70
Neuroscience Letters, 120 (1990) 70--73 ElsevierScientificPublishers Ireland Ltd.
NSL 07323
Glutamate-like immunoreactivity in neurons of the laterodorsal tegmental and pedunculopontine nuclei in the rat J.R. Clements a n d S. G r a n t School of Life and Health Sciences and Department of Psychology, University of Delaware, Newark, DE 19716 (U.S.A.)
(Received 15 June 1990;Revisedversion received26 July 1990;Accepted8 August 1990) Key words: Glutamate; Acetylcholine;Parkinson's disease; Immunocytochemistry;Pedunculopontinenucleus; Laterodorsal tegmental nucleus;
Reticular nuclei Studies of the pedunculopontine(PPT) and laterodorsaltegmental(LDT) nuclei in the mesopontinetegmentumhave emphasizedthe organization and projectionsof the cholinergicneurons. We report here that exhibitingglutamate immunoreactivityare present in both the LDT and PPT. These glutamaterglc neurons are interspersedamong the cholinergicneurons within both nuclei with no apparent segregation.Thesedata raise the possibility that excitatoryamino acids contribute to the functionsof the LDT and PPT.
The pedunculopontine (PPT) and laterodorsal tegmental (LDT) nuclei in the mesopontine tegmentum contain two prominent clusters of cholinergic neurons, respectively designated Ch5 and Ch6 [16, 21, 28]. In addition, cholinergic neurons are also found scattered between the L D T and PPT in the parabrachial region. It is clear that non-cholinergic neurons are also present in the PPT and L D T [13, 20, 21]. A wide range of peptides have been found in L D T and PPT neurons, with some co-localized in cholinergic neurons [5, 26-28]. However, classical transmitters such as monoamines and y-aminobutyric acid (GABA) have not been detected in either the PPT or L D T [12]. Since physiological studies have suggested that there may be an excitatory amino acid component to efferent projections from the L D T and PPT [2, 11, 22], we examined whether neurons in the L D T and PPT exhibit glutamate-like immunoreactivity. We report here that there is a large population of glutamate-like immunoreactive (GLI) neurons in the dorsal pontine tegrnentum, and that GLI neurons are interspersed within clusters of cholinergic neurons in both the LDT and PPT. Eight male Sprague-Dawley rats were used in this study. Rats were anesthetized with chloral hydrate (Sigma, St. Louis, MO) and perfused via the ascending aorta with 100 ml of warm 0.9% saline containing 200 IU of heparin and 0.2% sodium nitrite (Sigma) followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate Correspondence: S. Grant, Department of Psychology,220 Wolf Hall, University of Delaware, Newark, DE 19716,U.S.A.
0304-3940/90/$03.50 © 1990ElsevierScientificPublishers Ireland Ltd.
buffer (pH 7.4, 4°C). Brains were removed, blocked, and post-fixed for 6 h at 4°C. Tissue sections (50/tm) through the mesopontine tegmentum were cut on either a vibrating or freezing microtome. Alternate sections were processed for glutamate immunohistochemistry and Nicotinamide Adenine Dinucleotide Phosphate diaphorase (NADPH-d) histochemistry. As N A D P H - d has been shown to be a reliable and selective marker for cholinergic neurons in the L D T and PPT, the distribution of neurons exhibiting intense N A D P H - d staining was used to define the boundaries of the Ch5 and Ch6 cell groups [28]. N A D P H - d histochemistry was performed using the procedure of Scherer-Singler modified by the addition of 0.025% MgC12 [24]. Alternate sections for glutamate immunohistochemistry were incubated in 1.5% normal horse serum for 30 min prior to a 12 h incubation in a 1:75,000 dilution of an anti-glutamate monoclonal antibody in 10 mM phosphate-buffered saline (PBS; pH 7.5). The glutamate antibody has previously been described and characterized [15]. It exhibits minimal cross-reactivities (13%) with other amino acids (GABA, lysine, taurine, aspartate and cysteine sulfinate), and no detectable reactivity on immunoblots for large molecules such as G A D (glutamic acid decarboxylase). The presence of GLI in the tissue was visualized using a modification of the avidin-biotin immunoperoxidase method [10] with a Vectastain Elite goat anti-mouse kit (Vector Labs Inc., Burlingame, CA). The diaminobenzidene reaction product was intensified by immersing the tissue sections in a solution of reduced 1% osmium tetroxide for 1 h. Control
71
aq
:
•
aq
bv
41,
_
o
4
"
Fig. 1. Rat midbrain sections immunostained for glutamate-like immunoreactivity (GLI). The dashed lines outline the borders of the medial longitudinal fasciculus (A, C) and superior cerebellar peduncle (B, D). A, B: note the intensely immunoreactive populations of glutamate neurons within the LDT (A), PPT (B), and intermediate parabrachial region (arrowhead, A). C, D: alternate sections stained for NADPH-d histochemistry clearly indicate that populations of cholincrgic neurons are located within some of the same regions in which GLI neurons are found, aq, cerebral aqueduct; bv, blood vessel.
tissue sections, where either the primary or secondary antibody was omitted, or where the glutamate antibody was preabsorbed with either a glutamate-bovine serum albumin (BSA) conjugate were also processed. All immunocytochemistry was done at 20°C. All tissue sections from both the immunocytochemical and histochemical protocols were rinsed in phosphatebuffered saline (PBS), mounted on gelatin-coated slides, dehydrated rapidly in ethanol, cover-slipped with Permount (Fisher Scientific Co., Fair Lawn, N J) and examined. GLI neurons were more extensively distributed throughout the mesopontine tegmentum than NADPHd positive neurons, but distinct populations of GLI neurons were present within the confines of both the LDT and PPT. Alternate sections stained for glutamate-like immunoreactivity and NADPH-d clearly show that GLI neurons occurred within the same regions as cholinergic neurons (Fig. 1). GLI neurons within the boundaries of the LDT (Fig.
1A) ranged from 9 to 22 pm in diameter and exhibited a variety of morphological types. Within individual GLI neurons, reaction product was not only confined to the neuronal perikarya, but also extended into the neuronal processes (Fig. 2A). GLI neurons present in the region of the PPT extended from the cuneiform nucleus into the superior cerebellar peduncle (Fig. 1B). These GLI neurons were somewhat smaller than those in the LDT, ranging in size from 8 to 15/~m in diameter. GLI neurons similar in size to those in the PPT were also found immediately below the ventro-lateral edge of the central gray and extending into the parabrachial region between the LDT and PPT (Fig. 1A). Some neurons in surrounding brainstem nuclei also exhibited glutamate-like immunoreactivity, including the raphe nuclei (dorsal raphe, raphe pontis, median raphe), the trigeminal motor nucleus and the reticulotegmental nucleus (not shown). No immunostaining was observed in control tissue sections where either the primary or secondary antibody
72
Fig, 2. High-magnificationviewof GLI neuronswithinthe PPT. A: someGLI neuronalperikarya(asterisks)have GLI processes(arrows) extending into the neuropil. B: glutamate-likeimmunoreactivitywas not presentwhenthe GLU-2antibodywas pre-adsorbedwith a glutamate~BSAconjugate. was omitted (not shown), or the primary antibody was pre-absorbed with a glutamate conjugate (Fig. 2B). In this study, GLI neurons were found extensively distributed throughout the mesopontine tegmentum. Of particular interest were the GLI neurons found intercalated among cholinergic (intensely NADPH-d-positive) neurons in the LDT and PPT. Within the confines of the LDT/PPT, there was no spatial segregation of glutamatergic and cholinergic (NADPH-d-positive) neurons. GLI neurons were also found in areas surrounding clusters of NADPH-d neurons. In particular the regions immediately medial to the PPT may correspond to the non-cholinergic 'midbrain extrapyramidal area' and the physiologically defined mesencephalic locomotor center described by others [7, 13, 20]. It has been widely demonstrated that anti-glutamate antibodies accurately identify neurons and nerve terminals known from physiological studies to be excitatory [4, 14, 18]. Although glutamatergic neurons have previously been identified in other brain regions (i.e. the ventral globus pallidus and the nucleus of the hypoglossal nerve) that contain dense concentrations of cholinergic neurons [17], to our knowledge this represents the first anatomical demonstration of a potential excitatory amino acid transmitter component to the LDT and PPT. Further studies designed to determine whether glutamate-like immunoreactivity is present in the terminal projections of these neurons will be required to confirm whether glutamate is serving as a transmitter in PPT or LDT neurons. It also remains to be determined whether LDT and PPT neurons co-contain acetylcholine and glutamate, as has been found for acetylcholine and some neuropeptides in the LDT and PPT [5, 26, 27], and acetylcholine and GABA in the basal forebrain [6, 12]. Indeed, in preliminary studies done in our laboratory,
some LDT and PPT neurons were both GLI and histochemically positive for NADPH-d [3]. Based on existing evidence, it seems likely that these putative glutamatergic neurons contribute to the known efferent projections of the PPT and LDT. Anatomical studies have described noncholinergic projections from the PPT and LDT to the spinal cord, medulla, basal forebrain, septum, thalamus and substantia nigra [1, 8, 9, 19, 21, 25]. Also, an excitatory non-cholinergic component to synaptic responses to PPT and LDT stimulation in the thalamus, the major projection site of the LDT and PPT, has been described [11]. There is also anatomical and physiological evidence that dopamine neurons in the substantia nigra receive both a cholinergic and a glutamatergic projection from the PPT/LDT [1, 2, 9, 22, 23]. The presence of two neurochemically distinct neuronal populations is consistent with in vitro intracellular studies of LDT/PPT neurons in which two neuronal populations were distinguished on the basis of intrinsic membrane conductances [14, 29]. One of these neuronal populations corresponded to cholinergic neurons. While the transmitter of the second population has not been determined, the present results raise the possibility that the non-cholinergic population is glutamatergic. These findings, along with our results, suggest that a glutamatergic component parallels the cholinergic projections from the PPT and LDT. Tract tracing experiments are now needed to confirm that the glutamate-immunoreactive neurons described here are the source of excitatory amino acid input from the PPT/LDT. It has been widely suggested that the Ch5 and Ch6 cholinergic groups are an essential component of a reticular activating system [11, 28]. The PPT has also been suggested as the anatomical substrate of the mesencephalic locomotor center [7]. Our data suggest that an
73 excitatory a m i n o acid system arising f r o m the P P T a n d L D T m a y also c o n t r i b u t e to the general regulation o f arousal, l o c o m o t i o n , b e h a v i o r a l state, a n d sensory responsiveness. These results also raise the possibility that d y s f u n c t i o n o f this glutamatergic i n p u t to the s u b s t a n t i a nigra m a y c o n t r i b u t e to the death o f d o p a m i n e r g i c neurons in P a r k i n s o n ' s a n d other n e u r o d e g e n e r a t i v e diseases [1]. W e t h a n k Dr. J, M a d l , C o l o r a d o State University, for p r o v i d i n g the a n t i - g l u t a m a t e a n t i b o d y a n d g l u t a m a t e BSA conjugate. W e also gratefully acknowledge D. Highfield a n d D. T o t h for their expert assistance. This work was s u p p o r t e d by: N I H F I R S T A w a r d DE08185 (J.R.C.); N I M H A w a r d M H 4 5 6 1 0 (S.J.G.); the State o f Delaware, a n d I C I P h a r m a c e u t i c a l s (S.J.G.). 1 Beninato, M. and Spencer, R., Cholinergic projection to the rat substantia nigra from the pedunculopontine tegmental nucleus, Brain Res., 412 (1987) 169-174. 2 Clarke, P., Hommer, D., Pert, A. and Skirboll, L., Innervation of substantia nigra neurons by cholinergic afferents from pedunculopontine nucleus in the rat: neuroanatomical and electrophysiological evidence, Neuroscience, 23 (1987) 1011-1019. 3 Clements, J.R. and Grant, S.J., Glutamate and aeetylcholine are colocalized in the laterodorsal tegmental and pedunculopontine nuclei, Soc. Neurosci. Abstr., 16 (1990) in press. 4 Clements, J.R., Magnusson, K.R. and Beitz, A.J., Ultrastructural description of glutamate-, aspartate-, taurine- and glycine-like immunoreactive terminals from five rat brain regions, J. Electron Microsc. Tech. in press. 5 Crawley, J., Olschowka, J., Diz, D. and Jacobowitz, D., Behavioral investigation of the coexistence of substance P, corticotropin releasing factor, and acetylcholinesterase in lateral dorsal tegmental neurons projecting to the medial frontal cortex of the rat, Peptides, 6 (1985) 891-901. 6 Fisher, R., Buchwald, N., Hull, C. and Levine, M., GABAergic basal forebrain neurons project to the neocortex: the localization of glutamic acid decarboxylase and choline acetyltransferase in felinecorticipetal neurons, J. Comp. Neurol., 272 (1988) 489-502. 7 Garcia-Rill, E., Houser, C., Skinner, R., Smith, W. and Woodward, D., Locomotion inducing sites in the vicinity of the pedunculopontine nucleus, Brain Res. Bull., 18 (1987) 731-738. 8 Goldsmith, M. and Kooy, D., v.d., Separate non-cholinergic descending projections and cholinergic ascending projections from the nucleus tegmenti pedunculopontinus, Brain Res., 445 (1988) 386391. 9 Gould, E., Woolf, N. and Butcher, L., Cholinergic projections to the substantia nigra from the pedunculopontine and lateral dorsal tegmental nuclei, Neuroscience, 28 (1989) 611qi23. 10 Hsu, S., Raine, L. and Fanger, H., Use of avidin biotin peroxidase complex in immunoperoxidase techniques, J. Histochem. Cytochem., 29 (1981) 577-580. I 1 Hu, B., Steriade, M. and Deschenes, M., The effects of brainstem peribrachial stimulation on neurons of the lateral geniculate nucleus, Neuroscience, 31 (1989) 13-24. 12 Krosaka, T., Taucbi, M. and Dahl, J., Cholinergic neurons containing GABA-like and/or glutamic acid decarboxylase-like immunoreactivities in various brain regions of the rat, Exp. Brain Res., 70 (1988) 605-617. 13 Lee, H., Rye, D., Hallenger, A., Levey, A. and Wainer, B., Cholinergic vs. noncholinergic efferents from the mesopontine tegrnen-
turn to the extrapyramidal motor system nuclei, J. Comp. Neurol., 275 (1988) 469-492. 14 Leonard, C. and Llinas, R., In vitro study of pedunculopontine neurons and ACh actions. In M. Steriade and D. Biesold (Eds.), Brain Cholinergic Systems, Oxford University Press, New York, 1990, in press. 15 McDonald, A.J., Beitz, A.J., Larson, A.A., Kuriyama, R., Sellitto, C. and Madl, J.E., Co-localization of glutamate and tubulin in putative excitatory neurons of the hippocampus and amygdala: an immunohistochemical study using monoclonal antibodies, Neuroscience, 30 (1989) 405-421. 16 Mesulam, M., Mufson, E., Wainer, B. and Levey, A., Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Chl-Ch6), Neuroscience, 10 (1983) 1185-1201. 17 Ottersen, O. and Storm-Mathiesen, J., Glutamate- and GABAcontaining neurons in the mouse and rat brain, as demonstrated with a new immunocytochemicaltechnique, J. Comp. Neurol., 229 (1984) 374-392. 18 Petrusz, P. and Rustioni, A., Immunocytochemistry of excitatory amino acids in brain. In G. Bullock and P. Petrusz (Eds.), Techniques in Immunocytochemistry,Vol. 4, Academic Press, New York, 1989, pp. 253-272. 19 Rye, D., Lee, H., Saper, C. and Wainer, B., Medullary and spinal efferents of the pedunculopontine tegmental nucleus and adjacent mesopontine tegmentum in the rat, J. Comp. Neurol., 269 (1988) 315-341. 20 Rye, D., Saper, C., Lee, H. and Wainer, B., Pedunculopontine tegmental nucleus of the rat: cytoarchitecture, cytochemistry, and some extrapyramidal connections of the mesopontine tegmentum, J. Comp. Neurol., 259 (1987) 483-528. 21 Satoh, K. and Fibiger, H., Cholinergic neurons of the laterodorsal tegmental nucleus: Efferent and afferent connections, J. Comp. Neurol., 253 (1986) 277-302. 22 Scarnati, E., Prioria, A., Campana, E. and Pacitti, C., A microiontophoretic study of the nature of the putative synaptic neurotransmitter involved in the pedunculopontine substantia nigra pars compacta excitatory pathway in the rat, Exp. Brain Res., 62 (1986) 470-478. 23 Scarnati, E., Prioria, A., DiLoreto, S. and Pacitti, C., The reciprocal electrophysiological influence between the nucleus tegmenti pedunculopontinus and the substantia nigra in normal and decorticated rats, Brain Res., 423 (1987) 116-124. 24 Scherer-Singier, U., Vincent, S., Kimura, H. and McGeer, E., Demonstration of a unique population of neurons with NADPH diaphorase histochemistry, J. Neurosci. Methods, 9 (1983) 229234. 25 Semba, K., Reiner, P., McGeer, E. and Fibiger, H., Brainstem afferents to the magnocellular basal forebrain studied by axonal transport, immunohistochemistry,and electrophysiologyin the rat, J. Comp. Neurol., 267 (1988) 433-453. 26 Standaert, D., Saper, C,, Rye, D. and Wainer, B., Colocalization of atriopeptin like immunoreactivity with choline acetyltransferase and substance P like immunoreactivity in the pedunculopontine and laterodorsal tegmental nuclei in the rat, Brain Res., 382 (1986) 163-168. 27 Sutin, E. and Jacobowitz, D., Immunocytochemicallocalization of peptides and other neurochemicalsin the rat laterodorsal tegmental nucleus and adjacent area, J. Comp. Neurol., 270 (1988) 243-270. 28 Vincent, S., Satoh, K., Armstrong, D., Panula, P., Vale, W. and Fibiger, H., Neuropeptides and NADPH-diaphorase activity in the ascending cholinergic reticular system of the rat, Neuroscience, 17 (1986) 167-182. 29 Wilcox, K., Grant, S., Burkhardt, B. and Christoph, G., Electrophysiological properties of lateral dorsal tegmental neurons in vitro, Brain Res. Bull., 22 (1989) 557-560.
Neuroscience Letters, 120 (1990) 70--73 ElsevierScientificPublishers Ireland Ltd.
NSL 07323
Glutamate-like immunoreactivity in neurons of the laterodorsal tegmental and pedunculopontine nuclei in the rat J.R. Clements a n d S. G r a n t School of Life and Health Sciences and Department of Psychology, University of Delaware, Newark, DE 19716 (U.S.A.)
(Received 15 June 1990;Revisedversion received26 July 1990;Accepted8 August 1990) Key words: Glutamate; Acetylcholine;Parkinson's disease; Immunocytochemistry;Pedunculopontinenucleus; Laterodorsal tegmental nucleus;
Reticular nuclei Studies of the pedunculopontine(PPT) and laterodorsaltegmental(LDT) nuclei in the mesopontinetegmentumhave emphasizedthe organization and projectionsof the cholinergicneurons. We report here that exhibitingglutamate immunoreactivityare present in both the LDT and PPT. These glutamaterglc neurons are interspersedamong the cholinergicneurons within both nuclei with no apparent segregation.Thesedata raise the possibility that excitatoryamino acids contribute to the functionsof the LDT and PPT.
The pedunculopontine (PPT) and laterodorsal tegmental (LDT) nuclei in the mesopontine tegmentum contain two prominent clusters of cholinergic neurons, respectively designated Ch5 and Ch6 [16, 21, 28]. In addition, cholinergic neurons are also found scattered between the L D T and PPT in the parabrachial region. It is clear that non-cholinergic neurons are also present in the PPT and L D T [13, 20, 21]. A wide range of peptides have been found in L D T and PPT neurons, with some co-localized in cholinergic neurons [5, 26-28]. However, classical transmitters such as monoamines and y-aminobutyric acid (GABA) have not been detected in either the PPT or L D T [12]. Since physiological studies have suggested that there may be an excitatory amino acid component to efferent projections from the L D T and PPT [2, 11, 22], we examined whether neurons in the L D T and PPT exhibit glutamate-like immunoreactivity. We report here that there is a large population of glutamate-like immunoreactive (GLI) neurons in the dorsal pontine tegrnentum, and that GLI neurons are interspersed within clusters of cholinergic neurons in both the LDT and PPT. Eight male Sprague-Dawley rats were used in this study. Rats were anesthetized with chloral hydrate (Sigma, St. Louis, MO) and perfused via the ascending aorta with 100 ml of warm 0.9% saline containing 200 IU of heparin and 0.2% sodium nitrite (Sigma) followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate Correspondence: S. Grant, Department of Psychology,220 Wolf Hall, University of Delaware, Newark, DE 19716,U.S.A.
0304-3940/90/$03.50 © 1990ElsevierScientificPublishers Ireland Ltd.
buffer (pH 7.4, 4°C). Brains were removed, blocked, and post-fixed for 6 h at 4°C. Tissue sections (50/tm) through the mesopontine tegmentum were cut on either a vibrating or freezing microtome. Alternate sections were processed for glutamate immunohistochemistry and Nicotinamide Adenine Dinucleotide Phosphate diaphorase (NADPH-d) histochemistry. As N A D P H - d has been shown to be a reliable and selective marker for cholinergic neurons in the L D T and PPT, the distribution of neurons exhibiting intense N A D P H - d staining was used to define the boundaries of the Ch5 and Ch6 cell groups [28]. N A D P H - d histochemistry was performed using the procedure of Scherer-Singler modified by the addition of 0.025% MgC12 [24]. Alternate sections for glutamate immunohistochemistry were incubated in 1.5% normal horse serum for 30 min prior to a 12 h incubation in a 1:75,000 dilution of an anti-glutamate monoclonal antibody in 10 mM phosphate-buffered saline (PBS; pH 7.5). The glutamate antibody has previously been described and characterized [15]. It exhibits minimal cross-reactivities (13%) with other amino acids (GABA, lysine, taurine, aspartate and cysteine sulfinate), and no detectable reactivity on immunoblots for large molecules such as G A D (glutamic acid decarboxylase). The presence of GLI in the tissue was visualized using a modification of the avidin-biotin immunoperoxidase method [10] with a Vectastain Elite goat anti-mouse kit (Vector Labs Inc., Burlingame, CA). The diaminobenzidene reaction product was intensified by immersing the tissue sections in a solution of reduced 1% osmium tetroxide for 1 h. Control
71
aq
:
•
aq
bv
41,
_
o
4
"
Fig. 1. Rat midbrain sections immunostained for glutamate-like immunoreactivity (GLI). The dashed lines outline the borders of the medial longitudinal fasciculus (A, C) and superior cerebellar peduncle (B, D). A, B: note the intensely immunoreactive populations of glutamate neurons within the LDT (A), PPT (B), and intermediate parabrachial region (arrowhead, A). C, D: alternate sections stained for NADPH-d histochemistry clearly indicate that populations of cholincrgic neurons are located within some of the same regions in which GLI neurons are found, aq, cerebral aqueduct; bv, blood vessel.
tissue sections, where either the primary or secondary antibody was omitted, or where the glutamate antibody was preabsorbed with either a glutamate-bovine serum albumin (BSA) conjugate were also processed. All immunocytochemistry was done at 20°C. All tissue sections from both the immunocytochemical and histochemical protocols were rinsed in phosphatebuffered saline (PBS), mounted on gelatin-coated slides, dehydrated rapidly in ethanol, cover-slipped with Permount (Fisher Scientific Co., Fair Lawn, N J) and examined. GLI neurons were more extensively distributed throughout the mesopontine tegmentum than NADPHd positive neurons, but distinct populations of GLI neurons were present within the confines of both the LDT and PPT. Alternate sections stained for glutamate-like immunoreactivity and NADPH-d clearly show that GLI neurons occurred within the same regions as cholinergic neurons (Fig. 1). GLI neurons within the boundaries of the LDT (Fig.
1A) ranged from 9 to 22 pm in diameter and exhibited a variety of morphological types. Within individual GLI neurons, reaction product was not only confined to the neuronal perikarya, but also extended into the neuronal processes (Fig. 2A). GLI neurons present in the region of the PPT extended from the cuneiform nucleus into the superior cerebellar peduncle (Fig. 1B). These GLI neurons were somewhat smaller than those in the LDT, ranging in size from 8 to 15/~m in diameter. GLI neurons similar in size to those in the PPT were also found immediately below the ventro-lateral edge of the central gray and extending into the parabrachial region between the LDT and PPT (Fig. 1A). Some neurons in surrounding brainstem nuclei also exhibited glutamate-like immunoreactivity, including the raphe nuclei (dorsal raphe, raphe pontis, median raphe), the trigeminal motor nucleus and the reticulotegmental nucleus (not shown). No immunostaining was observed in control tissue sections where either the primary or secondary antibody
72
Fig, 2. High-magnificationviewof GLI neuronswithinthe PPT. A: someGLI neuronalperikarya(asterisks)have GLI processes(arrows) extending into the neuropil. B: glutamate-likeimmunoreactivitywas not presentwhenthe GLU-2antibodywas pre-adsorbedwith a glutamate~BSAconjugate. was omitted (not shown), or the primary antibody was pre-absorbed with a glutamate conjugate (Fig. 2B). In this study, GLI neurons were found extensively distributed throughout the mesopontine tegmentum. Of particular interest were the GLI neurons found intercalated among cholinergic (intensely NADPH-d-positive) neurons in the LDT and PPT. Within the confines of the LDT/PPT, there was no spatial segregation of glutamatergic and cholinergic (NADPH-d-positive) neurons. GLI neurons were also found in areas surrounding clusters of NADPH-d neurons. In particular the regions immediately medial to the PPT may correspond to the non-cholinergic 'midbrain extrapyramidal area' and the physiologically defined mesencephalic locomotor center described by others [7, 13, 20]. It has been widely demonstrated that anti-glutamate antibodies accurately identify neurons and nerve terminals known from physiological studies to be excitatory [4, 14, 18]. Although glutamatergic neurons have previously been identified in other brain regions (i.e. the ventral globus pallidus and the nucleus of the hypoglossal nerve) that contain dense concentrations of cholinergic neurons [17], to our knowledge this represents the first anatomical demonstration of a potential excitatory amino acid transmitter component to the LDT and PPT. Further studies designed to determine whether glutamate-like immunoreactivity is present in the terminal projections of these neurons will be required to confirm whether glutamate is serving as a transmitter in PPT or LDT neurons. It also remains to be determined whether LDT and PPT neurons co-contain acetylcholine and glutamate, as has been found for acetylcholine and some neuropeptides in the LDT and PPT [5, 26, 27], and acetylcholine and GABA in the basal forebrain [6, 12]. Indeed, in preliminary studies done in our laboratory,
some LDT and PPT neurons were both GLI and histochemically positive for NADPH-d [3]. Based on existing evidence, it seems likely that these putative glutamatergic neurons contribute to the known efferent projections of the PPT and LDT. Anatomical studies have described noncholinergic projections from the PPT and LDT to the spinal cord, medulla, basal forebrain, septum, thalamus and substantia nigra [1, 8, 9, 19, 21, 25]. Also, an excitatory non-cholinergic component to synaptic responses to PPT and LDT stimulation in the thalamus, the major projection site of the LDT and PPT, has been described [11]. There is also anatomical and physiological evidence that dopamine neurons in the substantia nigra receive both a cholinergic and a glutamatergic projection from the PPT/LDT [1, 2, 9, 22, 23]. The presence of two neurochemically distinct neuronal populations is consistent with in vitro intracellular studies of LDT/PPT neurons in which two neuronal populations were distinguished on the basis of intrinsic membrane conductances [14, 29]. One of these neuronal populations corresponded to cholinergic neurons. While the transmitter of the second population has not been determined, the present results raise the possibility that the non-cholinergic population is glutamatergic. These findings, along with our results, suggest that a glutamatergic component parallels the cholinergic projections from the PPT and LDT. Tract tracing experiments are now needed to confirm that the glutamate-immunoreactive neurons described here are the source of excitatory amino acid input from the PPT/LDT. It has been widely suggested that the Ch5 and Ch6 cholinergic groups are an essential component of a reticular activating system [11, 28]. The PPT has also been suggested as the anatomical substrate of the mesencephalic locomotor center [7]. Our data suggest that an
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