Brain Research, 532 (1990) 317-322 Elsevier
BRES 24304
31"7
Short Communications
Pontine cholinergic neurons simultaneously innervate two thalamic targets Priyattam J. Shiromani, Cheryl Floyd and Javier Vehizquez-Moctezuma Department of Psychiatry (V-116A), San Diego VA Medical Center and University of California, San Diego, La Jolla, CA 92161 (U.S.A.)
(Accepted 19 June) Key words: Thalamus; Brainstem; Immunocytochemistry; Acetylcholine; Rapid eye movement sleep
Cholinergic neurons located in the lateral dorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei have been shown to principally innervate the thalamus. In order to determine whether some of these neurons might simultaneously project to two thalamic targets we made microinjections of rhodamine-conjugated microbeads into the central-lateral nucleus of the thalamus and fluorescein isothiocynate (FITC)-conjugated microbeads into the dorso-lateral geniculate nucleus. We then determined whether both tracers were found in immunohistochemically identified cholinergic somata in the LDT and PPT nuclei. Results showed that some cholinergic and non-cholinergic neurons in the LDT and PPT nuclei projected to both thalamic sites. This finding extends our understanding of the projections of the LDT-PPT cholinergic neurons and further supports the role of these neurons in complex behaviors. The largest collection of cholinergic neurons, outside of the basal forebrain cholinergic system, are located in the dorsolateral pontine tegmentum, in the lateral dorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei. In recent years much attention has focused on the L D T - P P T cholinergic neurons because they might represent the ascending reticular activating system of Moruzzi and Magoun 13 and the ascending cholinergic system of Shute and Lewis 26. The L D T - P P T cholinergic neurons are hypothesized to activate the cortex, i.e. produce E E G desynchronization, by acting on intralaminar thalamic nuclei which then project to the cortex 3~°. The L D T - P P T cholinergic neurons are also implicated in"the generation of ponto-geniculate-occipital ( P G O ) waves, and these waves can be recorded from the dorsolateral geniculate nucleus 15,2°. Electrical stimulation of the L D T - P P T triggers P G O waves in the L G N and these waves can be blocked by nicotinic antagonists 5'21. Cortical E E G desynchronization and P G O waves occur regularly during each episode of rapid eye movement sleep ( R E M sleep). This phase of sleep has been shown to be triggered from the pons 6 and the L D T - P P T cholinergic neurons have been implicated 22'35. The coordinated and simultaneous occurrence of cortical E E G desynchronization and P G O waves during R E M sleep might be facilitated by the projection of single L D T - P P T cholinergic neurons to both the intralaminar thalamic nuclei and the L G N . The L D T - P P T cholinergic neurons
have been shown to heavily innervate the thalamus 11'
28,36. However, it is not known whether single L D T - P P T neurons project to both the intralaminar thalamic nuclei and the L G N . In order to examine this possibility, in this study we use two retrograde tracers, and in combination with immunohistochemistry to confirm the chemical identity of the cell, we demonstrate that some L D T - P P T cholinergic neurons simultaneously project to two targets in the thalamus. This finding further strengthens the role of pontine cholinergic neurons in complex behaviors. Three rats were deeply anesthetized and, in each rat, stereotaxic microinjections of rhodamine-conjugated latex miscrospheres were made into the fight medial dorso-lateral and central-lateral nuclei of the thalamus while fluorescein isothiocynate (FITC)-conjugated latex microspheres were microinjected into the right lateral geniculate nucleus (LGN). The beads were injected without any dilution. The beads have been shown to be transported retrogradely to neuronal cell bodies, and labelling due to transport occurring in damaged fibers of passage has been shown to be negligible 7. A micropipette (tip diameter 30-50 p m ) attached to a 1.0 pl Hamilton syringe was used to deliver single injections of the fluorescent-conjugated beads. A total volume of 100 nl was injected and the micropipette was left in place for 10 min after the injection. Seventy-two h after the infusion of the tracers the rats were anesthetized and the brains were fixed by intracardiac perfusion. The
Correspondence: P.J. Shiromani, Department of Psychiatry (V-116A), San Diego VA Medical Center, La Jolla, CA 92161, U.S.A.
318 rats were perfused with 50 ml of 0.9% saline and 150 ml 4% p a r a f o r m a l d e h y d e in 0.1 M phosphate buffer (PB). The brains were blocked, postfixed in the same fixative for 2 h and then transferred to a 30% sucrose solution. Sections were cut at a thickness of 50 ktm on an A m e r i c a n Optical freezing microtome and i m m e d i a t e l y processed for immunohistochemistry. One in 4 series of sections were m o u n t e d on gelatine coated slides and coverslipped with F l u o r o m o u n t . These sections were used to d e t e r m i n e whether retrogradely labelled cells were present and also to evaluate whether immunohistochemical p r o c e d u r e s hindered the quality of the rhodamine and F I T C - c o n j u g a t e d b e a d fluorescence. Sections 2, 3 and 4 in the series were i m m u n o l a b e l l e d with polyclonal antibodies against the acetylcholine-synthesizing enzyme choline acetyltransferase (CHAT). The sections were first rinsed in 0.1 M Tris-buffered saline (TBS, p H 7.4) containing 0.25% Triton-100 and 3% normal goat serum. The sections were incubated overnight at r o o m t e m p e r a t u r e in TBS-Triton with 1% normal goat serum and polyclonal C h A T antibody (Chemicon, 1:350 dilution). The following day after 3 rinses in TBS, the sections were placed for 2 h in an A M C A - c o n j u g a t e d secondary antibody (Jackson Labs, dilution 1:100) which allowed visualization of the ChAT-immunopositive neurons at the fluorescence level. The sections were rinsed 3 times in TBS and then m o u n t e d on gelatine coated slides and coverslipped with Fluoromount. The sections were stored at 4 °C to prevent fading of the fluorophores and microscopic examinations were made within a day or two after completion of immunohistochemical procedures. Thus, immunohistochemical procedures confirmed the chemical identity of the neuronal somata containing r h o d a m i n e or F I T C - c o n j u g a t e d latex beads. All sections were examined and photographed using a Leitz fluorescence microscope. The thalamic sections were counterstained with Cresyl violet and examined for location of the injection site. The rhodamine conjugated beads were found to be infused in the medial-dorsal (lateral division) and central-lateral thalamic nuclei while the FITC-conjugated beads were localized to the dorsal and medial divisions of the lateral geniculate nucleus (see Fig. 1 A - D ) . In the pons the largest collection of cholinergic p e r i k a r y a are located principally in the lateral dorsal
tegmental ( L D T ) and p e d u n c u l o p o n t i n e tegmental (PPT) nuclei 1'23. C h A T immunopositive cells in the L D T and PPT could be easily identified by the blue fluorescence (see Fig. 2A), while the F I T C and r h o d a m i n e fluorescence a p p e a r e d green and red, respectively (see Fig. 2B and C). U n d e r a p p r o p r i a t e filter settings we were able to detect neurons that were singly labelled, double-labelled or triple-labelled. The A M C A is excited at 350 nm, F I T C at 495 nm and r h o d a m i n e at 550 nm. In the L D T - P P T area we n o t e d a n u m b e r of s o m a t a containing the fluorescent latex beads. Larger numbers of bead-containing s o m a t a were seen ipsilateral to the injection site than were seen on the contralateral side. M o r e r h o d a m i n e - b e a d - c o n t a i n i n g somata were n o t e d in the dorso-lateral pons c o m p a r e d to F I T C - b e a d containing somata (see Fig. 2). This indicates that the centrallateral thalamic area receives a heavier input from the L D T - P P T area c o m p a r e d to the L G N . We noted that a n u m b e r of blue fluorescent CHATpositive cells also contained either F I T C or r h o d a m i n e conjugated beads. The presence of red fluorescent rhodamine beads in ChAT-positive L D T - P P T s o m a t a indicated that the neuron p r o j e c t e d to the central-lateral nucleus of the thalamus (see Fig. 2C) while the projection to the L G N was identified by the presence of green fluorescent beads in ChAT-positive L D T - P P T somata (see Fig. 2B). Studies have shown that in rats the L D T - P P T neurons project to the thalamus 2,4,11,28,36 However, we found that a few L D T - P P T CHATpositive cells contained both r h o d a m i n e and F I T C conjugated beads and this indicated that the n e u r o n p r o i e c t e d to both the central-lateral nucleus of the thalamus and L G N (see Fig. 2). These triple-labelled cells were found to be diffusely represented within the L D T - P P T and no clearly defined topographic distribution was evident. We estimate that about 5% of L D T - P P T cholinergic neurons project simultaneously to both the central-lateral nucleus of the thalamus and the L G N . The side ipsilateral to the injection contained m o r e triplelabelled neurons c o m p a r e d to the contralateral side. We noted some somata in the L D T - P P T which were not ChAT-positive but contained either r h o d a m i n e or fluorescein-conjugated beads. This indicates that both the central-lateral nucleus of the thalamus and the L G N
Fig. 1. Schematic diagrams of the location of the retrograde injection sites in the thalamus (A-D). Blackened areas indicate the presence of heavy concentration of the retrograde tracers, and the heavy black lines indicate the outer edges where some tracers could be found. Insert E depicts a schematic of the pontine brainstem at level Bregma -9.3, and the inset outlines the portion of the pontine tegmentum which is represented by a photomicrograph in Fig. 2. The figures are adapted from the rat atlas of Paxinos and Watson 17. Abbreviations: CL = central-lateral nucleus of the thalamus; DLG = dorsolateral geniculate; IntG = intergeniculate leaflet; lc = locus coeruleus; LD = lateral dorsal nucleus; ldt = lateral dorsal tegmental nucleus; LHb = lateral habenula; LP = lateral posterior nucleus; MD = medio-dorsal nucleus; MDL = medio-dorsal nucleus, lateral division; MG = medial geniculate nucleus; me5 = mesencephalic trigeminal nucleus; ppt = pedunculopontine tegmental nucleus; scp = superior cerebellar peduncle; VLGMC = ventrolateral geniculate nucleus, magnocellular division; VLGPC = ventrolateral geniculate nucleus, parvocellular division; 5 = motor nucleus of the fifth nerve; IV = fourth ventricle.
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320 receix~e input from non-cholinerbic L D T - P P T neurons.
Previously, Wolf and Butcher 36 used C h A T i m m u n o -
Also there were a few n e u r o n s that contained both types of fluorescent beads which were not ChAT-positive. Therefore, some non-cholinergic L D T - P P T neurons simultaneously project to both the central-lateral thalamic
cytochemistry in c o n j u n c t i o n with the retrograde tracers propidium iodide and true blue and showed that, in rats, some L D T - P P T cholinergic n e u r o n s had axon collaterals projecting to medial septum/vertical limb of the diagonal band, hypothalamus and some medial, posterior and ventral thalamic nuclei. However, they did not examine
nucleus and LGN.
collateralization to the intralaminar thalamic nuclei and LGN. This study demonstrated that single L D T - P P T cholinergic and non-cholinergic neurons project to both the central-lateral nucleus of the thalamus and the LGN. This finding adds to data from other studies in rats, cats and monkeys which have established that cholinergic and non-cholinergic n e u r o n s from the L D T - P P T primarily ascend and innervate various thalamic nuclei including the lateral geniculate nucleus (LGN) 2"4'11"16'27'28"32.
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Collectively these findings place the pontine cholinergic n e u r o n s on par with the noradrenergic locus coeruleus (LC) n e u r o n s which have also been shown to innervate dual targets. The LC which is sandwiched between the L D T - P P T cholinergic n e u r o n s has been shown to have bifurcating axons which innervate the cortex, thalamus, hippocampus, cerebellum and/or spinal cord 9'14"18'29. In
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the rat, the LC consists entirely of catecholamine neurons 3 while the chemical make-up of the L D T - P P T nuclei is much more heterogenous and some neurotransmitters and peptides are k n o w n to coexist 33'34. In this
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study, we found some non-cholinergic L D T - P P T neurons which projected to two thalamic sites. In future studies, it will be important to determine the chemical identity of these neurons and also provide a detailed topographical
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Fig. 2. Photomicrographs depict fluorescent choline acetyltransferase-immunopositive (top panel) and retrogradely labelled somata in the lateral dorsal tegmental (ldt) and pedunculo-pontine tegmental (ppt) area of the pontine brainstem. A section of the dorsolateral pons was photographed using ultraviolet excitation for AMCA (7-amino-4-methylcoumarin-3-acetic acid), FITC and rhodamine fluorophores. Panel A shows somata in the LDT and PPT which were found to be ChAT-positive. The blue fluorescence results from the excitation of the AMCA-conjugated secondary antibody which forms a complex with the polyclonal ChAT primary antibody. Panel B depicts somata containing retrogradely labelled FITC-conjugated latex beads and this indicates a projection from the LDT-PPT to the lateral geniculate nucleus. Panel C shows somata filled with rhodamine-conjugated latex beads and this indicates projection to the central-lateral nucleus of the thalamus. The heavy arrowheads designate neurons that are triple-labelled and this indicates that some LDT-PPT ChAT-positive neurons project to both the medial thalamus and the LGN. Small double arrows in the upper left corner of panel B mark 3 neurons that also are visible in panel A but not in panel C. This indicates that these neurons are cholinergic and project to the LGN. On the other hand, small double arrows in panel C (middle of the photomicrograph) depict collection of neurons which are ChAT-positive as seen in panel A, and some of these neurons are not seen in panel B when using an FITC-filter. We note that some rhodamine-filled somata are very bright and they bleed through and appear orange under an FITC filter (middle of panel B). Abbreviations are as in Fig. 1.
321 distribution of cholinergic and non-cholinergic bifurcating L D T - P P T neurons, similar to that which has already b e e n provided for the LC 8'1°. These findings continue to emphasize the role of the dorsolateral pontine t e g m e n t u m in the coordination of various sensory, motor and limbic functions, and in the coordination of events related to sleep-wake states. As noted earlier, the L D T - P P T cholinergic projection to the intralaminar thalamic nuclei has been implicated in producing cortical E E G desynchronization during wakefulness and R E M sleep 31, while the cholinergic projection to the L G N might be involved in the generation of P G O waves 15.20. A majority of the L D T - P P T cholinergic n e u r o n s project to the thalamus, and we now find that about 5% of these cells have bifurcating axons which project to two thalamic targets. We believe that infiltration of thalamic sites implicated in regulating cortical desynchrony and P G O wave activity by a small n u m b e r of L D T - P P T cholinergic neurons might be sufficient to
1 Armstrong, D., Saper, C., Levey, A., Wainer, B. and Terry, R., Distribution of cholinergic neurons in the rat brain demonstrated by the immunocytochemical localization of choline acetyltransferase, J. Comp. Neurol., 216 (1983) 53-68. 2 DeLima, A.D. and Singer, W., The brainstem projection to the lateral geniculate nucleus in the cat: identification of cholinergic and monoaminergic elements, J. Comp. Neurol., 259 (1987) 92-121. 3 Foote, S.L., Bloom, EE. and Aston-Jones, G., Nucleus locus coeruleus: new evidence of anatomical and physiological specificity, Physiol. Rev., 252 (1983) 117-127. 4 Hoover, D.B. and Baisden, R.H., Localization of putative cholinergic neurons innervating the anteroventral thalamus, Brain Res. Bull., 5 (1980) 519-524. 5 Hu, B., Bouhassira, D., Steriade, M. and Deschenes, M., The blockage of ponto-geniculo-occipitat waves in the cat lateral geniculate nucleus by nicotinic antagonists, Brain. Research, 473 (1988) 394-397. 6 Jouvet, M., Recherches sur les structures nerveuses et les mechanismes responsales des differentes du sommeil physiologique, Arch. ltal. Biol., 100 (1962) 125-206. 7 Katz, L.C., Burkhalter, A. and Dreyer, W.J., Fluorescent latex microspheres as a retrograde neuronal marker for in vivo and in vitro studies of visual cortex, Nature, 310 (1984) 498-500. 8 Loughlin, S.E., Foote, S.L. and Bloom, F.E., Efferent projections of nucleus locus coeruleus: topographical organization of cells of origin demonstrated by three-dimensional reconstruction, Neuroscience, 18 (1986) 291-306. 9 Loughlin, S.E., Foote, S.L. and Fallon, J.H., Locus coeruleus projections to cortex: topography, morphology and collateralization, Brain Res. Bull., 9 (1982) 287-294. 10 Loughlin, S.E., Foote, S.L. and Grzanna, R., Efferent projections of nucleus locus coeruleus: morphologic subpopulations have different efferent targets, Neuroscience, 18 (1986) 307-319. 11 Mesulam, M.M., Mufson, E.J., Wainer, B.H. and Levey, A.I., Central cholinergic pathways in the rat: an overview based on an alternative nomenclature, Neuroscience, 10 (1983) 1185-1201. 12 Mitani, A., Ito, K., Hallanger, A.E., Wainer, B.H., Kataoka, K. and McCarley, R.W., Cholinergic projections from the laterodorsal and pedunculopontine tegmental nuclei to the pontine gigantocellular tegmental field in the cat, Brain Research, 451 (1988) 397-402. 13 Moruzzi, G. and Magoun, H.W., Brain stem reticular formation
facilitate the coordination and simultaneous occurrence of P G O waves and cortical desynchrony during R E M sleep. However, activation of the larger group of thalamic projecting L D T - P P T cholinergic n e u r o n s might be required to produce transitions b e t w e e n sleep and wakefulness. Additionally, we suggest that since it has been determined that some cells from the L D T - P P T descend into the pontine and medullary reticular formation 12,19,24,25.37, the interaction b e t w e e n thalamic projecting and caudally projecting L D T - P P T n e u r o n s may also be responsible for orchestrating the occurrence of muscle atonia within R E M sleep. It remains to be determined whether some L D T - P P T cholinergic n e u r o n s project to both forebrain and medullary sites.
We would like to thank Dr. Kent Keyser for use of the fluorescence microscope and technical advice. This project was funded by grants from American Narcolepsy Association, NIHNS25212, VAMC Research Service and NIMH-38738.
and activation of the EEG, Electroenceph. Clin. Neurophysiol., 1 (1949) 455-473. 14 Nagai, R., Satoh, K., Imamoto, K. and Maeda, T., Divergent projections of catecholamine neurons of the locus coeruleus as revealed by fluorescent retrograde double labeling technique, Neurosci. Lett., 23 (1981) 117-123. 15 Nelson, J.P., McCarley, R.W. and Hobson, J.A., REM sleep burst neurons, PGO waves, and eye movement information, J. Neurophysiol., 50 (1983) 784-797. 16 Pare, D., Smith, Y., Parent, A. and Steriade, M., Projections of brainstem core cholinergic and non-cholinergic neurons of cat to intralaminar and reticular thalamic nuclei, Neuroscience, 25 (1988) 69-86. 17 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic, New York, 1986. 18 Room, P., Postema, F. and Korf, J., Divergent axon collaterals of rat locus coeruleus neurons: demonstration by a fluorescent double labeling technique, Brain Research, 221 (1981) 219-230. 19 Rye, D.B, Lee, H.J., Saper, C.B. and Wainer, B.H., Medullary and spinal efferents of the pedunculopontine tegmental nucleus and adjacent mesopontine tegmental in the rat, J. Comp. Neurol., 269 (1988) 315-341. 20 Sakai, K. and Jouvet, M., Brain stem PGO on cells projecting directly to the cat dorsal lateral geniculate nucleus, Brain Research, 194 (1980) 500-505. 21 Sakai, K., Petitjean, E and Jouvet, M., Effects of pontomesencephalic lesions and electrical stimulation upon pgo waves and emps in unanesthetized cats, Electroenceph. Clin. Neurophysiol., 41 (1976) 49-63. 22 Shiromani, P., Gillin, J.C. and Henriksen, S.J., Acetylcholine and the regulation of REM sleep: basic mechanisms and clinical implication for affective illness and narcolepsy, Annu. Rev. Pharmacol. Toxicol., 27 (1987) 137-156. 23 Shiromani, P.J., Armstrong, D.M., Berkowitz, A., Jeste, D.V. and Gillin, J.C., Distribution of choline acetyltransferase immunoreactive somata in the feline brainstem: implications for REM sleep generation, Sleep, 11 (1988) 1-16. 24 Shiromani, P.J., Armstrong, D.M. and Gillin, J.C., Cholinergic neurons from the dorsolateral pons project to the medial pons: a WGA-HRP and choline acetyltransferase immunohistochemical study, Neurosci. Lett.,, 95 (1988) 19-23. 25 Shiromani, P.J., Lai, Y.Y. and Siegel, J.M., Descending projections from the dorsolateral pontine tegmentum to the para-
322
26
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median reticular nucleus of the caudal medulla in the cat, Brain Research, 517 (1990) 224-228. Shute, C.C.D. and Lewis, P.R., The ascending ch~linergic reticular system: neocortical, olfactory and subcortical projections, Brain, 90 (1967) 497-520. Smith, Y., Pare, D., Deschenes, M., Parent, A. and Steriade, M., Cholinergic and non-cholinergic projections from the upper brainstem core to the visual thalamus in the cat, Exp. Brain Res., 70 (1988) 166-180. Sofroniew, M.V., Priestley, J.V., Consolazione, A., Eckenstein, F. and Cuello, A.C., Cholinergic projections from the midbrain and pons to the thalamus in the rat, identified by combined retrograde tracing and choline acetyltransferase immunohistochemistry, Brain Research, 329 (1985) 213-223. Steindler, D., Locus coeruleus neurons have axons that branch to the forebrain and cerebellum, Brain Research, 223 (1981) 367-373. Steriade, M., New vistas on the morphology, chemical transmitters and physiological actions of the ascending brainstem reticular system, Arch. Ital. Biol., 126 (1988) 225-238. Steriade, M. and McCarley, R.W., Brainstem Control of Wakefulness and Sleep, Plenum, New York, 1990.
32 Steriade, M., Pare, D., Parent, A. and Smith, Y., Projections of cholinergic and non-cholinergic neurons of the brainstem core to relay and associational thalamic nuclei in the cat and macaque monkey, Neuroscience, 25(1) (1988) 47-67. 33 Sutin, E.L., Immunocytochemical and in-situ Hybridization Histochemical Analysis of the Laterodorsal Tegmental Nucleus and Adjacent Nuclei, Doctoral Dissertation, University of California, San Diego, CA, 1989. 34 Vincent, S.R., Satoh, K., Armstrong, D.M. and Fibiger, H.C., Substance P in the ascending cholinergic reticular system, Nature, 306 (1983) 688-691. 35 Webster, H.H. and Jones, B.E., Neurotoxic lesions of the dorsolateral pontomesencephalic tegmentum-cholinergic cell area in the cat. II. Effects upon sleep-waking states, Brain Rbsearch, 458 (1988) 285-302. 36 Woolf, N.J. and Butcher, L.L., Cholinergic systems in the rat brain: III. Projections from the pontomesencephalic tegmentum to the thalamus, tectum, basal ganglia, and basal forebrain, Brain Res. Bull., 16 (1986) 603-637. 37 Woolf, N.J. and Butcher, L.L., Cholinergic systems in the rat brain: IV. Descending projections of the pontomesencephalic tegmentum, Brain Res. Bull., 23 (1989) 519-540.
BRES 24304
31"7
Short Communications
Pontine cholinergic neurons simultaneously innervate two thalamic targets Priyattam J. Shiromani, Cheryl Floyd and Javier Vehizquez-Moctezuma Department of Psychiatry (V-116A), San Diego VA Medical Center and University of California, San Diego, La Jolla, CA 92161 (U.S.A.)
(Accepted 19 June) Key words: Thalamus; Brainstem; Immunocytochemistry; Acetylcholine; Rapid eye movement sleep
Cholinergic neurons located in the lateral dorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei have been shown to principally innervate the thalamus. In order to determine whether some of these neurons might simultaneously project to two thalamic targets we made microinjections of rhodamine-conjugated microbeads into the central-lateral nucleus of the thalamus and fluorescein isothiocynate (FITC)-conjugated microbeads into the dorso-lateral geniculate nucleus. We then determined whether both tracers were found in immunohistochemically identified cholinergic somata in the LDT and PPT nuclei. Results showed that some cholinergic and non-cholinergic neurons in the LDT and PPT nuclei projected to both thalamic sites. This finding extends our understanding of the projections of the LDT-PPT cholinergic neurons and further supports the role of these neurons in complex behaviors. The largest collection of cholinergic neurons, outside of the basal forebrain cholinergic system, are located in the dorsolateral pontine tegmentum, in the lateral dorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei. In recent years much attention has focused on the L D T - P P T cholinergic neurons because they might represent the ascending reticular activating system of Moruzzi and Magoun 13 and the ascending cholinergic system of Shute and Lewis 26. The L D T - P P T cholinergic neurons are hypothesized to activate the cortex, i.e. produce E E G desynchronization, by acting on intralaminar thalamic nuclei which then project to the cortex 3~°. The L D T - P P T cholinergic neurons are also implicated in"the generation of ponto-geniculate-occipital ( P G O ) waves, and these waves can be recorded from the dorsolateral geniculate nucleus 15,2°. Electrical stimulation of the L D T - P P T triggers P G O waves in the L G N and these waves can be blocked by nicotinic antagonists 5'21. Cortical E E G desynchronization and P G O waves occur regularly during each episode of rapid eye movement sleep ( R E M sleep). This phase of sleep has been shown to be triggered from the pons 6 and the L D T - P P T cholinergic neurons have been implicated 22'35. The coordinated and simultaneous occurrence of cortical E E G desynchronization and P G O waves during R E M sleep might be facilitated by the projection of single L D T - P P T cholinergic neurons to both the intralaminar thalamic nuclei and the L G N . The L D T - P P T cholinergic neurons
have been shown to heavily innervate the thalamus 11'
28,36. However, it is not known whether single L D T - P P T neurons project to both the intralaminar thalamic nuclei and the L G N . In order to examine this possibility, in this study we use two retrograde tracers, and in combination with immunohistochemistry to confirm the chemical identity of the cell, we demonstrate that some L D T - P P T cholinergic neurons simultaneously project to two targets in the thalamus. This finding further strengthens the role of pontine cholinergic neurons in complex behaviors. Three rats were deeply anesthetized and, in each rat, stereotaxic microinjections of rhodamine-conjugated latex miscrospheres were made into the fight medial dorso-lateral and central-lateral nuclei of the thalamus while fluorescein isothiocynate (FITC)-conjugated latex microspheres were microinjected into the right lateral geniculate nucleus (LGN). The beads were injected without any dilution. The beads have been shown to be transported retrogradely to neuronal cell bodies, and labelling due to transport occurring in damaged fibers of passage has been shown to be negligible 7. A micropipette (tip diameter 30-50 p m ) attached to a 1.0 pl Hamilton syringe was used to deliver single injections of the fluorescent-conjugated beads. A total volume of 100 nl was injected and the micropipette was left in place for 10 min after the injection. Seventy-two h after the infusion of the tracers the rats were anesthetized and the brains were fixed by intracardiac perfusion. The
Correspondence: P.J. Shiromani, Department of Psychiatry (V-116A), San Diego VA Medical Center, La Jolla, CA 92161, U.S.A.
318 rats were perfused with 50 ml of 0.9% saline and 150 ml 4% p a r a f o r m a l d e h y d e in 0.1 M phosphate buffer (PB). The brains were blocked, postfixed in the same fixative for 2 h and then transferred to a 30% sucrose solution. Sections were cut at a thickness of 50 ktm on an A m e r i c a n Optical freezing microtome and i m m e d i a t e l y processed for immunohistochemistry. One in 4 series of sections were m o u n t e d on gelatine coated slides and coverslipped with F l u o r o m o u n t . These sections were used to d e t e r m i n e whether retrogradely labelled cells were present and also to evaluate whether immunohistochemical p r o c e d u r e s hindered the quality of the rhodamine and F I T C - c o n j u g a t e d b e a d fluorescence. Sections 2, 3 and 4 in the series were i m m u n o l a b e l l e d with polyclonal antibodies against the acetylcholine-synthesizing enzyme choline acetyltransferase (CHAT). The sections were first rinsed in 0.1 M Tris-buffered saline (TBS, p H 7.4) containing 0.25% Triton-100 and 3% normal goat serum. The sections were incubated overnight at r o o m t e m p e r a t u r e in TBS-Triton with 1% normal goat serum and polyclonal C h A T antibody (Chemicon, 1:350 dilution). The following day after 3 rinses in TBS, the sections were placed for 2 h in an A M C A - c o n j u g a t e d secondary antibody (Jackson Labs, dilution 1:100) which allowed visualization of the ChAT-immunopositive neurons at the fluorescence level. The sections were rinsed 3 times in TBS and then m o u n t e d on gelatine coated slides and coverslipped with Fluoromount. The sections were stored at 4 °C to prevent fading of the fluorophores and microscopic examinations were made within a day or two after completion of immunohistochemical procedures. Thus, immunohistochemical procedures confirmed the chemical identity of the neuronal somata containing r h o d a m i n e or F I T C - c o n j u g a t e d latex beads. All sections were examined and photographed using a Leitz fluorescence microscope. The thalamic sections were counterstained with Cresyl violet and examined for location of the injection site. The rhodamine conjugated beads were found to be infused in the medial-dorsal (lateral division) and central-lateral thalamic nuclei while the FITC-conjugated beads were localized to the dorsal and medial divisions of the lateral geniculate nucleus (see Fig. 1 A - D ) . In the pons the largest collection of cholinergic p e r i k a r y a are located principally in the lateral dorsal
tegmental ( L D T ) and p e d u n c u l o p o n t i n e tegmental (PPT) nuclei 1'23. C h A T immunopositive cells in the L D T and PPT could be easily identified by the blue fluorescence (see Fig. 2A), while the F I T C and r h o d a m i n e fluorescence a p p e a r e d green and red, respectively (see Fig. 2B and C). U n d e r a p p r o p r i a t e filter settings we were able to detect neurons that were singly labelled, double-labelled or triple-labelled. The A M C A is excited at 350 nm, F I T C at 495 nm and r h o d a m i n e at 550 nm. In the L D T - P P T area we n o t e d a n u m b e r of s o m a t a containing the fluorescent latex beads. Larger numbers of bead-containing s o m a t a were seen ipsilateral to the injection site than were seen on the contralateral side. M o r e r h o d a m i n e - b e a d - c o n t a i n i n g somata were n o t e d in the dorso-lateral pons c o m p a r e d to F I T C - b e a d containing somata (see Fig. 2). This indicates that the centrallateral thalamic area receives a heavier input from the L D T - P P T area c o m p a r e d to the L G N . We noted that a n u m b e r of blue fluorescent CHATpositive cells also contained either F I T C or r h o d a m i n e conjugated beads. The presence of red fluorescent rhodamine beads in ChAT-positive L D T - P P T s o m a t a indicated that the neuron p r o j e c t e d to the central-lateral nucleus of the thalamus (see Fig. 2C) while the projection to the L G N was identified by the presence of green fluorescent beads in ChAT-positive L D T - P P T somata (see Fig. 2B). Studies have shown that in rats the L D T - P P T neurons project to the thalamus 2,4,11,28,36 However, we found that a few L D T - P P T CHATpositive cells contained both r h o d a m i n e and F I T C conjugated beads and this indicated that the n e u r o n p r o i e c t e d to both the central-lateral nucleus of the thalamus and L G N (see Fig. 2). These triple-labelled cells were found to be diffusely represented within the L D T - P P T and no clearly defined topographic distribution was evident. We estimate that about 5% of L D T - P P T cholinergic neurons project simultaneously to both the central-lateral nucleus of the thalamus and the L G N . The side ipsilateral to the injection contained m o r e triplelabelled neurons c o m p a r e d to the contralateral side. We noted some somata in the L D T - P P T which were not ChAT-positive but contained either r h o d a m i n e or fluorescein-conjugated beads. This indicates that both the central-lateral nucleus of the thalamus and the L G N
Fig. 1. Schematic diagrams of the location of the retrograde injection sites in the thalamus (A-D). Blackened areas indicate the presence of heavy concentration of the retrograde tracers, and the heavy black lines indicate the outer edges where some tracers could be found. Insert E depicts a schematic of the pontine brainstem at level Bregma -9.3, and the inset outlines the portion of the pontine tegmentum which is represented by a photomicrograph in Fig. 2. The figures are adapted from the rat atlas of Paxinos and Watson 17. Abbreviations: CL = central-lateral nucleus of the thalamus; DLG = dorsolateral geniculate; IntG = intergeniculate leaflet; lc = locus coeruleus; LD = lateral dorsal nucleus; ldt = lateral dorsal tegmental nucleus; LHb = lateral habenula; LP = lateral posterior nucleus; MD = medio-dorsal nucleus; MDL = medio-dorsal nucleus, lateral division; MG = medial geniculate nucleus; me5 = mesencephalic trigeminal nucleus; ppt = pedunculopontine tegmental nucleus; scp = superior cerebellar peduncle; VLGMC = ventrolateral geniculate nucleus, magnocellular division; VLGPC = ventrolateral geniculate nucleus, parvocellular division; 5 = motor nucleus of the fifth nerve; IV = fourth ventricle.
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320 receix~e input from non-cholinerbic L D T - P P T neurons.
Previously, Wolf and Butcher 36 used C h A T i m m u n o -
Also there were a few n e u r o n s that contained both types of fluorescent beads which were not ChAT-positive. Therefore, some non-cholinergic L D T - P P T neurons simultaneously project to both the central-lateral thalamic
cytochemistry in c o n j u n c t i o n with the retrograde tracers propidium iodide and true blue and showed that, in rats, some L D T - P P T cholinergic n e u r o n s had axon collaterals projecting to medial septum/vertical limb of the diagonal band, hypothalamus and some medial, posterior and ventral thalamic nuclei. However, they did not examine
nucleus and LGN.
collateralization to the intralaminar thalamic nuclei and LGN. This study demonstrated that single L D T - P P T cholinergic and non-cholinergic neurons project to both the central-lateral nucleus of the thalamus and the LGN. This finding adds to data from other studies in rats, cats and monkeys which have established that cholinergic and non-cholinergic n e u r o n s from the L D T - P P T primarily ascend and innervate various thalamic nuclei including the lateral geniculate nucleus (LGN) 2"4'11"16'27'28"32.
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Collectively these findings place the pontine cholinergic n e u r o n s on par with the noradrenergic locus coeruleus (LC) n e u r o n s which have also been shown to innervate dual targets. The LC which is sandwiched between the L D T - P P T cholinergic n e u r o n s has been shown to have bifurcating axons which innervate the cortex, thalamus, hippocampus, cerebellum and/or spinal cord 9'14"18'29. In
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the rat, the LC consists entirely of catecholamine neurons 3 while the chemical make-up of the L D T - P P T nuclei is much more heterogenous and some neurotransmitters and peptides are k n o w n to coexist 33'34. In this
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Fig. 2. Photomicrographs depict fluorescent choline acetyltransferase-immunopositive (top panel) and retrogradely labelled somata in the lateral dorsal tegmental (ldt) and pedunculo-pontine tegmental (ppt) area of the pontine brainstem. A section of the dorsolateral pons was photographed using ultraviolet excitation for AMCA (7-amino-4-methylcoumarin-3-acetic acid), FITC and rhodamine fluorophores. Panel A shows somata in the LDT and PPT which were found to be ChAT-positive. The blue fluorescence results from the excitation of the AMCA-conjugated secondary antibody which forms a complex with the polyclonal ChAT primary antibody. Panel B depicts somata containing retrogradely labelled FITC-conjugated latex beads and this indicates a projection from the LDT-PPT to the lateral geniculate nucleus. Panel C shows somata filled with rhodamine-conjugated latex beads and this indicates projection to the central-lateral nucleus of the thalamus. The heavy arrowheads designate neurons that are triple-labelled and this indicates that some LDT-PPT ChAT-positive neurons project to both the medial thalamus and the LGN. Small double arrows in the upper left corner of panel B mark 3 neurons that also are visible in panel A but not in panel C. This indicates that these neurons are cholinergic and project to the LGN. On the other hand, small double arrows in panel C (middle of the photomicrograph) depict collection of neurons which are ChAT-positive as seen in panel A, and some of these neurons are not seen in panel B when using an FITC-filter. We note that some rhodamine-filled somata are very bright and they bleed through and appear orange under an FITC filter (middle of panel B). Abbreviations are as in Fig. 1.
321 distribution of cholinergic and non-cholinergic bifurcating L D T - P P T neurons, similar to that which has already b e e n provided for the LC 8'1°. These findings continue to emphasize the role of the dorsolateral pontine t e g m e n t u m in the coordination of various sensory, motor and limbic functions, and in the coordination of events related to sleep-wake states. As noted earlier, the L D T - P P T cholinergic projection to the intralaminar thalamic nuclei has been implicated in producing cortical E E G desynchronization during wakefulness and R E M sleep 31, while the cholinergic projection to the L G N might be involved in the generation of P G O waves 15.20. A majority of the L D T - P P T cholinergic n e u r o n s project to the thalamus, and we now find that about 5% of these cells have bifurcating axons which project to two thalamic targets. We believe that infiltration of thalamic sites implicated in regulating cortical desynchrony and P G O wave activity by a small n u m b e r of L D T - P P T cholinergic neurons might be sufficient to
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facilitate the coordination and simultaneous occurrence of P G O waves and cortical desynchrony during R E M sleep. However, activation of the larger group of thalamic projecting L D T - P P T cholinergic n e u r o n s might be required to produce transitions b e t w e e n sleep and wakefulness. Additionally, we suggest that since it has been determined that some cells from the L D T - P P T descend into the pontine and medullary reticular formation 12,19,24,25.37, the interaction b e t w e e n thalamic projecting and caudally projecting L D T - P P T n e u r o n s may also be responsible for orchestrating the occurrence of muscle atonia within R E M sleep. It remains to be determined whether some L D T - P P T cholinergic n e u r o n s project to both forebrain and medullary sites.
We would like to thank Dr. Kent Keyser for use of the fluorescence microscope and technical advice. This project was funded by grants from American Narcolepsy Association, NIHNS25212, VAMC Research Service and NIMH-38738.
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