Neuroscience Letters, 120 (1990) 197-200 ElsevierScientificPublishers Ireland Ltd.
197
NSL 07354
Parvalbumin in rat superior colliculus R.-B. Illing, D . M . V o g t a n d W.B. S p a t z Unitfor Morphological Brain Research, Universit~its-HNO-Klinik,Freiburg (F.R.G.)
(Received 16 July 1990;Revisedversion received24 August 1990;Accepted24 August 1990) Key words." Superiorcolliculus;Parvalbumin; Acetylcholinesterase;Cytochromeoxidase; Compartmentalarchitecture;Rat
Parvalbumin-like immunoreactivity(PA-LI) has been studied in sections of the superior colliculus(SC) of the rat and its distribution compared to the patterns of acetylcholinesterase(ACHE)and cytochromeoxidase(CO) staining. In the intermediatelayers it was found that PA-LI is spatially associated with AChE only in the medial part of the SC, but assumes a complementarydistribution further laterally.There was a positivecorrelation between PA-LI and CO. We conclude that the patterns of PA-LI and CO are not systematicallyrelated to collicular input known to be associated with the AChE-rich zones, but may reflectadherenceto channel separation beyond the terminal fieldsof clustered afferents.
A structural periodicity has been found on several levels of the superior colliculus (SC), superimposing the topographical organization o f its laminae. Such a periodic architecture is particularly prominent in the intermediate layers as visualized by acetylcholinesterase (ACHE) histochemistry [6, 11]. There is a detailed correspondence between the distributions of AChE-activity and choline acetyltransferase (ChAT)-like immunohistochemistry in both cat [5] and rat [9] SC. For the intermediate layers, this spatial association justifies to speak o f a cholinergic domain. The cholinergic domain represents a matrix to which other substances (such as N A D P H diaphorase [11]) and the terminal fields of some major collicular afferents are spatially related. Although much correlative work of this kind has been done in cat [6, 7], the situation in rodents, where it has been investigated, apparently conforms to the same structural design [12]. The pattern of AChE-staining does not seem to be the only matrix imposing a horizontal periodicity on certain collicular layers. It has been reported from rat [10] and mouse [13] that the heterogeneous distribution of cytochrome oxidase (CO) activity does not match the heterogeneous pattern of AChE staining in the intermediate layers. We here report that the immunohistochemical demonstration of parvalbumin reveals a rich pattern in all collicular layers, with a changing affiliation to the matrix of high AChE activity. Parvalbumin (PA) belongs to the family of calciumbinding proteins [2, 3]. Compared to other calcium-bindCorrespondence: R.-B. Illing, Unit for MorphologicalBrain Research, Universitfits-HNO-Klinik,Killianstr. 5, D-7800 Freiburg, F.R.G.
0304-3940/90/$03.50 © 1990ElsevierScientificPublishers Ireland Ltd.
ing proteins, such as calmodulin, its distribution has been shown to be more restricted so as to provide certain neurons with particular skills in the handling of calcium. The hypothesis has been put forward that PA is associated with GABAergic neurons [1], but it was found that this correspondence has important exceptions [8]. We have studied the distribution of PA in the colliculi of five albino rats. Under deep anesthesia (60 mg sodium pentobarbital) the animals were perfused with 4% paraformaldehyde and 0.01% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4. The colliculi were cut on a cryostat into 20/tm thick frontal or parasagittal sections and parallel series were stained for AChE activity [4], parvalbumin-like immunoreactivity (PA-LI), and CO-activity [14]. For immunocytochemistry, we used a monoclonal antibody (235) raised against PA (kindly provided by Dr. M.R. Celio) and applied the Avidin-Biotin technique (Vecta Stain). Selected sections were treated for the simultaneous visualization of AChE activity and immunoreactivity according to the protocol previously published [5]. In the SC, PA-LI showed a characteristic distribution (Fig. 1A). Stratum griseum superficiale (SGS) was homogeneously stained. This staining was attributed to a rich varicose neuropil as well as a population of vertically oriented neurons of like morphology, which were more numerous in the vicinity of stratum opticum (SO) than close to the surface. In contrast to the staining pattern in the superficial layers, the pattern of immunoreactivity in the intermediate layers was clustered (Fig. 1A). This pattern may appear as a row of patches or as a fiber net with various holes of different sizes. The patch-and-
198
D
Fig. 1. Pair of adjacent frontal sections through rat SC stained for PA-LI and AChE activity, respectively; medial is to the right. A: distribution of PA-LI. Immunoreactive elements are rich in all collicular layers, but while SGS was homogeneously stained, PA-LI in the intermediate layers occurred in clusters. Arrowhead points to a cluster of high PA-LI, arrow marks a sparsely stained zone. B: adjacent section stained for AChE activity. Again, there is a contrast between homogenous and patchy staining. Arrow and arrowhead point to areas corresponding to the zones labeled in A. Note that at the medially marked zone (arrowheads) there is a matching increase of density in both stains, while in the area further laterally high AChE-activity corresponds to low levels of PA-LI. Bar = 0.5 mm.
hole matrix clearly extended ventrally and dorsally beyond the intermediate layers. This gave rise to a periodic architecture in SO. Many neurons of differing size (soma diameter 10-35/zm) and morphology were seen in SO as well as in the intermediate and deep layers. Associated with the patches of immunoreactive neuropil were numerous small somata (diameter about 10/~m) which were mostly found to reside inside these patches. The distribution of neuronal somata other than those of the small cells gave no indication of a consistent position relative to the patches. The immunoreactive neuropil of SGS formed a distinct ventral border at the transition to SO. This border coincided with the border of high to low levels of ACHEstaining between SGS and SO (Fig. 1), indicating a matching occupation of SGS by both substances. On the level of the intermediate layers, the two patterns cease to maintain a simple spatial relationship. Richly immunoreactive patches may or may not superimpose with areas of high AChE activity (Fig. 1). Conversely, zones devoid of immunoreactive neuropil may or may not show high AChE activity. This was particularly obvious in doublestained sections (not illustrated). The sections depicted in Fig. 1 gave the impression that between PA-LI and AChE activity there exists a positive correlation in the medial part of the SC but a negative correlation along its longitudinal middle axis; laterally, a diffuse superposition was observed. This changing affiliation was confirmed in series of parasagittal sections (Fig. 2). Fig. 2A-C shows 3 adjoining para-
sagittal sections through the medial SC, stained for ACHE, PA-LI, and CO, respectively. In this part of SC, all three stains showed matching patterns. A set of sections along the middle axis is shown is Fig. 2D-F. At this level, zones of high AChE activity mostly fill gaps left in the pattern of PA-LI. Again, these results could be confirmed in double-stained material. Islands of high CO activity are easily appreciable in medial SC (Fig. 2C). The pattern is more diffuse in the middle and lateral colliculus, but where zones of higher CO activity could be discerned, they were associated with zones of high PA-LI rather than increased AChE activity (Fig. 2E,F). Similar to the SC of other mammals, the intensity of AChE staining in the intermediate layers weakened along a caudo-rostrai gradient (Fig. 2D). This trend was not matched but complemented by the distribution of PA-LI, which showed highest densities in the rostral SC and decreased caudally (Fig. 2E). The lack of a consistent relationship between the patterns of AChE staining and PA-LI forms a sharp contrast to the spatial superposition of AChE-rich zones with other systems. The structural matrix defined by the cholinergic domain of the intermediate collicular layers reflects a spatial separation of the terminal fields of major collicular afferents [6, 7]. Thus, there appears to be an inconsistent affiliation of PA-LI to the afferent systems known to be associated with the cholinergic systems or to distribute systematically outside the cholinergic domain. Despite the possibility that complementarity between PA and AChE may, in the medial part of the
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C
Fig. 2. Two sequences of sagittal sections through rat SC; rostral is to the right. A ~ : sequence through the medial SC. D-F: sequence along the longitudinal middle axis. A, D: patterns of AChE staining. B, E: Patterns of PA-LI in adjacent sections. C, F: subsequent sections stained for CO. In each sequence, arrowheads point to corresponding zones. In A to C, elevated levels of staining in the intermediate layers occurred in all three sections in corresponding zones, but in D - F patches of high AChE-staining correspond to zones of low PA-LI, with the pattern of CO largely following the distribution of PA-LI. Bar = 1 mm.
200
intermediate layers, be preserved at the synaptic level, we suspect that these afferents meet different environments in different parts of the SC. The structural differentiations observed in the multimodal layers of the SC into at least two groups of compartments is likely to be functionally meaningful. The systems represented by high levels of PA and ACHE, respectively, are interlaced in the multimodal collicular layers apparently to interact in specific ways. Local differences in their spatial relationship may point to shifts in signal processing, leading to different collicular outputs depending on where in the topographical array the activity occurred. The association of a population of small neurons with the patches of immunoreactive neuropil in the intermediate layers suggests that the PA compartments may at least in part be constituted by intrinsic neurons. Our observations therefore raise the possibility that the pattern of PA-LI indicates intrinsic collicular compartments. The finding that these compartments relate to zones of elevated oxidative metabolism may be taken to indicate that PA marks particularly active subcircuits of the SC. We thank Ms. M. Rudolf for expert technical assistance, Drs. M. Frotscher and M.R. Celio for gifts of antibody, and the Deutsche Forschungsgemeinschaft (SFB 325, Tp B1) for financial support. 1 Celio, M.R., Parvalbumin in most gamma-aminobutyric acid-containing neurons of the rat cerebral cortex, Science, 231 (1986) 995997. 2 Celio, M.R., Calbindin D-28k and parvalbumin in the rat nervous system, Neuroscience, 35 (1990) 375-475.
3 Cello, M.R. and Heizmann, C.W., Calcium-binding protein parvalbumin as a neuronal marker, Nature, 293 (1981) 300-302. 4 Geneser-Jensen, F.A. and Blackstad, T.W., Distribution of acetyl cholinesterase in the hippocampal region of the guinea pig, Z. Zell~ forsch., 114 (1971) 460-481. 5 Illing, R.-B., Choline acetyltransferase-like immunoreactivity in the superior colliculus of the cat and its relation to the pattern of acetylcholinesterase staining, J. Comp. Neurol., 296 (1990) 32-46. 6 Illing, R.-B. and Graybiel, A.M., Convergence of afferents from frontal cortex and substantia nigra onto acetyleholinesterase-rich patches of the cat's superior colliculus, Neuroscience, 14 (1985) 455-482. 7 Illing, R.-B. and Graybiel, A.M., Complementary and non-matching afferent compartments in the cat's superior collieulus: Innervation of the acetylcholinesterase-poor domain of the intermediate gray layer, Neuroscience, 18 (1986) 373-394. 8 Jones, E.G. and Hendry, S.H.C., Differential calcium binding protein immunoreactivity distinguishes classes of relay neurons in monkey thalamic nuclei, Eur. J. Neurosci., 1 (1989) 222-246. 9 Schnurr, B., Spatz, W.B. and Illing, R.-B., Organization of the superior colliculus. I. Modular and nonmodular architecture of cholinergic elements. In N. Eisner and G. Roth (Eds.), Proc. 18th G6ttingen Neurobiol. Conf., p. 346. 10 Wallace, M.N., Lattice of high oxidative metabolism in the intermediate gray layer of the rat and hamster superior colliculus, Neurosci. Lett., 70 (1986) 320-325. 11 Wallace, M.N., Spatial relationship of NADPH-diaphorase and acetylcholinesterase lattices in rat and mouse superior colliculus, Neuroscience, 19 (1986) 381-391. 12 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. 13 Wiener, ST, Laminar distribution and patchiness of cytochrome oxidase in mouse superior colliculus, J. Comp. Neurol., 244 (1986) 137-148. 14 Wong-Riley, M.T.T., Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry, Brain Res., 171 (1979)11-28.
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NSL 07354
Parvalbumin in rat superior colliculus R.-B. Illing, D . M . V o g t a n d W.B. S p a t z Unitfor Morphological Brain Research, Universit~its-HNO-Klinik,Freiburg (F.R.G.)
(Received 16 July 1990;Revisedversion received24 August 1990;Accepted24 August 1990) Key words." Superiorcolliculus;Parvalbumin; Acetylcholinesterase;Cytochromeoxidase; Compartmentalarchitecture;Rat
Parvalbumin-like immunoreactivity(PA-LI) has been studied in sections of the superior colliculus(SC) of the rat and its distribution compared to the patterns of acetylcholinesterase(ACHE)and cytochromeoxidase(CO) staining. In the intermediatelayers it was found that PA-LI is spatially associated with AChE only in the medial part of the SC, but assumes a complementarydistribution further laterally.There was a positivecorrelation between PA-LI and CO. We conclude that the patterns of PA-LI and CO are not systematicallyrelated to collicular input known to be associated with the AChE-rich zones, but may reflectadherenceto channel separation beyond the terminal fieldsof clustered afferents.
A structural periodicity has been found on several levels of the superior colliculus (SC), superimposing the topographical organization o f its laminae. Such a periodic architecture is particularly prominent in the intermediate layers as visualized by acetylcholinesterase (ACHE) histochemistry [6, 11]. There is a detailed correspondence between the distributions of AChE-activity and choline acetyltransferase (ChAT)-like immunohistochemistry in both cat [5] and rat [9] SC. For the intermediate layers, this spatial association justifies to speak o f a cholinergic domain. The cholinergic domain represents a matrix to which other substances (such as N A D P H diaphorase [11]) and the terminal fields of some major collicular afferents are spatially related. Although much correlative work of this kind has been done in cat [6, 7], the situation in rodents, where it has been investigated, apparently conforms to the same structural design [12]. The pattern of AChE-staining does not seem to be the only matrix imposing a horizontal periodicity on certain collicular layers. It has been reported from rat [10] and mouse [13] that the heterogeneous distribution of cytochrome oxidase (CO) activity does not match the heterogeneous pattern of AChE staining in the intermediate layers. We here report that the immunohistochemical demonstration of parvalbumin reveals a rich pattern in all collicular layers, with a changing affiliation to the matrix of high AChE activity. Parvalbumin (PA) belongs to the family of calciumbinding proteins [2, 3]. Compared to other calcium-bindCorrespondence: R.-B. Illing, Unit for MorphologicalBrain Research, Universitfits-HNO-Klinik,Killianstr. 5, D-7800 Freiburg, F.R.G.
0304-3940/90/$03.50 © 1990ElsevierScientificPublishers Ireland Ltd.
ing proteins, such as calmodulin, its distribution has been shown to be more restricted so as to provide certain neurons with particular skills in the handling of calcium. The hypothesis has been put forward that PA is associated with GABAergic neurons [1], but it was found that this correspondence has important exceptions [8]. We have studied the distribution of PA in the colliculi of five albino rats. Under deep anesthesia (60 mg sodium pentobarbital) the animals were perfused with 4% paraformaldehyde and 0.01% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4. The colliculi were cut on a cryostat into 20/tm thick frontal or parasagittal sections and parallel series were stained for AChE activity [4], parvalbumin-like immunoreactivity (PA-LI), and CO-activity [14]. For immunocytochemistry, we used a monoclonal antibody (235) raised against PA (kindly provided by Dr. M.R. Celio) and applied the Avidin-Biotin technique (Vecta Stain). Selected sections were treated for the simultaneous visualization of AChE activity and immunoreactivity according to the protocol previously published [5]. In the SC, PA-LI showed a characteristic distribution (Fig. 1A). Stratum griseum superficiale (SGS) was homogeneously stained. This staining was attributed to a rich varicose neuropil as well as a population of vertically oriented neurons of like morphology, which were more numerous in the vicinity of stratum opticum (SO) than close to the surface. In contrast to the staining pattern in the superficial layers, the pattern of immunoreactivity in the intermediate layers was clustered (Fig. 1A). This pattern may appear as a row of patches or as a fiber net with various holes of different sizes. The patch-and-
198
D
Fig. 1. Pair of adjacent frontal sections through rat SC stained for PA-LI and AChE activity, respectively; medial is to the right. A: distribution of PA-LI. Immunoreactive elements are rich in all collicular layers, but while SGS was homogeneously stained, PA-LI in the intermediate layers occurred in clusters. Arrowhead points to a cluster of high PA-LI, arrow marks a sparsely stained zone. B: adjacent section stained for AChE activity. Again, there is a contrast between homogenous and patchy staining. Arrow and arrowhead point to areas corresponding to the zones labeled in A. Note that at the medially marked zone (arrowheads) there is a matching increase of density in both stains, while in the area further laterally high AChE-activity corresponds to low levels of PA-LI. Bar = 0.5 mm.
hole matrix clearly extended ventrally and dorsally beyond the intermediate layers. This gave rise to a periodic architecture in SO. Many neurons of differing size (soma diameter 10-35/zm) and morphology were seen in SO as well as in the intermediate and deep layers. Associated with the patches of immunoreactive neuropil were numerous small somata (diameter about 10/~m) which were mostly found to reside inside these patches. The distribution of neuronal somata other than those of the small cells gave no indication of a consistent position relative to the patches. The immunoreactive neuropil of SGS formed a distinct ventral border at the transition to SO. This border coincided with the border of high to low levels of ACHEstaining between SGS and SO (Fig. 1), indicating a matching occupation of SGS by both substances. On the level of the intermediate layers, the two patterns cease to maintain a simple spatial relationship. Richly immunoreactive patches may or may not superimpose with areas of high AChE activity (Fig. 1). Conversely, zones devoid of immunoreactive neuropil may or may not show high AChE activity. This was particularly obvious in doublestained sections (not illustrated). The sections depicted in Fig. 1 gave the impression that between PA-LI and AChE activity there exists a positive correlation in the medial part of the SC but a negative correlation along its longitudinal middle axis; laterally, a diffuse superposition was observed. This changing affiliation was confirmed in series of parasagittal sections (Fig. 2). Fig. 2A-C shows 3 adjoining para-
sagittal sections through the medial SC, stained for ACHE, PA-LI, and CO, respectively. In this part of SC, all three stains showed matching patterns. A set of sections along the middle axis is shown is Fig. 2D-F. At this level, zones of high AChE activity mostly fill gaps left in the pattern of PA-LI. Again, these results could be confirmed in double-stained material. Islands of high CO activity are easily appreciable in medial SC (Fig. 2C). The pattern is more diffuse in the middle and lateral colliculus, but where zones of higher CO activity could be discerned, they were associated with zones of high PA-LI rather than increased AChE activity (Fig. 2E,F). Similar to the SC of other mammals, the intensity of AChE staining in the intermediate layers weakened along a caudo-rostrai gradient (Fig. 2D). This trend was not matched but complemented by the distribution of PA-LI, which showed highest densities in the rostral SC and decreased caudally (Fig. 2E). The lack of a consistent relationship between the patterns of AChE staining and PA-LI forms a sharp contrast to the spatial superposition of AChE-rich zones with other systems. The structural matrix defined by the cholinergic domain of the intermediate collicular layers reflects a spatial separation of the terminal fields of major collicular afferents [6, 7]. Thus, there appears to be an inconsistent affiliation of PA-LI to the afferent systems known to be associated with the cholinergic systems or to distribute systematically outside the cholinergic domain. Despite the possibility that complementarity between PA and AChE may, in the medial part of the
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C
Fig. 2. Two sequences of sagittal sections through rat SC; rostral is to the right. A ~ : sequence through the medial SC. D-F: sequence along the longitudinal middle axis. A, D: patterns of AChE staining. B, E: Patterns of PA-LI in adjacent sections. C, F: subsequent sections stained for CO. In each sequence, arrowheads point to corresponding zones. In A to C, elevated levels of staining in the intermediate layers occurred in all three sections in corresponding zones, but in D - F patches of high AChE-staining correspond to zones of low PA-LI, with the pattern of CO largely following the distribution of PA-LI. Bar = 1 mm.
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intermediate layers, be preserved at the synaptic level, we suspect that these afferents meet different environments in different parts of the SC. The structural differentiations observed in the multimodal layers of the SC into at least two groups of compartments is likely to be functionally meaningful. The systems represented by high levels of PA and ACHE, respectively, are interlaced in the multimodal collicular layers apparently to interact in specific ways. Local differences in their spatial relationship may point to shifts in signal processing, leading to different collicular outputs depending on where in the topographical array the activity occurred. The association of a population of small neurons with the patches of immunoreactive neuropil in the intermediate layers suggests that the PA compartments may at least in part be constituted by intrinsic neurons. Our observations therefore raise the possibility that the pattern of PA-LI indicates intrinsic collicular compartments. The finding that these compartments relate to zones of elevated oxidative metabolism may be taken to indicate that PA marks particularly active subcircuits of the SC. We thank Ms. M. Rudolf for expert technical assistance, Drs. M. Frotscher and M.R. Celio for gifts of antibody, and the Deutsche Forschungsgemeinschaft (SFB 325, Tp B1) for financial support. 1 Celio, M.R., Parvalbumin in most gamma-aminobutyric acid-containing neurons of the rat cerebral cortex, Science, 231 (1986) 995997. 2 Celio, M.R., Calbindin D-28k and parvalbumin in the rat nervous system, Neuroscience, 35 (1990) 375-475.
3 Cello, M.R. and Heizmann, C.W., Calcium-binding protein parvalbumin as a neuronal marker, Nature, 293 (1981) 300-302. 4 Geneser-Jensen, F.A. and Blackstad, T.W., Distribution of acetyl cholinesterase in the hippocampal region of the guinea pig, Z. Zell~ forsch., 114 (1971) 460-481. 5 Illing, R.-B., Choline acetyltransferase-like immunoreactivity in the superior colliculus of the cat and its relation to the pattern of acetylcholinesterase staining, J. Comp. Neurol., 296 (1990) 32-46. 6 Illing, R.-B. and Graybiel, A.M., Convergence of afferents from frontal cortex and substantia nigra onto acetyleholinesterase-rich patches of the cat's superior colliculus, Neuroscience, 14 (1985) 455-482. 7 Illing, R.-B. and Graybiel, A.M., Complementary and non-matching afferent compartments in the cat's superior collieulus: Innervation of the acetylcholinesterase-poor domain of the intermediate gray layer, Neuroscience, 18 (1986) 373-394. 8 Jones, E.G. and Hendry, S.H.C., Differential calcium binding protein immunoreactivity distinguishes classes of relay neurons in monkey thalamic nuclei, Eur. J. Neurosci., 1 (1989) 222-246. 9 Schnurr, B., Spatz, W.B. and Illing, R.-B., Organization of the superior colliculus. I. Modular and nonmodular architecture of cholinergic elements. In N. Eisner and G. Roth (Eds.), Proc. 18th G6ttingen Neurobiol. Conf., p. 346. 10 Wallace, M.N., Lattice of high oxidative metabolism in the intermediate gray layer of the rat and hamster superior colliculus, Neurosci. Lett., 70 (1986) 320-325. 11 Wallace, M.N., Spatial relationship of NADPH-diaphorase and acetylcholinesterase lattices in rat and mouse superior colliculus, Neuroscience, 19 (1986) 381-391. 12 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. 13 Wiener, ST, Laminar distribution and patchiness of cytochrome oxidase in mouse superior colliculus, J. Comp. Neurol., 244 (1986) 137-148. 14 Wong-Riley, M.T.T., Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry, Brain Res., 171 (1979)11-28.
