- Email: [email protected]
Nicotinamide-adenine dinucleotide phosphate diaphorase activity matches acetylcholinesterase-rich patches in the medial thalamic nuclei of the cat
165
Brain Research, 625 (1993) 165-168 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 25857
Nicotinamide-adenine dinucleotide phosphate diaphorase activity matches acetylcholinesterase-rich patches in the medial thalamic nuclei of the cat Elisa Mengual, Jos6 Luis Velayos, Fernando Reinoso-Su~rez
*
Departamento de Morfolog[a, Facultad de Medicina, Universidad Aut6noma de Madrid, At, da. del Arzobispo Morcillo, s / n, 28029 Madrid, Spain (Accepted 6 July 1993)
Key words: Acetylcholinesterase; Medial thalamus; Ascending cholinergic reticular system; Nitric oxide; Histochemistry; Nicotinamide-adenine dinucleotide phosphate diaphorase
Patches of high nicotinamide-adenine dinucleotide phosphate diaphorase (NADPH-d) activity were found in the thalamic nuclei of cats. These patches matched acetylcholinesterase (AChE)-rich patches within the medial thalamus, patches were NADPH-d negative. There were also patches of NADPH-d activity in the lateral habenula, but these staining. These results suggest that the functional role of discrete thalamic regions may require the joint presence enzymatic activities.
The existence of inhomogeneities in the distribution of acetylcholinesterase (ACHE) activity within the medial thalamic nuclei has been pointed out by several authors in different mammalian species 2'4'9A°'12. Thus, in the cat, patches of dense AChE staining with irregular contour and ill-defined borders, are visible within a moderately stained matrix. In caudal and intermediate portions, these AChE-rich patches are scattered within the midline region and on the border line between the mediodorsal nucleus (MD) and the intralaminar nuclei; rostrally, the patches are located within MD. This pattern has been consistent in all the animals studied 13. In addition, correspondence between the distribution patterns for AChE and nicotinamide-adenine dinucleotide phosphate diaphorase (NADPH-d) activities have been reported in several nervous system structures in different species 16'22. In the present paper we have histochemically compared NADPH-d and AChE enzymatic activities in the cat thalamus, in order to examine whether there is any relation in the thalamic distribution of the two enzymes. Three adult cats of either sex were deeply anesthetized with pentobarbital (35 mg/kg, i.p.) and per-
* Corresponding author. Fax: (34) (1) 315 0075.
mediodorsal and midline whereas other AChE~rich did not match the AChE of AChE and NADPH-d
fused transcardially with 600 ml of saline, followed by 3 1 of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.3) and increasing concentrations of buffered sucrose (5, 10, 20%). The brains were then removed and soaked in 30% buffered sucrose. After 48 h, they were cut on a freezing microtome in the coronal plane at 50 /zm, and adjacent series were histochemically processed for NADPH-d and ACHE, as well as for Nissl stain to allow the delineation of nuclear boundaries. In order to visualize NADPH-d activity, freefloating sections were incubated in Tris buffer (0.1 M, pH 8) containing 1 mM /3-NADPH (Sigma), 1 mM Nitroblue Tetrazolium (Sigma), and 0.3% Triton X-100, for 30-60 min, then rinsed in PB, mounted on subbed slides, dehydrated and coverslipped with DePeX. AChE histochemistry was developed according to a slight modification of the Geneser-Jensen and Blackstad protocol 7. NADPH-d histochemistry in the thalamus revealed the existence of several patches of high activity in the area comprised by MD and the midline nuclei. These patches tended to vary from section to section in size and intensity of staining, as well as in their location and number (Fig. 1A-F). Nevertheless, we could identify a consistent pattern in all the cases studied. Thus, in the caudal pole of MD, NADPH-d activity usually
166 appeared as a single patch in the midline nuclei; rostrally, several small patches appeared between the rhomboid nucleus and M D in addition to a patch that was preferentially located in the midline. These small patches could be linked to one another or to the midline patch, forming a band that ventrally bordered the M D (Fig. 1A). In more rostral portions, this band was laterally displaced (Fig. 1C,E). In rostral portions of MD, two patches were visible in the medial thalamus, one at each side of the midline (Fig. 1E). In the MD rostral pole, the patches were concentrated in the central region of the nucleus. Patches of N A D P H - d staining were also found in the lateral habenula (Fig. 1A), and occasionally in the lateral portion of the medial habenula. At a higher magnification, these patches could be identified as dense clusters of thin varicose fibres (Fig. 1G) Thus, NADPH-d-positive fibres with varicosities were easily distinguishable at the patch borders, where the axonal staining was less dense. Isolated thin varicose and nonvaricose fibres were also visible throughout the whole thalamus, as well as several thin bundles of stained fibres of passage. Moderately stained cells, disperse or aggregated in clusters (not illustrated), as well as a few scattered, intensely stained neurons (Fig. 1H), were also detectable in several thalamic nuclei. Remarkably, when these sections were compared with and laid over the corresponding adjacent A C h E stained sections, the N A D P H - d patches were found to completely match AChE-rich patches within the mediodorsal and midline thalamic nuclei, showing an overall correspondence in the location, size and intensity of staining. The intensity of the N A D P H - d activity was reflected by that of the A C h E staining (Fig. 1A-F). This gradation in the intensity of the staining and subsequent correspondence between N A D P H - d and A C h E activities, was also evident within the same patch (Fig. 1C,D and Fig. 1E-F). In contrast, the patches of N A D P H - d staining within the lateral habenula did not show correspondence with A C h E activity (Fig. 1A,B). Conversely, other regions within the medial thalamus displaying rich A C h E activity, like the
Fig. 1. A-F: dark-field microphotographs of three pairs of adjacent coronal sections through the cat medial thalamus, stained for NADPH-d (A,C,E) and AChE (B,D,F) and spaced 750 /xm apart. Arrowheads point to matching patches in the medial thalamus. Arrows in A,B and in C-F point, respectively, to LH and to the central medial nucleus, in which no correspondence between NADPH-d and AChE was detected. G,H: bright-field microphotographs of two patches of NADPH-d activity within the medial thalamus (G), and an intensely NADPH-d stained neuron in MD (H). Asterisks in E and F mark matching blood vessels. Abbreviations: MD, mediodorsal nucleus; LH, lateral habenula. Bar = 1 mm (A-F) and 250 ~zm(G-H).
167 central medial nucleus (Fig. 1C-F), did not show any N A D P H - d activity. This histochemical study is the first to find patches of N A D P H - d activity in the cat thalamus, something which has not been reported in previous studies on the distribution of this enzyme TM. This lack of staining might be due to the fixation solution containing glutaraldehyde: according to recent studies, this may result in incomplete labelling of structures 1. In contrast, similar studies in the rat 21' carried out without glutaraldehyde, report very few thin varicose processes in MD and dense plexuses of fine fibres in the central lateral and rhomboid nuclei. Second, and also noteworthy, is the striking correspondence between the N A D P H - d activity in the cat medial thalamus and AChE-rich patches in the same region. Correspondence between N A D P H - d and AChE histochemical activities has already been reported in the cat striatum ~6 and also in the intermediate layers of the rat superior colliculus 22, in close relationship with the arrangement of afferent fibres or with the distribution of specific ones 16'23. These studies suggest that N A D P H - d could be a specific marker of given pathways within the nervous system. The thalamic region comprised within the MD and the midline nuclei, is richly innervated by the ascending cholinergic reticular system which arises in the dorsolateral mesopontine tegmentum 5'x7'19. AChE may be a reliable marker for cholinergic innervation 5 in these thalamic nuclei. Since cholinergic neurons of the dorsolateral mesopontine tegmentum are characteristically NADPH-d-positive 2°, it very much seems that the NADPH-d stained patches in the medial thalamus are a result of the innervation arising from these cholinergic/NADPH-d-positive neurons. In contrast, the lateral habenula (LH) does not receive afferent inputs from the brainstem ~8. The patches of N A D P H - d activity in L H might be accounted for by projections from NADPH-d-positive neurons in the diagonal band of Broca and substantia innominata, in the basal forebrain 14 (and personal observations) where retrogradely labelled neurons are found after H R P injections in L H TM. Unlike brainstem cholinergic neurons, the cholinergic neurons from the basal forebrain do not colocalize NADPH-d in the monkey and human brain although a few do in the rat brain 8. It has recently been shown that NADPH-diaphorase is a nitric oxide synthase H and nitric oxide acts in a neurotransmitter-like fashion in the innervation of some peripheral structures 3'~5 as well as in the central nervous system 6. In this sense, N A D P H - d reactivity might be postulated to be a specific marker for a pathway that, essentially arising in the cholinergic neu-
rons of the mesopontine reticular formation, innervated only some cellular subsets of the medial thalamus by means of nitric oxide. This biochemically restricted innervation might imply a certain segregation of information towards discrete thalamic regions. Identification of the functional organization underlying the matching distribution of NADPH-d and AChE histochemical activities, undoubtedly requires further study. We thank Dr. C. Estrada for her kind donation of some reactive products, Dr. J.R. Alonso for his technical advice, Dr. J.M. Gim6nez-Amaya for his commentaries on the manuscript, C. G6mez, J. Hern~indez-Claumarchirant, P. Romero and R. S~inchez-Lozano for their technical assistance in the histochemical preparations and G. de la Fuente and B. Rodrlguez for their help in the experimental work. E.M. is a recipient of a fellowship from the Caja de Madrid (1990). This work was supported by CAICYT PB 90-0220. 1 Ar~valo, R., Alonso, J.R., Brifi6n, J.G., Porteros, A. and Aij6n, J., NADPH-diaphorase histochemical technique in the teleost brain, Zool. Jb. Anat., in press. 2 Brandel, A.J.-P., Hirsch, E.C., Hersh, L.B. and Javoy-Agid, F., Compartmental ordering of cholinergic innervation in the mediodorsal nucleus of the thalamus in human brain, Brain Res., 515 (1990) 117-125. 3 Bult, H., Boeckxstaens, G.E., Pelckmans, P.A., Jordaens, F.H., Van Maercke, Y.M. and Herman, A.G., Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurotransmitter, Nature, 345 (1990) 346-347. 4 Cabaliero-Bleda, M., Fernandez, B. and Puelles, L., Comparative mapping of acetylcholinesterase and reduced nicotinamide adenine dinucleotide diaphorase in the rabbit dorsal thalamus, Acta Anat., 140 (1991) 224-235. 5 Fitzpatrick, D., Diamond, I.T. and Raczkowski, D., Cholinergic and monoaminergic innervation of the cat's thalamus: comparison of the lateral geniculate nucleus with other principal sensory nuclei, J. Comp. Neurol., 288 (1989) 647-675. 6 Garthwaite, J., Charles, S.L. and Chess-Williams, R., Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain, Nature, 336 (1988) 385-388. 7 Geneser-Jensen, F.A. and Blackstad, J.W., Distribution of acetylcholinesterase in the hippocampal region of the guinea pig. I. Entorhinal area, parasubiculum and presubiculum, Z. Zellforsch. mikrosk. Anat., 114 (1971) 460-481. 8 Geula, C., Shatz, C.R. and Mesulam, M.-M., Differential localization of NADPH-diaphorase and calbindin-D 28k within the cholinergic neurons of the basal forebrain, striatum and brainstem in the rat, monkey, baboon and human, Neuroscience, 54 (1993) 461-476. 9 Graybiel, A.M. and Berson, D.M., Histochemical identification and afferent connections of subdivisions in the lateralis posterior-pulvinar complex and related thalamic nuclei in the cat, Neuroscience, 5 (1980) 1175-1238. 10 Hirai, T. and Jones, E.G., A new parcellation of the human thalamus on the basis of histochemical staining, Brain Res. Ret,., 14 (1989) 1-34. 11 Hope, B.T., Michael, G.J., Knigge, K.M. and Vincent, S.R., Neuronal NADPH diaphorase is a nitric oxide synthase, Proc. Natl. Acad. Sci. USA, 88 (1991) 2811-2814. 12 Jones, E.G., The Thalamus, Plenum Press, New York, 1985, 232 Pp. 13 Mengual, E. and Velayos, J.L., Anatomical and histochemical study of the mediodorsal nucleus and adjacent thalamic nuclei in the cat, Eur. Z Neurosci. Suppl., 4 (1991) 145. 14 Mizukawa, K., Vincent, S.R., McGeer, P.L. and McGeer, E.G., Distribution of NADPH-diaphorase-positive cells and fibers in the cat central nervous system, J. Comp. Neurol., 279 (1989) 281-311.
168 15 Ramagopal, M.V. and Leighton, H.J., Effects of NG-monomethyl-L-arginine on field stimulation-induced decreases in cytosolic Ca 2 + levels and relaxation in the rat anococcygeus muscle, Eur. J. PharmacoL, 174 (1989) 297-299. 16 Sandell, J.H., Graybiel, A.M. and Chesselet, M.-F., A new enzyme marker for striatal compartmentalization: NADPH diaphorase activity in the caudate nucleus and putamen of the cat, J. Cornp. Neurol., 243 (1986) 326-334. 17 Steriade, M., Par6, 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 (1988) 47-67. 18 Varela-Sim6, G., Conexiones aferentes de los nftcleos mediales del tdlamo y de la hab~nula del gato, estudiadas por el m~todo del transporte axonal retrdgrado de la peroxidasa, Doctoral Thesis, Universidad Aut6noma de Madrid, 1985.
19 Velayos, J.L. and Reinoso-Sufirez, F., Topographic organization of the brainstem afferents to the mediodorsal thalamic nucleus, J. Comp. Neurol., 206 (1982) 17-27. 20 Vincent, S.R., Satoh, K., Armstrong, D.M. and Fibiger, H.C., NADPH-diaphorase: a selective histochemical marker for the cholinergic neurons of the pontine reticular formation, Neurosci. Lett., 43 (1983) 31-36. 21 Vincent, S.R. and Kimura, H., Histochemical mapping of nitric oxide synthase in the rat brain, Neuroscience, 46 (1992) 755-784. 22 Wallace, M.N. Spatial relationship of NADPH-diaphorase and acetylcholinesterase lattices in the rat and mouse superior colliculus, Neuroscience, 19 (1986) 381-391. 23 Wallace, M.N. and Fredens, K., Relationship of afferent inputs to the lattice of high NADPH-diaphorase activity in the mouse superior colliculus, Exp. Brain Res., 78 (1989) 435-445.
Brain Research, 625 (1993) 165-168 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 25857
Nicotinamide-adenine dinucleotide phosphate diaphorase activity matches acetylcholinesterase-rich patches in the medial thalamic nuclei of the cat Elisa Mengual, Jos6 Luis Velayos, Fernando Reinoso-Su~rez
*
Departamento de Morfolog[a, Facultad de Medicina, Universidad Aut6noma de Madrid, At, da. del Arzobispo Morcillo, s / n, 28029 Madrid, Spain (Accepted 6 July 1993)
Key words: Acetylcholinesterase; Medial thalamus; Ascending cholinergic reticular system; Nitric oxide; Histochemistry; Nicotinamide-adenine dinucleotide phosphate diaphorase
Patches of high nicotinamide-adenine dinucleotide phosphate diaphorase (NADPH-d) activity were found in the thalamic nuclei of cats. These patches matched acetylcholinesterase (AChE)-rich patches within the medial thalamus, patches were NADPH-d negative. There were also patches of NADPH-d activity in the lateral habenula, but these staining. These results suggest that the functional role of discrete thalamic regions may require the joint presence enzymatic activities.
The existence of inhomogeneities in the distribution of acetylcholinesterase (ACHE) activity within the medial thalamic nuclei has been pointed out by several authors in different mammalian species 2'4'9A°'12. Thus, in the cat, patches of dense AChE staining with irregular contour and ill-defined borders, are visible within a moderately stained matrix. In caudal and intermediate portions, these AChE-rich patches are scattered within the midline region and on the border line between the mediodorsal nucleus (MD) and the intralaminar nuclei; rostrally, the patches are located within MD. This pattern has been consistent in all the animals studied 13. In addition, correspondence between the distribution patterns for AChE and nicotinamide-adenine dinucleotide phosphate diaphorase (NADPH-d) activities have been reported in several nervous system structures in different species 16'22. In the present paper we have histochemically compared NADPH-d and AChE enzymatic activities in the cat thalamus, in order to examine whether there is any relation in the thalamic distribution of the two enzymes. Three adult cats of either sex were deeply anesthetized with pentobarbital (35 mg/kg, i.p.) and per-
* Corresponding author. Fax: (34) (1) 315 0075.
mediodorsal and midline whereas other AChE~rich did not match the AChE of AChE and NADPH-d
fused transcardially with 600 ml of saline, followed by 3 1 of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.3) and increasing concentrations of buffered sucrose (5, 10, 20%). The brains were then removed and soaked in 30% buffered sucrose. After 48 h, they were cut on a freezing microtome in the coronal plane at 50 /zm, and adjacent series were histochemically processed for NADPH-d and ACHE, as well as for Nissl stain to allow the delineation of nuclear boundaries. In order to visualize NADPH-d activity, freefloating sections were incubated in Tris buffer (0.1 M, pH 8) containing 1 mM /3-NADPH (Sigma), 1 mM Nitroblue Tetrazolium (Sigma), and 0.3% Triton X-100, for 30-60 min, then rinsed in PB, mounted on subbed slides, dehydrated and coverslipped with DePeX. AChE histochemistry was developed according to a slight modification of the Geneser-Jensen and Blackstad protocol 7. NADPH-d histochemistry in the thalamus revealed the existence of several patches of high activity in the area comprised by MD and the midline nuclei. These patches tended to vary from section to section in size and intensity of staining, as well as in their location and number (Fig. 1A-F). Nevertheless, we could identify a consistent pattern in all the cases studied. Thus, in the caudal pole of MD, NADPH-d activity usually
166 appeared as a single patch in the midline nuclei; rostrally, several small patches appeared between the rhomboid nucleus and M D in addition to a patch that was preferentially located in the midline. These small patches could be linked to one another or to the midline patch, forming a band that ventrally bordered the M D (Fig. 1A). In more rostral portions, this band was laterally displaced (Fig. 1C,E). In rostral portions of MD, two patches were visible in the medial thalamus, one at each side of the midline (Fig. 1E). In the MD rostral pole, the patches were concentrated in the central region of the nucleus. Patches of N A D P H - d staining were also found in the lateral habenula (Fig. 1A), and occasionally in the lateral portion of the medial habenula. At a higher magnification, these patches could be identified as dense clusters of thin varicose fibres (Fig. 1G) Thus, NADPH-d-positive fibres with varicosities were easily distinguishable at the patch borders, where the axonal staining was less dense. Isolated thin varicose and nonvaricose fibres were also visible throughout the whole thalamus, as well as several thin bundles of stained fibres of passage. Moderately stained cells, disperse or aggregated in clusters (not illustrated), as well as a few scattered, intensely stained neurons (Fig. 1H), were also detectable in several thalamic nuclei. Remarkably, when these sections were compared with and laid over the corresponding adjacent A C h E stained sections, the N A D P H - d patches were found to completely match AChE-rich patches within the mediodorsal and midline thalamic nuclei, showing an overall correspondence in the location, size and intensity of staining. The intensity of the N A D P H - d activity was reflected by that of the A C h E staining (Fig. 1A-F). This gradation in the intensity of the staining and subsequent correspondence between N A D P H - d and A C h E activities, was also evident within the same patch (Fig. 1C,D and Fig. 1E-F). In contrast, the patches of N A D P H - d staining within the lateral habenula did not show correspondence with A C h E activity (Fig. 1A,B). Conversely, other regions within the medial thalamus displaying rich A C h E activity, like the
Fig. 1. A-F: dark-field microphotographs of three pairs of adjacent coronal sections through the cat medial thalamus, stained for NADPH-d (A,C,E) and AChE (B,D,F) and spaced 750 /xm apart. Arrowheads point to matching patches in the medial thalamus. Arrows in A,B and in C-F point, respectively, to LH and to the central medial nucleus, in which no correspondence between NADPH-d and AChE was detected. G,H: bright-field microphotographs of two patches of NADPH-d activity within the medial thalamus (G), and an intensely NADPH-d stained neuron in MD (H). Asterisks in E and F mark matching blood vessels. Abbreviations: MD, mediodorsal nucleus; LH, lateral habenula. Bar = 1 mm (A-F) and 250 ~zm(G-H).
167 central medial nucleus (Fig. 1C-F), did not show any N A D P H - d activity. This histochemical study is the first to find patches of N A D P H - d activity in the cat thalamus, something which has not been reported in previous studies on the distribution of this enzyme TM. This lack of staining might be due to the fixation solution containing glutaraldehyde: according to recent studies, this may result in incomplete labelling of structures 1. In contrast, similar studies in the rat 21' carried out without glutaraldehyde, report very few thin varicose processes in MD and dense plexuses of fine fibres in the central lateral and rhomboid nuclei. Second, and also noteworthy, is the striking correspondence between the N A D P H - d activity in the cat medial thalamus and AChE-rich patches in the same region. Correspondence between N A D P H - d and AChE histochemical activities has already been reported in the cat striatum ~6 and also in the intermediate layers of the rat superior colliculus 22, in close relationship with the arrangement of afferent fibres or with the distribution of specific ones 16'23. These studies suggest that N A D P H - d could be a specific marker of given pathways within the nervous system. The thalamic region comprised within the MD and the midline nuclei, is richly innervated by the ascending cholinergic reticular system which arises in the dorsolateral mesopontine tegmentum 5'x7'19. AChE may be a reliable marker for cholinergic innervation 5 in these thalamic nuclei. Since cholinergic neurons of the dorsolateral mesopontine tegmentum are characteristically NADPH-d-positive 2°, it very much seems that the NADPH-d stained patches in the medial thalamus are a result of the innervation arising from these cholinergic/NADPH-d-positive neurons. In contrast, the lateral habenula (LH) does not receive afferent inputs from the brainstem ~8. The patches of N A D P H - d activity in L H might be accounted for by projections from NADPH-d-positive neurons in the diagonal band of Broca and substantia innominata, in the basal forebrain 14 (and personal observations) where retrogradely labelled neurons are found after H R P injections in L H TM. Unlike brainstem cholinergic neurons, the cholinergic neurons from the basal forebrain do not colocalize NADPH-d in the monkey and human brain although a few do in the rat brain 8. It has recently been shown that NADPH-diaphorase is a nitric oxide synthase H and nitric oxide acts in a neurotransmitter-like fashion in the innervation of some peripheral structures 3'~5 as well as in the central nervous system 6. In this sense, N A D P H - d reactivity might be postulated to be a specific marker for a pathway that, essentially arising in the cholinergic neu-
rons of the mesopontine reticular formation, innervated only some cellular subsets of the medial thalamus by means of nitric oxide. This biochemically restricted innervation might imply a certain segregation of information towards discrete thalamic regions. Identification of the functional organization underlying the matching distribution of NADPH-d and AChE histochemical activities, undoubtedly requires further study. We thank Dr. C. Estrada for her kind donation of some reactive products, Dr. J.R. Alonso for his technical advice, Dr. J.M. Gim6nez-Amaya for his commentaries on the manuscript, C. G6mez, J. Hern~indez-Claumarchirant, P. Romero and R. S~inchez-Lozano for their technical assistance in the histochemical preparations and G. de la Fuente and B. Rodrlguez for their help in the experimental work. E.M. is a recipient of a fellowship from the Caja de Madrid (1990). This work was supported by CAICYT PB 90-0220. 1 Ar~valo, R., Alonso, J.R., Brifi6n, J.G., Porteros, A. and Aij6n, J., NADPH-diaphorase histochemical technique in the teleost brain, Zool. Jb. Anat., in press. 2 Brandel, A.J.-P., Hirsch, E.C., Hersh, L.B. and Javoy-Agid, F., Compartmental ordering of cholinergic innervation in the mediodorsal nucleus of the thalamus in human brain, Brain Res., 515 (1990) 117-125. 3 Bult, H., Boeckxstaens, G.E., Pelckmans, P.A., Jordaens, F.H., Van Maercke, Y.M. and Herman, A.G., Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurotransmitter, Nature, 345 (1990) 346-347. 4 Cabaliero-Bleda, M., Fernandez, B. and Puelles, L., Comparative mapping of acetylcholinesterase and reduced nicotinamide adenine dinucleotide diaphorase in the rabbit dorsal thalamus, Acta Anat., 140 (1991) 224-235. 5 Fitzpatrick, D., Diamond, I.T. and Raczkowski, D., Cholinergic and monoaminergic innervation of the cat's thalamus: comparison of the lateral geniculate nucleus with other principal sensory nuclei, J. Comp. Neurol., 288 (1989) 647-675. 6 Garthwaite, J., Charles, S.L. and Chess-Williams, R., Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain, Nature, 336 (1988) 385-388. 7 Geneser-Jensen, F.A. and Blackstad, J.W., Distribution of acetylcholinesterase in the hippocampal region of the guinea pig. I. Entorhinal area, parasubiculum and presubiculum, Z. Zellforsch. mikrosk. Anat., 114 (1971) 460-481. 8 Geula, C., Shatz, C.R. and Mesulam, M.-M., Differential localization of NADPH-diaphorase and calbindin-D 28k within the cholinergic neurons of the basal forebrain, striatum and brainstem in the rat, monkey, baboon and human, Neuroscience, 54 (1993) 461-476. 9 Graybiel, A.M. and Berson, D.M., Histochemical identification and afferent connections of subdivisions in the lateralis posterior-pulvinar complex and related thalamic nuclei in the cat, Neuroscience, 5 (1980) 1175-1238. 10 Hirai, T. and Jones, E.G., A new parcellation of the human thalamus on the basis of histochemical staining, Brain Res. Ret,., 14 (1989) 1-34. 11 Hope, B.T., Michael, G.J., Knigge, K.M. and Vincent, S.R., Neuronal NADPH diaphorase is a nitric oxide synthase, Proc. Natl. Acad. Sci. USA, 88 (1991) 2811-2814. 12 Jones, E.G., The Thalamus, Plenum Press, New York, 1985, 232 Pp. 13 Mengual, E. and Velayos, J.L., Anatomical and histochemical study of the mediodorsal nucleus and adjacent thalamic nuclei in the cat, Eur. Z Neurosci. Suppl., 4 (1991) 145. 14 Mizukawa, K., Vincent, S.R., McGeer, P.L. and McGeer, E.G., Distribution of NADPH-diaphorase-positive cells and fibers in the cat central nervous system, J. Comp. Neurol., 279 (1989) 281-311.
168 15 Ramagopal, M.V. and Leighton, H.J., Effects of NG-monomethyl-L-arginine on field stimulation-induced decreases in cytosolic Ca 2 + levels and relaxation in the rat anococcygeus muscle, Eur. J. PharmacoL, 174 (1989) 297-299. 16 Sandell, J.H., Graybiel, A.M. and Chesselet, M.-F., A new enzyme marker for striatal compartmentalization: NADPH diaphorase activity in the caudate nucleus and putamen of the cat, J. Cornp. Neurol., 243 (1986) 326-334. 17 Steriade, M., Par6, 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 (1988) 47-67. 18 Varela-Sim6, G., Conexiones aferentes de los nftcleos mediales del tdlamo y de la hab~nula del gato, estudiadas por el m~todo del transporte axonal retrdgrado de la peroxidasa, Doctoral Thesis, Universidad Aut6noma de Madrid, 1985.
19 Velayos, J.L. and Reinoso-Sufirez, F., Topographic organization of the brainstem afferents to the mediodorsal thalamic nucleus, J. Comp. Neurol., 206 (1982) 17-27. 20 Vincent, S.R., Satoh, K., Armstrong, D.M. and Fibiger, H.C., NADPH-diaphorase: a selective histochemical marker for the cholinergic neurons of the pontine reticular formation, Neurosci. Lett., 43 (1983) 31-36. 21 Vincent, S.R. and Kimura, H., Histochemical mapping of nitric oxide synthase in the rat brain, Neuroscience, 46 (1992) 755-784. 22 Wallace, M.N. Spatial relationship of NADPH-diaphorase and acetylcholinesterase lattices in the rat and mouse superior colliculus, Neuroscience, 19 (1986) 381-391. 23 Wallace, M.N. and Fredens, K., Relationship of afferent inputs to the lattice of high NADPH-diaphorase activity in the mouse superior colliculus, Exp. Brain Res., 78 (1989) 435-445.