Developmental changes in the localization of the transplasma membrane NADH-dehydrogenases in the rat brain

DEVELOPMENTAL BRAIN RESEARCH

ELSEVIER

Developmental Brain Research 89 (1995) 253-263

Research report

Developmental changes in the localization of the transplasma membrane NADH-dehydrogenases in the rat brain Yue Yong, Jean-Luc Dreyer * Department of Biochemistry, Universityof Fribourg, CH-1700Fribourg, Switzerland Accepted 19 July 1995

Abstract The function of transplasma membrane oxidoreductases (PMO's) has been further studied by means of investigating the postnatal (PN) developmental changes in the tissue localization of six isoenzymes previously characterized (see accompanying paper). The changes were followed in the midbrain for PMO-I, -II, and -V and in the brainstem for PMO-III, -IV and -VI. PMO-I is not observed before PN5 and develops as long vertical fibers located mainly in the pontine nucleus and in the dorsal raphe nucleus until it merges all over the midbrain except for the aqueduct and the superior colliculus after PN10. At that stage it is highly expressed in the trigeminal nucleus and the dorsal raphe, but its expression then strongly decreases and PMO-I disapears almost completely later on. Similarly PMO-II only develops around PN5, first in the dorsal and caudal linear raphe and later on (at PN7) also in the pontine nucleus and in the median raphe; at PN10 PMO-II gradually had vanished from these areas and strongly developed in the dorsal raphe and in the mesencephalic trigeminal nucleus. Later on PMO-II alos decreases from these areas. PMO-III slowly develops within the gigantocellular reticular nucleus from PN1 to PN5 and later on reaches the facial nucleus (after PN5), the density of PMO-III in these regions at PN10 being much higher than in the adult. PMO-IV follows a similar developmental pattern in the midbrain, with an optimal density around PN10 also. PMO-V appears only at about PN5, first within the dorsal raphe in parallel fibers and in multipolar neurons. It disappears from the fibers around PN10 and remains present in neurons up to adulthood. PMO-VI appears at early stages within the gigantocellular reticular nucleus and after PN5 within the central gray in vertical fibers. At later stages PMO-VI is found in the spinal trigeminal nucleus, at first within the neuropil then in multipolar neurons that remain present up to adulthood. These datas suggested that the different isoenzymes are expressed at various stages in specific areas. The role of PMO's in neuronal development is discussed.

Keywords: Plasma membrane redox; Cellular activation; Cytotoxicity;Neurogenesis; Brain development

1. Introduction Transplasma membrane NADH-dehydrogenases serve to transfer electrons from the intracellular pool of reduced nicotinamide nucleotide to various types of extracellular electron acceptors [21]. The enzymes have been reported to have possible implications in regulation of cell growth and differentiation [6], as well as in energy transduction and postsynaptic membrane signal transduction [3]. Cell proliferation and a more oxidising state (e.g. a higher N A D + / N A D H ratio) in the cytosol are associated in some mammalian cell types [7,31]. Several studies have shown that electron flow through the plasma membrane oxidoreductases stimulates growth and cell proliferation [4]. Although six related NADH-dehydrogenases could be puri-

* Corresponding author. Fax: (41) (37) 299735. 0165-3806/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD1 0165 -3 806(95 )00125 -5

fied from rat brain synaptic plasma membranes [8], their real functions in the central nervous system (CNS), especially in neurite outgrowth, synaptogenesis and development, have not been charaterized so far. In the companion study we have described the detailed tissue distribution in adult brain of these six isoenzymes [30]. In the present study, we examine whether the expression of these enzymes coincides with particular developmental events checked at respresenting levels.

2. Material and methods

2.1. Production and characterization of antibodies The transplasma membrane NADH-dehydrogenases were purified from synaptic membrane of rat brain nerve ending according to Dreyer [8] and polyclonal anti-dehy-

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drogenase antibodies against the six enzymes were prepared as described in the previous paper [30]. 2.2. Tissue preparing

Postnatal rats at the first, third, fifth, seventh and tenth day, as well as adult Sprague-Dawley rat (300-350 g) have been used. Tissue preparation followed the procedure described in the previous paper [30,22]. 2.3. lmmunohistochemistry

Immunohistochemical preparations have been performed according to the methods described previously using the avidin-biotinylated-peroxydase complex method (see accompanying paper [30]). Immunohistochemical controls were carried out with either PBS Triton X-100 or with the pre-immune rabbit serum.

3. Results

The investigation was carried out at postnatal days 1 to 15 (PN1 to PN15) by checking those selected areas wich in the adult brain display greatest differences in the tissue distibution among the six isoenzymes (see previous paper). For investigating the developmental changes pertaining to the expression of Dehydrogenase I and II we focused on the midbrain level at the various postnatal stages (Fig. 1). Dehydrogenase I is not detectable in PN1 sections and only faintly present within a few short and thin fibers from the neuropil in PN3 sections (Fig. la,b). But in PN5 sections, many long and thick neural fibers with a distribution vertical to the long axis of the brain emerged mainly within the oral part of the pontine nucleus. Densely stained, short and tightly ranged fibrous neuropil can be observed in the dorsal raphe nucleus. Lightly stained punctuate neuropil is also observed in the mesencephalic trigeminal nuleus (Fig. lc). The same areas become much denser stained at PN7. Meanwhile, the fibrous neuropil in the oral part of the pontine nucleus becomes also more obvious (Fig. ld) and densely stained punctate neuropil develops within the pontine reticular nucleus (Fig. ld). The area around the aqueduct was negatively stained. The same level at PN10 showed very deep staining all over the mid-brain section except in the area around the aqueduct and in the superior colliculus. The mesencephalic trigeminal nucleus displays very deeply stained punctate neuropil with many neural fibers easily seen in the external side and only a few neural fibers in the internal side of that nucleus (Fig. le). Similarly, densely stained punctate neuropil was present in the dorsal raphe nucleus including many reaction product granules (Fig. le). In sections of the same level from the adult rat brain, absolutely no similar fibrous neuropilar staining could be found, but only lightly stained non-cell form puncta is present instead (Fig. If).

The same areas, i.e. the midbrain level, were also chosen for Dehydrogenase II. At PN1 and PN3 no positive labelling was found (Fig. 2a,b). But at PN5 some fibrous, moderately stained neuropil can be seen in the dorsal and caudal linear raphe nucleus (Fig. 2c). At PN7 not only did the positive staining in that region become more intense, but similar fibrous neuropil also had developed in the oral part of the pontine reticular nucleus as well as in the pontine nucleus, in the median raphe nucleus and in the retrorubral nucleus. At that time the areas around the aqueduct and the superior colliculus were very lightly or even negatively stained (Fig. 2d). Astonishingly PN10 sections showed a different pattern: the densely labeled fibrous neuropil found at PN5 and PN7 had disappeared, while lightly stained punctuate neuropil appeared in the mesencephalic trigeminal nucleus (Fig. 2e) and in the dorsal raphe nucleus. At that time lightly stained neural fibers can be distingushed in the external side of the dorsal raphe nucleus and the oral part of the pontine reticular nucleus. At a yet later stage, i.e. in the adult rat brain, only lightly stained non-cell structure puncta can be found, similar to those present at PN10, yet no positive fibrous neuropil can be observed (Fig. 2f). Sections from the brainstem were chosen for observing the developmental changes related to Dehydrogenase III and IV. In the case of Dehydrogenase III, PN1 and PN3 sections exhibit only few, very lightly labelled neurons within the gigantocellular reticular nucleus (Fig. 3a,b). In PN5 sections densely labeled, medium-sized, multipolar neurons appear within the facial nucleus. At this stage the neurons within the gigantocellular reticular nucleus were stained denser than those of the previous days (Fig. 3c) and displayed reaction product granules within their cytoplasma. In addition long and thick fibrous neuropil was moderately labeled in the same area with a regular distribution parallel to the coronal axis of the brain (Fig. 3d). This fibrous neuropil did not exist in the same region at PN10, but many large, densely labeled multipolar neurons appeared instead. Surprisingly the density of the neurons in this region was much higher at PN10 than in the adult rat brain (Fig. 3e). No reticular structures were found in this region at PN10, whereas such structures are clearly found in the same area in adult rat brain, together with a similar but much more dense neuronal staining. The gigantocellular reticular neurons in the adult brain were large and mutipolar and displayed many reaction granules throughout their cytoplasma (Fig. 3f). The brainstem was also a representative areas for our observations pertaining to Dehydrogenase IV. At PNI very lightly stained punctuate neuropil can be found scattered only within the reticular structures of the gigantocellular reticular nucleus (Fig. 4a). The facial nucleus was moderately labelled at PN3 (Fig. 4b). Gigantocellular reticular neurons together with some reticular structures were positively but lightly stained (Fig. 4c). At PN5 no significant changes were observed, except that the ceils in this region

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were more densely labeled (Fig. 4d). At PN7 fibrous neuropil developed (similar to those observed for Dehydrogenases I, II and III) and displays long and thick fibers with a distribution parallel to the coronal axis of the brain (Fig. 4d). At PN7 and PN10 positive gigantocellular reticular neurons of medium-size with a multipolar shape are observed. Again the density of these neurons in the area of central gray is much higher at these stages than those found in adult rat brain (Fig. 4d,e,f). We chose the level of the midbrain for investigating the developmental changes pertaining to Dehydrogenase V. At PN1 and PN3 only non-cell neuropilar formation can be found in that region (Fig. 5a,b). Lightly stained, mediumsized and pyramidally shaped neurons are present in the area of the dorsal raphe nucleus at PN5 (Fig. 5c). Later on, at PN7, a larger density of moderately stained fibrous neuropil was labeled in the dorsal raphe nucleus and displayed long and thick fibers with a distribution mainly parallel to the coronal axis of the brain, as well as many neurons (Fig. 5d). By contrast at PN10 only some lightly labeled small multipolar neurons are found, and no fibrous neuropil (Fig. 5e). These neurons were quite similar in dimension and shape to those found in adult rat brain (Fig. 5f). The brainstem was chosen for observations related to the developmental changes of Dehydrogenase VI. PN1 sections showed some very lightly stained, fibrous neuropil within the gigantocellular reticular nucleus (Fig. 6a). In the same region long and thin fibrous staining within the neuropil can be seen at PN3 (Fig. 6b). Similar but more densely stained neural fibers are present in the central gray at PN5, the fibers being distributed vertically to sagitally to the brain (Fig. 6c). Many reaction product granules occur within the fibrous neuropil. In addition fibrous and puncta staining with a higher density can be found in the same region at PN7 (Fig. 6d). The non-cell formed punctuate neuropil was very densely stained in the oral part of the spinal trigeminal nucleus, whereas in the region of the central gray mainly fibrous neuropil was found (Fig. 6d). But at PN10 the fibrous neuropil had almost vanished, and some large, multipolar neurons were positively labelled that contained many reaction granules in their cytoplasma (Fig. 6f). At PN10 the labeling had disapeared to a greater extent, no fibrous neuropil was any longer present, and in the adult brain only some large, multipolar neurons were positively labelled.

4. Discussion

Plasma membrane oxidoreductases (PMO's) have been demonstrated in all cell types but their real function is not known [3,6-8,20]. They are involved in a number of processes related to cell activation, e.g. control of cell growth and development, control of ion-transporter or

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exchanger, control of cell adhesion - - probably by modulating thiol-disulfide exchange of extracellular matrix proteins [7,8]. They are related to diaphorases (quinone reductases), however their biochemical properties (broader substrate specificity, cofactor specificity greater for NADH than NADPH, smaller MW), indicate that they are unrelated to NO-synthase or NADPH-diaphorases. Anti-NOsynthase antibodies do not react with PMO"s [31]. The influence of PMO's on central processes such as cellular signalling and ion mobilization, also in differentiation and cell growth, potentially bears a great significance. From this and the previous paper the following conclusions can be drawn: PMO's are present allover the different brain areas and therefore they constitute a family of enzymes largely expressed in all cell types. PMO-I and -II display a tissue distribution markedly different from PMO-III to -VI, the former being localised in fibers and the latter rather in cell bodies or associated with endocytotic granules. The different isoenzymes are expressed at various stages in specific areas. • PMO-I is observed in the parietal cortex, in CA1 of the hippocampus, in the caudate putamen, the auditory nuclei, the vestibular nuclei and the cerebellar cortex. PMO-I is not observed before PN5 and develops as long vertical fibers located mainly in the pontine nucleus and in the dorsal raphe nucleus,/mtil it merges all over the midbrain except for the aqueduct and the superior colliculus after PN10. At that stage it is highly expressed in the trigeminal nucleus and the dorsal raphe, but its expression then strongly decreases and PMO-I disapears almost completely later on. • PMO-II is mainly located in the dendate gyrus and the cerebellar cortex. It only develops around PN5, first in the dorsal and caudal linear raphe and later on, after PN7, also in the pontine nucleus and in the median raphe. However at PN10 PMO-II gradually had vanished from these areas and strongly developed in the dorsal raphe and in the mesencephalic trigeminal nucleus. At later stages the expression of PMO-II decreases from these areas. • PMO-III and PMO-IV are present in the piriform cortex, in both CA1 and CA2, in the basal ganglia, the supraoptic nucleus, the hypothalamus, the brainstem, in motor nuclei (oculomotor, facial, hypoglossal and ambiguus nuclei), and in the cerebellar cortex. PMO-III slowly develops within the gigantocellular reticular nucleus from PN1 to PN5 and later on reaches the facial nucleus after PN5, the density of PMO-III in these regions at PN10 being much higher than in the adult. PMO-IV follows a similar developmental pattern in the midbrain, with an optimal density around PN10 also. • PMO-V is poorly present in most brain areas apart of the Purkinje layer of the cerebellar cortex. It appears only at about PN5, first within the dorsal raphe in parallel fibers and in multipolar neurons. It disappears

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from the fibers around PN10 and remains present in neurons up to adulthood. PMO-VI is present in the oriens layer and in the striatum radiatum of the hippocampus, in the hypothalamus, in the mesencephalic trigeminal nucleus, in some nuclei of the brainstem and in the cerebellar cortex. It appears at early stages within the gigantocellular reticular nucleus and after PN5 within the central gray in vertical fibers. At later stages PMO-VI is found in the spinal trigeminal nucleus, at first within the neuropil then in multipolar neurons that remain present up to adulthood. From these studies it appears that the major regions expressing PMO's are the dorsal raphe, the trigeminal nucleus, the superior colliculus, the central gray, the midbrain and the cerebellum. Detailed investigations on PMO's may lead to a better understanding of clinical implications of PMO's in events related to recorded pathologies. 4.1. Dorsal raphe

The midbrain periaqueductal gray including the dorsal raphe neucleus contains the largest collection of serotonin-containing neurons in the brain [1] influencing the limbic system and affective-motivational aspect of the pain-related neural system [16]. Analgesia can be produced by stimulation of that region [17] through direct projections to nociceptors in the primary sensory nuclei. A considerable number of neurons in the periaqueductal gray and dorsal raphe nucleus send projections fibers to the trigeminal sensory complex and subserve suppression of the activity of nociceptive neurons in the trigeminal system. NADPH diaphorase has been found to colocalize with 5-HT neurons in the dorsomedial and ventromedial subgroups [4]. Most 5-HT neurons project differentially to forebrain regions, a few cells also show collateralization to the MPFC and Acb [5] that may integrate cognitive and motor activities. Since PMO's are closely related (but definitely distinct) to NADPH diaphorase, part of these functions may possibly attributed to them. On the other hand, descending projections, via the raphe magnus or directly, modulate the responses caused by noxious stimulation of the spinal dorsal horn neurons. In ascending projections, it directly modulates the responses of pain sensitive neurons in the thalamus. 4.2. Trigeminal nucleus and superior colliculus

The primary sensory mesencephalic trigeminal neurones are considered to have purely proprioceptive functions and may upregulate galanin and neuropeptide Y [2]. 5-HT axons exert a direct action on enkephalinergic interneurons in the spinal trigeminal nucleus [18]. Also neuropeptides (gal, NPY and Substance P and VIP) play a role in adaptive processes after peripheral nerve injury [26]. These

functions are clearly affected under oxydative stress and oxygen radicals. Many neurons in the intertrigeminal region send their axons to the lateral part of the superior colliculus (SC) especially in the stratum grisum intermedium [27,29]. This area plays an important role first in the transformation from visual and auditory signals to motor commands (to generate saccadic eye movements towards targets [24]) but also in sensory integration (performed in the deep layers wich has promotor functions [11,12,21]). The SC receives dense cholinergic inputs from three brainstem nuclei (the pedunculopontine tegmental nucleus, the lateral dorsal tegmental nucleus and the parabigeminal nucleus). The tegmental inputs project densely to the intermediate gray layer and sparsely to the superficial layers [14]. All these regions exhibit abundant NADPH-diaphorase activity. As mentioned above this activity is in part due to PMO's. Postnatal development of NADPH-diaphorase activity in the superior colliculus and the ventral lateral geniculate nucleus displays two different developmental patterns [10]. The first pattern, observed in the deep layers of the SC shows a transient activity during the first week which progressively decreases during the following 2 weeks. The second pattern is observed in the superficial layers of the SC and in the LGv and becomes positive during the first week, increases during the second and third weeks reaching the adult pattern at the fourth week, in contrast to what we observe with PMO's. In both cases however the developmental chronology displays an insideout pattern. It has been suggested that NAD(P)H-diaphorase activity may play different roles at different stages of the developing nervous system [10]. In the neocortex the development of NADPH diaphorase activity correlates with the laminar differentiation, suggesting an important role in the maturation of cortical neurons and in the establishment of functional connections [28]. This raises the possibility of a similar function for the various PMO's. Numerous studies have demonstrated that diaphorase-staining neurons are notably resistant to destruction in Huntington's and Alzheimer's diseases, vacuolar stroke and NMDA neurotoxicity [9,13,19,23]. Oxidative damage plays an important role in neurodegenerative disease [15]. The similarity in localization of diaphorase and NO-synthase-staining neurons in the brain is striking. Diaphorase can derive from many NAD(P)H-utilizing enzymes, most diaphorases in brain tissues being unrelated to NO-synthase [25] but presumably are related to PMO's.

Acknowledgements The authors are indebted to Mrs Chr. Deforel-Poncet for skilled technical assistance. The present work was supported by Grant 31-27733.89 from the Swiss National Foundation.

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