) 6. Tungsten electrodes were used to record neurons in the RF in l0 cats. Such electrodes were also used for stimulation, recording of visually evoked responses, and placing of electrolytic lesions in the P G N - L G N . Retrogradely labeled neurons in the brain stem were found in 4 of 5 cats, in which small amounts of H R P (0.02, 0.05 or 0.15/A) were injected unilaterally in the PGN. Labeled neurons were found bilaterally, about 7 0 ~ on the ipsilateral side between frontal planes A2 and P4 (Fig. IA). In the rostral end of this region, A2 and A1 (B), the cells were spread in and around the lateral parts of the dccussating brachium conjunctivum (BC). Around APO (C), the location extends more medially, with the highest density just dorsal to the BC. Some cells were also found in the BC and a few ventral to it. Further caudally, at P1 (D), the location is similar but with an increasing number of cells ventromedial to BC. This group of labeled cells extends caudally into the locus coeruleus complex (LC). At frontal planes P2-P4 this is the only location of labeled cells except for a few scattered around the BC. Labeled cells were also found in nucleus raphe centralis linearis and raphe dorsalis (n.R.). Fig. I is from an experiment (0.05/tl) with about 800 latzeled brain stem cells. In the other 3 experiments the number of cells were assessed as 2800, 250 and 100 (0.05, 0.15 and 0.02 /~1, respectively), including cells in the RF (61~), n.R (20~/) and LC (16~,); percentages are from the experiment with the largest numI~er of labeled cells. Labeled brain stem neurons were also found in 3 of 4 cats in which small H RP injections (0.02, 0.05 and 0.1/~1) were placed in the LGN. In one cat one injection was in lamina A and another in lamina AI. in the other cats the injections were in lamina A1 and the dense injection zones of the injection sites were confined to this lamina, as has been illustrated (Figs. 1 and 2 in ref. 1). In all cats the diffuse zone of the injection site covered a large part of the PGN. The distribution of labeled cells in the brain stem was similar to that after injections in the PGN and agreed with that previously reported 5,1°. The labeled cells were found in the RF (58 ~), n.R (21 ~ ) and LC (16 ~). The largest number of brain stem neurons, about 3000, were found in the cat which received one of the injections in lamina A (0. I/~1). In the other two experiments about 400 (0.1 /~l) and 20 (0.02/~1) brain stem cells were labeled. All PGN and L G N injections gave many labeled cells in the nucleus of the optic tract (n. OT). In the experiments with 2800 (PGN injection) and 3000 (LGN injection) labeled brain stem neurons, we found, in addition, a large number of labeled cells in nucleus parabigeminalis. To interpret the H R P results, it is important to recognize that retrograde labeling occurs from terminals far out in the diffuse injection zone 1. Further evidence for such uptake has now been obtained, since retinal ganglion cells projecting to
436 lamina A in the dorsal part of the L G N were labeled with all P G N injections. We can therefore neither exclude labeling of brain stem neurons after injections in the PGN from the diffuse zone extending into LGN, nor a corresponding labeling from the diffuse zone in the P G N after localized L G N injections. Our H R P experiments allow the conclusion (cf. legend Fig. l) that a large number of brain stem neurons project to the P G N - L G N complex (including the reticular nucleus of thalamus dorsal to the PGN). It is noteworthy that the number of labeled brain stem neurons varied over a large range, including absence of labeling in two experiments, in which labeling in other afferent systems to the P G N - L G N complex (retina, visual cortex, n. OT) was normal. It is not known if the H R P uptake is activity dependent0:9: if so, the poor labeling might be due to depressed activity in the RF. Whatever factor is responsible, it may explain why Leger et al. 1° did not find labeled neurons in the brain stem after H R P injections smaller than 0.2 #l of 30 ~ HRP. In the electrophysiological experiments. 107 cells in the brain stem were antidromically activated from the P G N - L G N region with thresholds below 200 uA, often as low as 5-10/zA. Fixed latency at near threshold, collision with spontaneous spikes, and ability to follow high frequency stimulation, were used as criteria for antidromic activation. Bilateral stimulation was systematically investigated in two experiments. Of 60 identified neurons, 34 projected only ipsilaterally, 22 only contralateratly and 4 bilaterally. The cells were recorded between frontal planes A 0.5 and P1. mainly close
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Fig. 2. A: extracellular recordings from a RF neuron, showing antidromic activation from the PGN at threshold stimulation and shortening of antidromic latency with stronger stimulation. Each record consists of 3 superimposed traces. Stimulus strengths are indicated in #A. The cell had low threshold points for antidromic activation throughout the PGN but not in the laminae of the LGN. B: distribution of the longest antidromic latencies for ipsilaterally projecting RF neurons activated from the PGN-LGN complex. Bilaterally projecting cedts are specially indicated. C: threshold profiles for two RF neurons obtained from the track through the PON-LGN complex shown to the left in the parasagittal plane. Stars indicate lesions. One cell has low threshold points exclusively in the PGN (black dots) while the low threshold points for the other are restricted to the PGN (open circles).
437 to or in the medial part of the BC (Fig. IB). This is within the RF region containing labeled cells in Fig. 1. The histogram in Fig. 2B shows that in 57 ipsilaterally projecting cells the antidromic latency at near threshold stimulation ranged from 7 to 34 ms. With increasing strength of stimulation the antidromic latency usually decreased in discrete steps, often of 1-4 ms and occasionally of as much as 10-12 ms (Fig. 2A). It is assumed that the longest latency spikes originated in distal branches and that the latency shortening is caused by stimulationof successively more proximal branches. Very long latency spikes tended to disappear after repeated tracking or strong stimulation. For a few cells we found regions where the antidromic latency was short (3-10 ms) and did not decrease with increasing strength. These spikes presumably originated in the stem axon or a major branch. In individual cells the latency was more than doubled from short latency sites to other sites of antidromic activation. For comparison of antidromic latencies with latencies of synaptic effects evoked from the RF in the P G N - L G N , the slow preterminal conduction is a decisive factor; for this reason the histogram in Fig. 2B gives the longest latency encountered for each cell. For 14 of the cells, we used microstimulation at closely spaced sites along one or more complete tracks through the PGN and the LGN. Fig. 2B shows threshold profiles for two cells. One has low threshold points throughout the PGN but not in the LGN, while for the other the low threshold points are restricted to the LGN. Ten of the RF cells were of the former type and 3 of the latter. One cell had low threshold points both in the LGN and the ventral half of the PGN. The results show the existence of one population of RF cells projecting to the PGN and another to the LGN. Preliminary observations show that individual RF cells can be antidromically activated at different rostro-caudal levels in the PGN or the LGN, suggesting extensive axonal branching and termination without regard to the retinotopic organization of the LGN and the PGN. The RF neurons projecting to the PGN may be inhibitory and responsible for the inhibition of recurrent IPSPs in principal cells from the RF 2. The shortest antidromic latencies in Fig. 2 are compatible with this hypothesis; they are similar to the latencies of the inhibition of perigeniculate neurons evoked from the rostral RF 2. The long antidromic latencies in Fig. 2 may contribute to the long duration of the IPSP in the perigeniculate neurons. Correspondingly, RF neurons projecting to the LGN might mediate inhibition to intrageniculate interneurons and give reticular suppression of feed-forward inhibition in principal cells. If this interpretation is correct, our results suggest that feed-back and feed-forward inhibition may be differentially controlled. However, the large number of neurons in the RF projecting to the P G N - L G N complex revealed by the HRP study should be considered in view of the results showing extensive divergence for individual reticulo-perigeniculate and reticulo-geniculate neurons, which suggests lack of spatial selectivity in the control of the LGN. We can not exclude the possibility that a group of reticulo-geniculate neurons projects to both the PGN and the LGN (cf. above) and further analysis is required to find out if subgroups exist among those cells projecting to the PGN or the LGN. It is also desirable to find out if the projection to the P G N - L G N complex from
438 t h e R F is p r i v a t e o r s h a r e d w i t h o t h e r t h a l a m i c nuclei. Finally, it is w o r t h n o t i n g t h a t the R F is o n l y o n e o f several e x t r a r e t i n a l systems to the P G N - L G N
c o m p l e x . Besides
the well k n o w n p r o j e c t i o n f r o m the visual c o r t e x ~ we h a v e c o n f i r m e d the existence o f p r o j e c t i o n s f r o m r a p h e nuclei a, l o c u s c o e r u l e u s ~2, nucleus o f t h e o p t i c t r a c t s a n d n u c l e u s p a r a b i g e m i n a l i s 7. T h i s w o r k was s u p p o r t e d by t h e S w e d i s h M e d i c a l R e s e a r c h C o u n c i l ( P r o j e c t 04767).
1 Ahls6n, G., Retrograde labelling of retinogeniculate neurones in the cat by HRP uptake from the diffuse injection zone, Brain Research, 223 (1981) 374-380. 2 Ahls6n, G., Lindstr~Sm, S. and Lo, F.-S., Inhibition of perigeniculate neurones by brain stem stimulation, Neurosci. Left., Suppl. 5 (1980) $292. 3 Bobillier, P., Petitjean, F.. Salveri, D., Ligier. M. and Seguin, S.. Differential projections of the nucleus raphe dorsalis and nucleus raphe centralis as revealed by autoradiography, Brain Research, 85 (1975) 205-210. 4 Dubin, M. W. and Cleland, B. G., Organization of visual inputs to interneurons of lateral geniculate nucleus of the cat, J. NeurophysioL, 40 (1977) 410-427. 5 Gilbert, C. D. and Kelly, J. P., The projections of cells in different layers of the cat's visual cortex. J. comp. NeuroL, 163 (1975~ 81-106. 6 Graham. R. C. and Karno~vsky, M. J., The early stages of absorption of injected horseradish peroxidase in the proximal tubulus of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochern. Cytochem., 14 (1966) 291-302. 7 Graybiel, A. M., A satellite system of the superior colliculus: the parabigeminal nucleus and its projections to the superficial collicular layers, Brain Research, 145 (1978) 365-374. 8 Graybiel, A. M. and Berson. D. M., Autoradiographic evidence for a projection from the pretectal nucleus of the optic tract to the dorsal lateral geniculate complex in the eat. Brain Research, 195 (1980) 1-12. 9 Kristensson, K. and Olsson, T., Uptake and retrograde axonal transport of horseradish perox~dase in botulinum-intoxicated mice, Brain Research, 155 (! 978) 118-123. 10 Leger, L., Sakai, K., SalverL D., Touret, M. and Jouvet, M., Delineation of dorsal lateral geniculate afferents from the cat brain stem as visualized by the horseradish peroxidase techniqu,~. Brain Research, 93 (1975) 490-496. 11 Lindstr6m, S., Synaptic organization of inhibitory pathways to principal cells in the lateral geniculate nucleus of the cat, Brain Research, in press. 12 McBride, R. I. and Sutin, J., Projections of the locus coeruleus and adjacent pontine tegmentum in the cat, J. comp. Neurol., 165 (t976) 265-284. 13 Mesulam, M. M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 14 Nakai, Y. and Domino, E. F.. Reticular facilitation of visually evoked responses by optic tract stimulation before and after enucleation, Exp. Neurol., 22 (1968) 532-544. 15 Purpura, D. P., McMurtry, J. G. and Maekawa, K., Synaptic events in ventrolateral tbalamic neurons during suppression of recruiting responses by brain stern reticular stimulation, Brain Research, 1 (1966) 63-76. 16 Sakai, K. and Jouvet, M., Brain stem PGO-on cells projecting directly to the cat dorsal lateral geniculate nucleus. Brain Research, t94 (1980) 500-505. 17 Singer, W., The effect of mesencephalic reticular stimulation on intracellular potentials of cat lateral geniculate neurons, Brain Research, 61 (1973) 35-54. 18 Singer, W., Control of thalamic transmission by corticofugal and ascending reticular pathways in the visual system, Physiol. Rev., 57 (1977) 386-420. 19 Singer, W., Hollander, H. and Vanegas, H., Decreased peroxidase labeling of lateral geniculate neurons following deafferentation, Brain Research, 120 (1977) 133-137. 20 Szentagothai, J., Lateral geniculate body structure and eye movement, Bibl. ophthaL, 82 (1972) 178-188.
433
Projection of brain stem neurons to the perigeniculate nucleus and the lateral geniculate nucleus in the cat
GUNILLA AHLSI~N and FU-SUN LO* Department of Physiology, University of GSteborg, Box 33031, S-400 33 GSteborg (Sweden)
(Accepted December 31st, 1981) Key words: reticular formation - - retrograde horseradish peroxidase-labehng - - antidromic activation - - lateral geniculate nucleus - - perigeniculate nucleus
Small horseradish peroxidase injections in the perigeniculate nucleus (PGN) or the lateral geniculate nucleus (LGN) gave retrograde labeling of many cells in the pontomesencephalic reticular formation (RF), the nuclei raphe dorsalis and centralis linearis, locus coeruleus, nucleus of the optic tract and nucleus parabigeminalis. Antidromic stimulation was used to identify neurons in the RF projecting to the PGN-LGN complex. Threshold mapping through the PGN and the LGN shows separate projection from the reticular formation to the PGN and the LGN. As in other thalamic nuclei, transmission in the dorsal lateral geniculate nucleus ( L G N ) can be effectively facilitated by electrical stimulation in the reticular formation (RF) xs. It has been proposed that this effect is mainly due to disinhibition o f principal cells in the LGN14,17, as was first demonstrated for the ventrolateral thalamic nucleus (ref. 15). Principal cells in the L G N are inhibited both through a feed-forward pathway, via retinal ganglion cells and intrageniculate interneurons, and through a recurrent pathway including axon collaterals of principal cells and interneurons in the perigeniculate nucleus (PGN)4, al. Weak stimulation ( < 30 # A ) in the R F induces a long-lasting (about 100 ms) inhibition o f perigeniculate neurons with a latency o f 10-15 ms, as well as a corresponding reduction o f recurrent IPSPs in principal cells 2. A similar inhibition is evoked in intrageniculate interneurons assumed to mediate feed-forward inhibition (Ahls6n, Lindstr6m and Lo, unpublished observation), q-he latency o f the inhibition from the R F does not preclude a pathway with several relays, but might be due to slow conduction o f inhibitory reticular neurons projecting directly to P G N and L G N . It was therefore desirable to obtain information regarding the projection from the R F to the P G N and the L G N . Degeneration studies indicated the existence o f such a projection in the cat 20. More recently, the horseradish peroxidase (HRP) method revealed retrograde labeling o f cells in the pontomesencephalic R F after large H R P injections in the LGNS, 10. However, no labeled neurons were found in the brain stem after small H R P injections * Present address: Shanghai Brain Research Institute, 319 Yo-Yang Road, Shanghai, China. 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
434 (ref. 10). Since large injections give considerable diffusion outside P G N a n d LGN. It was desirable to reinvestigate the problem. It will be shown that small H RP injections in P G N or L G N m a y label a large n u m b e r of n e u r o n s in the RF. but o u r results also show that the H R P technique is n o t sufficiently discriminative to differentiate Izetween projections to P G N a n d L G N . A further analysis was therefore made using threshold m a p p i n g for a n t i d r o m i c s t i m u l a t i o n : since a previous study showed that strong s t i m u l a t i o n in the L G N gave a n t i d r o m i c activation of some brain stem cells t6. S
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Fig. 1. A-D: location of labeled cells in the brain stem after two 0.025 #1 injections of HRP (10 ~ in 1 M NaCI) m the PGN. Survival time 22 h. A: frontal sections through the centers of the two injection sites; DAB-procedure. Black areas represent densely HRP-stained central zones of the injection sites while dotted areas represent weaker stained peripheral areas (see ref. I). Note that the peripheral zone of the injection site covers not only the PGN but also a large part of the reticular nucleus of thalamus and extends into lamina A of the LGN. The correspondin~ contralaterat retinal labeling (cf. text) was not found in retinal regions projecting to more medial or lateral parts of the LGN, which makes it very unlikely that uptake and retrograde labeling has occurred from regions outside the dotted area. B-D: reconstructions of labeled cells in 3 consecutive 80 #m sections at A2 (B), APO ((3) arid PI (D). E and F: location of 49 cells antidromicaUy activated by stimulation in the PGN-LGN complex with thresholds below 200uA. Open circles represent cells with bilateral projection; E at P 0.3, F at P 1.0. Abbreviations: BC, brachium conjuuctivum; CNF, cuneiform nucleus; DR, dorsal raphe nuclei; [C. inferior colliculus; LGN, lateral geniculate nucleus; MLB, medial longitudinal bundle; PAG, periaqueductal gray; PGN, perigeniculate nucleus; RNT, reticular nucleus of thalamus: SC. superior colliculus; TV, ventral tegmental nucleus; lII, oculomotor nucleus.
435 The experiments were made on cats anesthetized with pentobarbital sodium. Small volumes (0.02-0.15/A usually divided in 2-3 injections) of 10 ~ H RP solution (Sigma type VI or Boehringer grade I, in 1 M NaC1) were pressure-injected from a glass micropipette into the PGN or the LGN. After 16-24 h survival time the brains were fixed 1 and relevant regions removed. Frontal sections, 80 /~m, were cut on a freeze microtome. The brain stem sections were reacted for H R P using the tetramethylbenzidine (TMB) method a3. The P G N - L G N sections and the retinae were processed with diaminobenzidine (DAB) 6. Tungsten electrodes were used to record neurons in the RF in l0 cats. Such electrodes were also used for stimulation, recording of visually evoked responses, and placing of electrolytic lesions in the P G N - L G N . Retrogradely labeled neurons in the brain stem were found in 4 of 5 cats, in which small amounts of H R P (0.02, 0.05 or 0.15/A) were injected unilaterally in the PGN. Labeled neurons were found bilaterally, about 7 0 ~ on the ipsilateral side between frontal planes A2 and P4 (Fig. IA). In the rostral end of this region, A2 and A1 (B), the cells were spread in and around the lateral parts of the dccussating brachium conjunctivum (BC). Around APO (C), the location extends more medially, with the highest density just dorsal to the BC. Some cells were also found in the BC and a few ventral to it. Further caudally, at P1 (D), the location is similar but with an increasing number of cells ventromedial to BC. This group of labeled cells extends caudally into the locus coeruleus complex (LC). At frontal planes P2-P4 this is the only location of labeled cells except for a few scattered around the BC. Labeled cells were also found in nucleus raphe centralis linearis and raphe dorsalis (n.R.). Fig. I is from an experiment (0.05/tl) with about 800 latzeled brain stem cells. In the other 3 experiments the number of cells were assessed as 2800, 250 and 100 (0.05, 0.15 and 0.02 /~1, respectively), including cells in the RF (61~), n.R (20~/) and LC (16~,); percentages are from the experiment with the largest numI~er of labeled cells. Labeled brain stem neurons were also found in 3 of 4 cats in which small H RP injections (0.02, 0.05 and 0.1/~1) were placed in the LGN. In one cat one injection was in lamina A and another in lamina AI. in the other cats the injections were in lamina A1 and the dense injection zones of the injection sites were confined to this lamina, as has been illustrated (Figs. 1 and 2 in ref. 1). In all cats the diffuse zone of the injection site covered a large part of the PGN. The distribution of labeled cells in the brain stem was similar to that after injections in the PGN and agreed with that previously reported 5,1°. The labeled cells were found in the RF (58 ~), n.R (21 ~ ) and LC (16 ~). The largest number of brain stem neurons, about 3000, were found in the cat which received one of the injections in lamina A (0. I/~1). In the other two experiments about 400 (0.1 /~l) and 20 (0.02/~1) brain stem cells were labeled. All PGN and L G N injections gave many labeled cells in the nucleus of the optic tract (n. OT). In the experiments with 2800 (PGN injection) and 3000 (LGN injection) labeled brain stem neurons, we found, in addition, a large number of labeled cells in nucleus parabigeminalis. To interpret the H R P results, it is important to recognize that retrograde labeling occurs from terminals far out in the diffuse injection zone 1. Further evidence for such uptake has now been obtained, since retinal ganglion cells projecting to
436 lamina A in the dorsal part of the L G N were labeled with all P G N injections. We can therefore neither exclude labeling of brain stem neurons after injections in the PGN from the diffuse zone extending into LGN, nor a corresponding labeling from the diffuse zone in the P G N after localized L G N injections. Our H R P experiments allow the conclusion (cf. legend Fig. l) that a large number of brain stem neurons project to the P G N - L G N complex (including the reticular nucleus of thalamus dorsal to the PGN). It is noteworthy that the number of labeled brain stem neurons varied over a large range, including absence of labeling in two experiments, in which labeling in other afferent systems to the P G N - L G N complex (retina, visual cortex, n. OT) was normal. It is not known if the H R P uptake is activity dependent0:9: if so, the poor labeling might be due to depressed activity in the RF. Whatever factor is responsible, it may explain why Leger et al. 1° did not find labeled neurons in the brain stem after H R P injections smaller than 0.2 #l of 30 ~ HRP. In the electrophysiological experiments. 107 cells in the brain stem were antidromically activated from the P G N - L G N region with thresholds below 200 uA, often as low as 5-10/zA. Fixed latency at near threshold, collision with spontaneous spikes, and ability to follow high frequency stimulation, were used as criteria for antidromic activation. Bilateral stimulation was systematically investigated in two experiments. Of 60 identified neurons, 34 projected only ipsilaterally, 22 only contralateratly and 4 bilaterally. The cells were recorded between frontal planes A 0.5 and P1. mainly close
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Fig. 2. A: extracellular recordings from a RF neuron, showing antidromic activation from the PGN at threshold stimulation and shortening of antidromic latency with stronger stimulation. Each record consists of 3 superimposed traces. Stimulus strengths are indicated in #A. The cell had low threshold points for antidromic activation throughout the PGN but not in the laminae of the LGN. B: distribution of the longest antidromic latencies for ipsilaterally projecting RF neurons activated from the PGN-LGN complex. Bilaterally projecting cedts are specially indicated. C: threshold profiles for two RF neurons obtained from the track through the PON-LGN complex shown to the left in the parasagittal plane. Stars indicate lesions. One cell has low threshold points exclusively in the PGN (black dots) while the low threshold points for the other are restricted to the PGN (open circles).
437 to or in the medial part of the BC (Fig. IB). This is within the RF region containing labeled cells in Fig. 1. The histogram in Fig. 2B shows that in 57 ipsilaterally projecting cells the antidromic latency at near threshold stimulation ranged from 7 to 34 ms. With increasing strength of stimulation the antidromic latency usually decreased in discrete steps, often of 1-4 ms and occasionally of as much as 10-12 ms (Fig. 2A). It is assumed that the longest latency spikes originated in distal branches and that the latency shortening is caused by stimulationof successively more proximal branches. Very long latency spikes tended to disappear after repeated tracking or strong stimulation. For a few cells we found regions where the antidromic latency was short (3-10 ms) and did not decrease with increasing strength. These spikes presumably originated in the stem axon or a major branch. In individual cells the latency was more than doubled from short latency sites to other sites of antidromic activation. For comparison of antidromic latencies with latencies of synaptic effects evoked from the RF in the P G N - L G N , the slow preterminal conduction is a decisive factor; for this reason the histogram in Fig. 2B gives the longest latency encountered for each cell. For 14 of the cells, we used microstimulation at closely spaced sites along one or more complete tracks through the PGN and the LGN. Fig. 2B shows threshold profiles for two cells. One has low threshold points throughout the PGN but not in the LGN, while for the other the low threshold points are restricted to the LGN. Ten of the RF cells were of the former type and 3 of the latter. One cell had low threshold points both in the LGN and the ventral half of the PGN. The results show the existence of one population of RF cells projecting to the PGN and another to the LGN. Preliminary observations show that individual RF cells can be antidromically activated at different rostro-caudal levels in the PGN or the LGN, suggesting extensive axonal branching and termination without regard to the retinotopic organization of the LGN and the PGN. The RF neurons projecting to the PGN may be inhibitory and responsible for the inhibition of recurrent IPSPs in principal cells from the RF 2. The shortest antidromic latencies in Fig. 2 are compatible with this hypothesis; they are similar to the latencies of the inhibition of perigeniculate neurons evoked from the rostral RF 2. The long antidromic latencies in Fig. 2 may contribute to the long duration of the IPSP in the perigeniculate neurons. Correspondingly, RF neurons projecting to the LGN might mediate inhibition to intrageniculate interneurons and give reticular suppression of feed-forward inhibition in principal cells. If this interpretation is correct, our results suggest that feed-back and feed-forward inhibition may be differentially controlled. However, the large number of neurons in the RF projecting to the P G N - L G N complex revealed by the HRP study should be considered in view of the results showing extensive divergence for individual reticulo-perigeniculate and reticulo-geniculate neurons, which suggests lack of spatial selectivity in the control of the LGN. We can not exclude the possibility that a group of reticulo-geniculate neurons projects to both the PGN and the LGN (cf. above) and further analysis is required to find out if subgroups exist among those cells projecting to the PGN or the LGN. It is also desirable to find out if the projection to the P G N - L G N complex from
438 t h e R F is p r i v a t e o r s h a r e d w i t h o t h e r t h a l a m i c nuclei. Finally, it is w o r t h n o t i n g t h a t the R F is o n l y o n e o f several e x t r a r e t i n a l systems to the P G N - L G N
c o m p l e x . Besides
the well k n o w n p r o j e c t i o n f r o m the visual c o r t e x ~ we h a v e c o n f i r m e d the existence o f p r o j e c t i o n s f r o m r a p h e nuclei a, l o c u s c o e r u l e u s ~2, nucleus o f t h e o p t i c t r a c t s a n d n u c l e u s p a r a b i g e m i n a l i s 7. T h i s w o r k was s u p p o r t e d by t h e S w e d i s h M e d i c a l R e s e a r c h C o u n c i l ( P r o j e c t 04767).
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