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The immunohistochemical localization of neuronal nitric oxide synthase in the basal forebrain of the dog
Journal of Chemical Neuroanatomy 31 (2006) 200–209 www.elsevier.com/locate/jchemneu
The immunohistochemical localization of neuronal nitric oxide synthase in the basal forebrain of the dog Laura Mene´ndez a, Daniel Insua a, Jose´ Luis Rois a, Germa´n Santamarina b, Marı´a Luisa Sua´rez b, Pedro Pesini a,* b
a Departamento de Anatomı´a, Facultad de Veterinaria, Universidad de Santiago, 27002 Lugo, Spain Departamento de Ciencias Clı´nicas, Facultad de Veterinaria, Universidad de Santiago, 27002 Lugo, Spain
Received 4 November 2005; received in revised form 18 January 2006; accepted 18 January 2006 Available online 20 February 2006
Abstract The present work describes for the first time the anatomical distribution of neuronal nitric oxide synthase (nNOS) immunoreactivity and NADPH-d activity in the basal forebrain of the dog. As in other species, small, intensely nNOS-immunoreactive cells were seen within the olfactory tubercle, caudate nucleus, putamen, nucleus accumbens and amygdala. In addition, a population of mixed large and small nNOS positive cells was found in the medial septum, diagonal band and nucleus basalis overlapping the distribution of the magnocellular cholinergic system of the basal forebrain. Our results show that the distribution of NOS containing neurons in these nuclei in the dog is more extensive and uniform than that reported in rodents and primates. When double labeling of nNOS and NADPH-d was performed in the same tissue section most neurons were double labeled. However, a considerable number of large perikarya in the diagonal band and nucleus basalis appeared to be single labeled for nNOS. Thought a certain degree of interference between the two procedures could not be completely excluded, these findings suggest that NADPH-d histochemistry, which is frequently used to show the presence of NOS, underestimates the potential of basal forebrains neurons to produce nitric oxide. In addition, a few neurons mainly localized among the fibers of the internal capsule, appeared to be labeled only for NADPH-d. These neurons could be expressing a different isoform of NOS, not recognized by our anti-nNOS antibody, as has been reported in healthy humans and AD patients. # 2006 Elsevier B.V. All rights reserved. Keywords: NADPH-d; ChAT; Telencephalon; Aging; Alzheimer’s disease; Animal models
1. Introduction The anatomical distribution of nitrergic neurons in the basal forebrain has been extensively studied in rodents and primates either by the labeling of NADPH-d activity, by immunohistochemistry with antibodies against nNOS or by in situ hybridization of the NOS mRNA (Ellison et al., 1987; Vincent and Kimura, 1992; Egberongbe et al., 1994; Rodrigo et al., 1994; Hashikawa et al., 1994; Satoh et al., 1995; Iwase et al., 1998; Yew et al., 1999; Downen et al., 1999). Far less attention has been devoted to the localization of these neurons in the carnivores (Mizukawa et al., 1989). In general, these works have provide evidence of extensive overlapping of the nitrergic neurons with the magnocellular cholinergic system of the basal forebrain,
* Corresponding author. Tel.: +34 982 285900; fax: +34 982 252195. E-mail address: [email protected] (P. Pesini). 0891-0618/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2006.01.002
though notable differences exist from one species to another. Thus, it has been reported that in the rat numerous cholinergic neurons in the diagonal band and anterior part of the nucleus basalis co-express nNOS whereas in primates the nitrergic and cholinergic neurons of the basal forebrain seem to constitute different populations (Ellison et al., 1987; Brauer et al., 1991; Pasqualotto and Vincent, 1991; Kitchener and Diamond, 1993; Geula et al., 1993; Sugaya and McKinney, 1994). One of the findings more consistently observed in the Alzheimer’s disease (AD) is the degeneration of the cholinergic neurons of the basal forebrain, which provide extensive innervation to the hippocampus and cerebral cortex (Vogels et al., 1990; Lyness et al., 2003). Since cortical cholinergic deficits are correlated with the severity of the dementia, numerous studies have focused on the mechanisms that may cause the degeneration of these basal forebrain neurons in the course of AD (Quirion et al., 1988; Wenk and Willard, 1998; Blusztajn and Berse, 2000; Terry and Buccafusco, 2003).
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Among the mechanisms involved, one of the most actively explored in recent years is the possible implication of the oxidative stress caused by an excessive production of nitric oxide (NO) (Law et al., 2001; Luth et al., 2002). The NO is a diffusible gas produced during the conversion of L-arginine to Lcitruline in a reaction catalyzed by nitric oxide synthase (NOS) which is coincident with NADPH-diaphorase (NADPH-d) (Vincent, 1995; Mayer, 1995). Nitrergic neurons constitutively express a neuronal isoform of NOS (nNOS) and the NO produced is used as a neurotransmitter under normal circumstances. Additionally, astrocytes and microglia can express an inducible isoform of NOS when they are activated in response to ischemia or inflammation (Murphy, 2000). Aberrant expression of these two NOS isoform has been demonstrated in the brain of AD patients leading to an excessive production of NO and the consequent formation of peroxinitrites that are highly neurotoxic (Luth et al., 2002). Other studies have shown that NO modulates acetylcholine release, but whether or not this has any pathological impact in AD is not known so far (for a review see Law et al., 2001). Nevertheless, it has been suggested that the presence of NOS positive neurons in close proximity to cholinergic neurons may be related to the selected vulnerability of the basal forebrain cholinergic system secondary to NO neurotoxicity (Law et al., 2001). The dog has been pointed out as a natural model especially adequate for the study of human brain aging and neurodegenerative diseases (Cummings et al., 1996b; Head et al., 2000; Studzinski et al., 2005). Most of the increasing interest aroused by the dog is based on the fact that it may suffer an age-related cognitive dysfunction that reproduces several aspects of AD (Cummings et al., 1993, 1996a; Colle et al., 2000; Head and Torp, 2002; Araujo et al., 2005). However, there is a dearth of data regarding the organization and neurochemical characteristics of the neuronal populations in the basal forebrain of this species. In particular and to the best of our knowledge, no report has been published on the distribution of the nitrergic neurons in the basal forebrain of the dog. Thus, in the present work we have used the labeling of nNOS immunoreactivity and NADPH-d staining to obtain a detailed map of these neurons in the canine basal forebrain. 2. Materials and methods Five adult dogs (5–11-years old) of either sex were used for this study. The dogs died or were euthanized (by the i.v. injection of 0.5 g of sodium pentobarbital) for a variety of reasons not related to neurological disease. Specifically, one dog died from congestive heart failure. The other four dogs were euthanized for worsening in their condition (refractory congestive heart failure, renal failure, generalized lymphoma and pyometra, respectively). The study was approved by the ethical committee of the University of Santiago and every effort was done to minimize the suffering of the animals. Necropsies were performed in all the dogs with the consent of their owners. The brains were removed within the two first hours after death and fixed in 4% paraformaldehyde in 0.1 M phosphate buffered saline pH 7.4 (PBS) for 48 h. Then, the basal forebrain was dissected out and submerged in 30% sucrose in PBS for 5 days. Serial sections (50 mm thickness) were cut in a cryostat and collected in PBS. Every tenth section was processed for immunostaining with the avidin–biotinperoxidase technique. Prior to the incubation with the first antibody, the freefloating sections were immersed for 10 min in PBS containing 3% hydrogen
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peroxide to deactivate endogenous peroxidase, washed two times in PBS and pre-incubated for 30 min in 10% normal donkey serum (Jackson ImmunoResearch Inc.) in 0.3% Triton-X-100 PBS. The sections were then incubated overnight in a 1/250 solution of the rabbit anti-nNOS polyclonal antibody (Chemicon Int.) in the same buffer and after two washes transferred to the secondary antibody for one hour (biotin conjugated donkey anti-rabbit IgG diluted 1/200, Jackson ImmunoResearch Inc.). The reaction was visualized incubating the section in 1/200 ExtrAvidin1-peroxidase (Sigma) for one hour. After washing in PBS, the tissue-bound peroxidase was developed by Trisbuffer containing 3,30 -diaminobenzidine tetrahydrochloride (20 mg/100 ml, Sigma) and 0.003% H2O2 for 10 min. The anti-nNOS antibody was prepared against recombinant human nNOS and it does not cross react with iNOS and eNOS by immunohistochemistry. The specificity of the labeling was controlled by replacing the anti-nNOS antibody with non-immune rabbit serum in the first incubation bath. No residual immunoreactivity was found in this case. A series of adjacent sections was used for the visualization of NADPH-d following the protocol of Vincent and Kimura (1992). Briefly, after washing in PBS the sections were incubated in 0.1 M phosphate buffer (PB) pH 7.4 containing 0.01% nitrobluetetrazolium (Sigma), 0.1% b-NADPH (Sigma) and 0.3% Triton-X-100 for 1 h at 37 8C. The reaction was stopped by two washing of 10 min in PB at room temperature. A third series from each dog was used for the double labeling of NADPH-d and nNOS immunoreactivity. The NADPH-d staining was carried out just before the development of the tissue-bound peroxidase within the immunostaining protocol. After the staining, the sections were rinsed in distilled water, mounted in albumin coated slides and air dried. The coverslips were then directly applied with glycerol. A fourth series was routinely stained for Nissl substance with cresyl violet to aid in the delimitation of the basal forebrain nuclei. The anatomical mapping of the labeled neurons was performed with an Olympus BX61 microscope equipped with digital camera and motorized stage controlled by StereoInvestigator software (MicroBrighField, Magdeburg). This equipment allows one to draw the contour and main anatomical references of each section at low magnification (2). Then, a complete scanning, onto the drawings of the sections is performed at higher magnification (10) to mark the position of the labeled neurons. Additionally, the distribution of the magnocellular cholinergic system of the basal telencephalon was mapped from another series of sections with anticholine acetyltransferase rabbit antisera (1:250 Chemicon. Int. Cat. # AB143) following the same above described immunohistochemical protocol. The boundaries of the different cholinergic populations in this region were delimited following the map published by St. Jacques et al. (1996) and then used as reference for the cartography of the nitrergic neurons.
3. Results Neurons immunoreactive to nNOS presented a brown labeling within the cytoplasm and the initial segment of their dendrites as their nuclei appeared unstained. No sex differences were found in the distribution of nNOS and NADPH-d. Positive neurons were widely distributed across the basal telencephalon as depicted in the drawings of Fig. 1A–F in which each dot represent one neuron in its precise localization. The main features of their anatomical distribution are emphasized in the following description. In general, many nNOS positive neurons were found within the nuclei occupied by the magnocellular cholinergic system of the basal telencephalon that is the septum, diagonal band and the nucleus basalis. In the rostral sections (Fig. 1A–C), a dense population appeared in the vertical and horizontal parts of the nucleus of the diagonal band. Additionally, a few labeled cells were found scattered in the region of the septum, mainly within the medial nucleus. The majority of the labeled cells in these
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areas were large (main diameter 24–34 mm), and round or polygonal in shape. Nevertheless, intermingled with them always appeared a number of smaller (main diameter 14– 24 mm), usually elongated or fusiform, labeled perikarya (Fig. 2A and B). Both types of labeled cells were found as well throughout the anterior, intermediate and posterior parts of the nucleus basalis (Fig. 2C–F). At rostral sections, the dense population of labeled cells within the anterior part of the nucleus basalis occupied a relatively broad band located between the ventral part of the caudate nucleus and the cortex of the olfactory tubercle. Caudal to the olfactory tubercle, the population in the anterior part of the nucleus basalis merged with that in the horizontal part of the diagonal band from which it could be delimited because of its lower density (Fig. 1C). The population of nNOS positive neurons within the intermediate part of the nucleus basalis was separated in two subgroups corresponding to its dorsal and ventral subdivisions caused by the passage of the ansa peduncularis from the thalamus to the insula and claustrum (Fig. 1D–E). Caudal to the ansa peduncularis, labeled neurons were densely packed within the posterior part of the nucleus basalis which protrudes as a wedge between the putamen and the external medullary lamina of the globus pallidum up to the level of the tail of the nucleus caudatus (Figs. 1F and 2F). Many small nNOS positive cells appeared within the nucleus caudatus, the putamen and the claustrum (Fig. 1A–F). Though the neurons in the caudate nucleus were distributed across its whole extent, the core of the nucleus always contained less labeled cells than its cortical portion along the internal capsule and the lateral ventricle. The labeled neurons in the putamen appeared uniformly distributed whereas in the claustrum they were predominately concentrated in the ventral part of the nucleus. In contrast, hardly any labeled cell was seen within the limits of the globus pallidum apart from a few that sporadically appeared interspersed in its external medullary lamina or between the fibers of the internal capsule. These cells were large and rounded in shape more similar to the labeled neurons in the adjacent nucleus basalis than to the cells found in the other divisions of the corpus striatum. Scattered small nNOS positive neurons were also found in the amygdala, the deepest layer of the olfactory tubercle and within the small islands of Calleja. However, no labeled neurons were seen within the main island of Calleja that is located between the diagonal band and the nucleus accumbens (Fig. 1B). Outside the nuclei of the basal telencephalon, labeled cells were found as well in the cortex and in several diencephalic structures including the paraventricular hypothalamic nucleus, the lateral hypothalamic area and the zona incerta but were not mapped in the present work. These neurons were small, most frequently fusiform, with a relatively big nucleus located in the center of the perikarya.
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The NADPH-d staining confirmed the existence of two types of nitrergic cells that could be clearly differentiated (Fig. 3A–D). The first type consisted of the relatively large, rounded or triangular neurons with short prolongations that constituted the majority of those found in the diagonal band and the nucleus basalis (Fig. 3A–C). These large neurons showed a moderate to low NADPH-d activity and used to appear lightly stained with this technique. The second type consisted of the smaller perikarya found in the nuclei of the corpus striatum and the olfactory tubercle as well as intermingled with the larger neurons in the diagonal band and the nucleus basalis (Fig. 3B– D). This second type stained intensely for NADPH-d and varied from multipolar to fusiform showing extensive dendritic trees. By other side, the distribution of NADPH-d labeled cells was similar to the obtained with nNOS immunohistochemistry and no major anatomical differences were noted. Nonetheless, the number of nNOS labeled neurons in the diagonal band and nucleus basalis was always appreciably higher than the number of NADPH-d stained cells in the same areas of the adjacent sections (compare Figs. 2B and 3A). The double staining of nNOS immunoreactivity and NADPH-d in the same tissue section gave similar results (data not shown). The immense majority of the small perikarya, if not all, appeared doubled labeled showing a blackish coloration that could be clearly differentiated from either the brown color showed by the nNOS immunoreactive cells or the bright-blue color of the NADPH-d positive cells seen in the respective single labeled adjacent sections. Many of the large perikarya appeared as well double-labeled. Usually, they were of a mixed brown-bluish color but some times the two colors were segregated in different zones of a particular cytoplasm. Additionally, a significant number of large perikarya in the diagonal band and nucleus basalis appeared to be single labeled for nNOS immunoreactivity and a few large neurons, scattered among the fibers of the internal capsule, were single-labeled for NADPH-d. 4. Discussion The anatomical distribution of the nitrergic neurons in the basal forebrain of rodents, cats and primates has been studied by immunohistochemistry, in situ hybridization or the visualization of the NADPH-d activity (Ellison et al., 1987; Mizukawa et al., 1989; Vincent and Kimura, 1992; Egberongbe et al., 1994; Rodrigo et al., 1994; Hashikawa et al., 1994; Satoh et al., 1995; Iwase et al., 1998; Yew et al., 1999; Downen et al., 1999). A common feature to all these species is the presence of numerous NOS containing neurons throughout the caudate nucleus, accumbens and putamen as well as in the olfactory tubercle and in several nuclei of the amygdala. In this regard, the dog does not appear to be an exception since we have found nNOS positive cells in all these areas. Furthermore, the
Fig. 1. (A)–(F) Schematic drawings of coronal sections depicting the distribution of nNOS (left side) and ChAT (right side) immunoreactive neurons in the basal forebrain of the dog. Each dot represents a neuron in its precise localization. AC, anterior commissure; AMg, amygdala; ASt, amygdalo-striatal transition area; CL, claustrum; DBh, diagonal band, horizontal limb; DBv, diagonal band, vertical limb; IC, internal capsule; FX, fornix; GP, globus pallidum; IC, island of Calleja; ICm, island of Calleja major; LV, lateral ventricle; NBa, nucleus basalis anterior; NBid, nucleus basalis intermediodorsal; NBiv, nucleus basalis intermedioventral; NBp, nucleus basalis posterior; NC, caudate nucleus; OPt, optic tract; OT, olfactory tubercle; PT, putamen.
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Fig. 2. nNOS immunoreactive neurons in the basal forebrain. In all photographs dorsal is to the top and medial to the right. (A) vertical limb of the diagonal band at the level of Fig. 1B. Note that labeled neurons are lacking in the Island of Calleja major which contains a very dense, dust-like positive labeling. (B) Horizontal limb of the diagonal band at the level of Fig. 1C. (C) Nucleus basalis anterior at the level of Fig. 1B. (D) Nucleus basalis intermediodorsal at the level of Fig. 1E. (E) Nucleus basalis intermedioventral at the level of Fig. 1E. (F) Nucleus basalis posterior at the level of Fig. 1F. Note that the labeled cells are concentrated between the putamen and the external medullary lamina of the globus pallidum (eml). Arrowheads in (A)–(F) point to small, less abundant perikarya. Scale bar in (F) represents 200 mm for all photographs.
morphology of the labeled neurons in these nuclei of the dog was comparable to that reported in other species. They appeared uniformly small and when visualized by means of NADPH-d histochemistry showed an intense staining of their perikarya and extensive dendritic trees. In addition, a population composed of large and small nNOS neurons were seen in other basal forebrain areas such as the medial septum, the diagonal band and the nucleus basalis. Both, large and small nitrergic neurons have been described in the diagonal band and nucleus basalis of the rat, cats and primates, including humans (Ellison et al., 1987; Mizukawa et al., 1989; Vincent and Kimura, 1992; Hashikawa et al., 1994; Downen et al., 1999). However, these previous reports point to the possible existence of several notable species-specific differences. Thus, it has been reported that in the rat dense clusters of large intensely stained NADPH-d positive neurons are present in the medial septum and the vertical and horizontal limb of the diagonal band whereas they are lacking in the region of the nucleus basalis (Vincent and Kimura, 1992). On the other hand, humans, baboon and monkey present numerous NOS positive neurons in the horizontal limb of the diagonal band and in the anterior part of the nucleus basalis but very few in the medial septum,
vertical limb of the diagonal band, and the intermediate and posterior divisions of the nucleus basalis (Geula et al., 1993; Egberongbe et al., 1994; Satoh et al., 1995). As seen from our results, the distribution of nNOS immunoreactive neurons in the dog’s forebrain appears to be more extensive and uniform across these nuclei than in rodents and primates since we have found numerous labeled cells in the vertical and horizontal limbs of the diagonal band and throughout the rostrocaudal extension of the nucleus basalis. Other authors have described the presence of NADPH-d neurons in the same areas of the cat forebrain, which suggests that this pattern could be stable throughout the carnivores (Mizukawa et al., 1989). However, it could not be completely excluded that some of the reported discrepancies between species were related with procedural differences in the processing of the tissue or in the methodology utilized for the visualization of the NOS containing neurons. In the present work we have used either nNOS immunohistochemistry or NADPH-d staining and no major anatomical differences were noted. However, the number of nNOS labeled neurons in the diagonal band and nucleus basalis was always appreciably higher than the number of NADPH-d stained cells in the same areas of the adjacent sections. Furthermore, when
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the two staining procedures were combined in the same tissue section, a significant number of the large neurons in the diagonal band and nucleus basalis appeared single labeled for nNOS immunoreactivity. Similarly, the presence of nNOS positive-NADPH-d negative neurons has been described in the hippocampus of rats and humans and in the basal forebrain of monkeys (Dinerman et al., 1994; Satoh et al., 1995; Doyle and Slater, 1997). Most probably, these observations are related with the processing of the samples since the conservation of NADPH-d activity in the brain is known to be highly dependent on the degree of fixation of the tissue (Dinerman et al., 1994; Vincent, 1995). In any case, our results show that NADPH-d histochemistry could significantly underestimate the number of
nNOS containing neurons in the basal forebrain, particularly when it is applied to not-perfused tissue obtained from necropsies. On the other hand, the single NADPH-d labeled neurons that we have seen scattered among the fibers of the internal capsule could be expressing a different isoform of NOS, not recognized by our anti-nNOS antibody, as has been reported in healthy humans and AD patients (Dinerman et al., 1994; Luth et al., 2002). The relation between the nitrergic and the magnocellular cholinergic neurons of the basal forebrain is an issue of considerable interest. In the dog, as in other species this system has been divided in four groups of neurons called ChAT-1 to ChAT-4 (Mesulam et al., 1983). The ChAT-1 group is localized
Fig. 3. NADPH-d positive neurons in the basal forebrain. In all photographs dorsal is to the top and medial to the right. (A) Large neurons in the horizontal limb of the diagonal band showed at the same magnification of Fig. 2B which correspond to the same region in an adjacent section stained for nNOS immunoreactivity. Note that the number of NADPH-d stained cells is clearly lesser than the number of nNOS immunoreactive neurons in Fig. 2B. (B) Enlargement of the framed area in (A) at the same magnification that (C) and (D). Arrowheads point to small perikarya. (C) Nucleus basalis anterior at the level of Fig. 1B. Double arrowheads point to large perikarya. (D) Nucleus caudatus at the level of Fig. 1C. No large labeled neurons were found in this nucleus. Scale bar in (D) represents 100 mm for (A) and 50 mm for (B)–(D).
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Fig. 3. (Continued ).
in the medial septum; the ChAT-2 and ChAT-3 are in the vertical and horizontal limbs of the diagonal band, respectively; the ChAT-4 is within the nucleus basalis usually subdivided in anterior, intermediodorsal, intermedioventral and posterior parts (Kimura et al., 1981; Mesulam et al., 1983, 1984; Satoh and Fibiger, 1985; Vincent and Reiner, 1987; Oh et al., 1992; Bruckner et al., 1992; Lauterborn et al., 1993; Rieck et al., 1995; St. Jacques et al., 1996; Ichikawa et al., 1997; Kasashima et al., 1998; Oda and Nakanishi, 2000). Double labeling studies in the rat have reported that a high percent of ChAT positive cells co-express NADPH-d in the diagonal band, but this proportion decreases in the anterior part of the nucleus basalis and became virtually zero toward its intermediate and caudal parts (Brauer et al., 1991; Pasqualotto and Vincent, 1991; Kitchener and Diamond, 1993; Geula et al., 1993; Sugaya and McKinney, 1994). Similar double labeling experiments in primates including humans, have shown that very few if any neurons of the basal magnocellular cholinergic system co-
express NADPH-d (Ellison et al., 1987; Geula et al., 1993). To our knowledge, such double-labeling studies have not been carried out in the carnivores. However, the comparison of the anatomical distribution of the NADPH-d positive and ChAT immunoreactive neurons in the basal forebrain of the cat reveals an extensive anatomical overlapping of these two neurotransmitters systems (Vincent and Reiner, 1987; Mizukawa et al., 1989). Agreeing with this, we have found in the dog significant numbers of large nNOS labeled cells in all the nuclei occupied by the magnocellular cholinergic groups as described by St. Jacques et al. (1996). This suggests that the carnivores could present a higher degree of co-expression of ChAT and nNOS throughout these areas, particularly in the neurons of the intermediate and posterior part of the nucleus basalis. Further experiments are underway in our laboratory to specifically asses this issue in the dog. It has been proposed that an over-expression of NO by neurons and neuroglia in the basal forebrain could contribute to
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the degeneration of the cholinergic magnocellular neurons that occurs in AD (Benzing and Mufson, 1995; Smith et al., 1997; Law et al., 2001). Indeed, augmented expression of the neuronal and inducible isoforms of NOS has been demonstrated in the brain of normal adults and AD patients (Benzing and Mufson, 1995; Yew et al., 1999; Luth et al., 2001, 2002). Contrasting with this, other experiments have shown that the expression of NADPH-d is linked to a protective effect of these neurons against ischemia, neurotoxicity and various neurodegenerative processes (Ferrante et al., 1985; Koh and Choi, 1988; Hyman et al., 1992; Behrens et al., 1996). Taken together, these findings could help to explain the particular vulnerability of the human basal forebrain cholinergic neurons in AD since most of them are NADPH-d negative and, at least within the vertical limb of the diagonal band and the anterior part of the nucleus basalis, co-exist with nitrergic cells. However, the protective effect of NADPH-d would differ from one cholinergic group to another as a consequence of the variable distribution of the nitrergic neurons. In addition, it should be considered that other major species-specific differences in the cytochemical phenotype of the basal forebrain cholinergic neurons could modify their response to aging or their vulnerability to neurodegenerative processes from one species to another (Geula et al., 1993). Thus, we must be cautious when using the results obtained in rodents to infer the response to aging of this neurotransmitter system in humans or carnivores. During the last decade, the dog has acquired an increasing interest for the study of brain aging. Numerous reports suggest that the canine could be a useful model to contrast relevant aspects of the cholinergic hypothesis of human brain aging and AD (Cummings et al., 1996b; Head et al., 2000; Colle et al., 2000; Head and Torp, 2002; Araujo et al., 2005). However, the response to normal or pathological aging of the different neuronal populations in the basal forebrain of the dog is almost completely unknown and more data are necessary to adequately validate the canine model for human cognitive aging and dementia. In summary, this work represents the first study on the distribution of nNOS immunoreactive cells in the basal forebrain of the dog. Small, intensely stained cells were distributed within the caudatus, accumbens, putamen, olfactory tubercle and amygdaloidal body following a pattern that seems to be well preserved through different species. In addition, a population of mixed large and small nNOS positive cells was found in the medial septum, diagonal band and nucleus basalis overlapping the distribution of the magnocellular cholinergic system of the basal forebrain. Regarding these nuclei, our results are in agreement with the distribution of NADPH-d neurons in the cat but differ notably from those reported in rodents and primates. These differences could translate into a different response of the basal forebrain magnocellular neurons to normal or pathological aging in the carnivores. Acknowledgements This work has been financed by the Spanish Ministerio de Ciencia y Tecnologı´a and FEDER (project no. SAF2003-
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The immunohistochemical localization of neuronal nitric oxide synthase in the basal forebrain of the dog Laura Mene´ndez a, Daniel Insua a, Jose´ Luis Rois a, Germa´n Santamarina b, Marı´a Luisa Sua´rez b, Pedro Pesini a,* b
a Departamento de Anatomı´a, Facultad de Veterinaria, Universidad de Santiago, 27002 Lugo, Spain Departamento de Ciencias Clı´nicas, Facultad de Veterinaria, Universidad de Santiago, 27002 Lugo, Spain
Received 4 November 2005; received in revised form 18 January 2006; accepted 18 January 2006 Available online 20 February 2006
Abstract The present work describes for the first time the anatomical distribution of neuronal nitric oxide synthase (nNOS) immunoreactivity and NADPH-d activity in the basal forebrain of the dog. As in other species, small, intensely nNOS-immunoreactive cells were seen within the olfactory tubercle, caudate nucleus, putamen, nucleus accumbens and amygdala. In addition, a population of mixed large and small nNOS positive cells was found in the medial septum, diagonal band and nucleus basalis overlapping the distribution of the magnocellular cholinergic system of the basal forebrain. Our results show that the distribution of NOS containing neurons in these nuclei in the dog is more extensive and uniform than that reported in rodents and primates. When double labeling of nNOS and NADPH-d was performed in the same tissue section most neurons were double labeled. However, a considerable number of large perikarya in the diagonal band and nucleus basalis appeared to be single labeled for nNOS. Thought a certain degree of interference between the two procedures could not be completely excluded, these findings suggest that NADPH-d histochemistry, which is frequently used to show the presence of NOS, underestimates the potential of basal forebrains neurons to produce nitric oxide. In addition, a few neurons mainly localized among the fibers of the internal capsule, appeared to be labeled only for NADPH-d. These neurons could be expressing a different isoform of NOS, not recognized by our anti-nNOS antibody, as has been reported in healthy humans and AD patients. # 2006 Elsevier B.V. All rights reserved. Keywords: NADPH-d; ChAT; Telencephalon; Aging; Alzheimer’s disease; Animal models
1. Introduction The anatomical distribution of nitrergic neurons in the basal forebrain has been extensively studied in rodents and primates either by the labeling of NADPH-d activity, by immunohistochemistry with antibodies against nNOS or by in situ hybridization of the NOS mRNA (Ellison et al., 1987; Vincent and Kimura, 1992; Egberongbe et al., 1994; Rodrigo et al., 1994; Hashikawa et al., 1994; Satoh et al., 1995; Iwase et al., 1998; Yew et al., 1999; Downen et al., 1999). Far less attention has been devoted to the localization of these neurons in the carnivores (Mizukawa et al., 1989). In general, these works have provide evidence of extensive overlapping of the nitrergic neurons with the magnocellular cholinergic system of the basal forebrain,
* Corresponding author. Tel.: +34 982 285900; fax: +34 982 252195. E-mail address: [email protected] (P. Pesini). 0891-0618/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2006.01.002
though notable differences exist from one species to another. Thus, it has been reported that in the rat numerous cholinergic neurons in the diagonal band and anterior part of the nucleus basalis co-express nNOS whereas in primates the nitrergic and cholinergic neurons of the basal forebrain seem to constitute different populations (Ellison et al., 1987; Brauer et al., 1991; Pasqualotto and Vincent, 1991; Kitchener and Diamond, 1993; Geula et al., 1993; Sugaya and McKinney, 1994). One of the findings more consistently observed in the Alzheimer’s disease (AD) is the degeneration of the cholinergic neurons of the basal forebrain, which provide extensive innervation to the hippocampus and cerebral cortex (Vogels et al., 1990; Lyness et al., 2003). Since cortical cholinergic deficits are correlated with the severity of the dementia, numerous studies have focused on the mechanisms that may cause the degeneration of these basal forebrain neurons in the course of AD (Quirion et al., 1988; Wenk and Willard, 1998; Blusztajn and Berse, 2000; Terry and Buccafusco, 2003).
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Among the mechanisms involved, one of the most actively explored in recent years is the possible implication of the oxidative stress caused by an excessive production of nitric oxide (NO) (Law et al., 2001; Luth et al., 2002). The NO is a diffusible gas produced during the conversion of L-arginine to Lcitruline in a reaction catalyzed by nitric oxide synthase (NOS) which is coincident with NADPH-diaphorase (NADPH-d) (Vincent, 1995; Mayer, 1995). Nitrergic neurons constitutively express a neuronal isoform of NOS (nNOS) and the NO produced is used as a neurotransmitter under normal circumstances. Additionally, astrocytes and microglia can express an inducible isoform of NOS when they are activated in response to ischemia or inflammation (Murphy, 2000). Aberrant expression of these two NOS isoform has been demonstrated in the brain of AD patients leading to an excessive production of NO and the consequent formation of peroxinitrites that are highly neurotoxic (Luth et al., 2002). Other studies have shown that NO modulates acetylcholine release, but whether or not this has any pathological impact in AD is not known so far (for a review see Law et al., 2001). Nevertheless, it has been suggested that the presence of NOS positive neurons in close proximity to cholinergic neurons may be related to the selected vulnerability of the basal forebrain cholinergic system secondary to NO neurotoxicity (Law et al., 2001). The dog has been pointed out as a natural model especially adequate for the study of human brain aging and neurodegenerative diseases (Cummings et al., 1996b; Head et al., 2000; Studzinski et al., 2005). Most of the increasing interest aroused by the dog is based on the fact that it may suffer an age-related cognitive dysfunction that reproduces several aspects of AD (Cummings et al., 1993, 1996a; Colle et al., 2000; Head and Torp, 2002; Araujo et al., 2005). However, there is a dearth of data regarding the organization and neurochemical characteristics of the neuronal populations in the basal forebrain of this species. In particular and to the best of our knowledge, no report has been published on the distribution of the nitrergic neurons in the basal forebrain of the dog. Thus, in the present work we have used the labeling of nNOS immunoreactivity and NADPH-d staining to obtain a detailed map of these neurons in the canine basal forebrain. 2. Materials and methods Five adult dogs (5–11-years old) of either sex were used for this study. The dogs died or were euthanized (by the i.v. injection of 0.5 g of sodium pentobarbital) for a variety of reasons not related to neurological disease. Specifically, one dog died from congestive heart failure. The other four dogs were euthanized for worsening in their condition (refractory congestive heart failure, renal failure, generalized lymphoma and pyometra, respectively). The study was approved by the ethical committee of the University of Santiago and every effort was done to minimize the suffering of the animals. Necropsies were performed in all the dogs with the consent of their owners. The brains were removed within the two first hours after death and fixed in 4% paraformaldehyde in 0.1 M phosphate buffered saline pH 7.4 (PBS) for 48 h. Then, the basal forebrain was dissected out and submerged in 30% sucrose in PBS for 5 days. Serial sections (50 mm thickness) were cut in a cryostat and collected in PBS. Every tenth section was processed for immunostaining with the avidin–biotinperoxidase technique. Prior to the incubation with the first antibody, the freefloating sections were immersed for 10 min in PBS containing 3% hydrogen
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peroxide to deactivate endogenous peroxidase, washed two times in PBS and pre-incubated for 30 min in 10% normal donkey serum (Jackson ImmunoResearch Inc.) in 0.3% Triton-X-100 PBS. The sections were then incubated overnight in a 1/250 solution of the rabbit anti-nNOS polyclonal antibody (Chemicon Int.) in the same buffer and after two washes transferred to the secondary antibody for one hour (biotin conjugated donkey anti-rabbit IgG diluted 1/200, Jackson ImmunoResearch Inc.). The reaction was visualized incubating the section in 1/200 ExtrAvidin1-peroxidase (Sigma) for one hour. After washing in PBS, the tissue-bound peroxidase was developed by Trisbuffer containing 3,30 -diaminobenzidine tetrahydrochloride (20 mg/100 ml, Sigma) and 0.003% H2O2 for 10 min. The anti-nNOS antibody was prepared against recombinant human nNOS and it does not cross react with iNOS and eNOS by immunohistochemistry. The specificity of the labeling was controlled by replacing the anti-nNOS antibody with non-immune rabbit serum in the first incubation bath. No residual immunoreactivity was found in this case. A series of adjacent sections was used for the visualization of NADPH-d following the protocol of Vincent and Kimura (1992). Briefly, after washing in PBS the sections were incubated in 0.1 M phosphate buffer (PB) pH 7.4 containing 0.01% nitrobluetetrazolium (Sigma), 0.1% b-NADPH (Sigma) and 0.3% Triton-X-100 for 1 h at 37 8C. The reaction was stopped by two washing of 10 min in PB at room temperature. A third series from each dog was used for the double labeling of NADPH-d and nNOS immunoreactivity. The NADPH-d staining was carried out just before the development of the tissue-bound peroxidase within the immunostaining protocol. After the staining, the sections were rinsed in distilled water, mounted in albumin coated slides and air dried. The coverslips were then directly applied with glycerol. A fourth series was routinely stained for Nissl substance with cresyl violet to aid in the delimitation of the basal forebrain nuclei. The anatomical mapping of the labeled neurons was performed with an Olympus BX61 microscope equipped with digital camera and motorized stage controlled by StereoInvestigator software (MicroBrighField, Magdeburg). This equipment allows one to draw the contour and main anatomical references of each section at low magnification (2). Then, a complete scanning, onto the drawings of the sections is performed at higher magnification (10) to mark the position of the labeled neurons. Additionally, the distribution of the magnocellular cholinergic system of the basal telencephalon was mapped from another series of sections with anticholine acetyltransferase rabbit antisera (1:250 Chemicon. Int. Cat. # AB143) following the same above described immunohistochemical protocol. The boundaries of the different cholinergic populations in this region were delimited following the map published by St. Jacques et al. (1996) and then used as reference for the cartography of the nitrergic neurons.
3. Results Neurons immunoreactive to nNOS presented a brown labeling within the cytoplasm and the initial segment of their dendrites as their nuclei appeared unstained. No sex differences were found in the distribution of nNOS and NADPH-d. Positive neurons were widely distributed across the basal telencephalon as depicted in the drawings of Fig. 1A–F in which each dot represent one neuron in its precise localization. The main features of their anatomical distribution are emphasized in the following description. In general, many nNOS positive neurons were found within the nuclei occupied by the magnocellular cholinergic system of the basal telencephalon that is the septum, diagonal band and the nucleus basalis. In the rostral sections (Fig. 1A–C), a dense population appeared in the vertical and horizontal parts of the nucleus of the diagonal band. Additionally, a few labeled cells were found scattered in the region of the septum, mainly within the medial nucleus. The majority of the labeled cells in these
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areas were large (main diameter 24–34 mm), and round or polygonal in shape. Nevertheless, intermingled with them always appeared a number of smaller (main diameter 14– 24 mm), usually elongated or fusiform, labeled perikarya (Fig. 2A and B). Both types of labeled cells were found as well throughout the anterior, intermediate and posterior parts of the nucleus basalis (Fig. 2C–F). At rostral sections, the dense population of labeled cells within the anterior part of the nucleus basalis occupied a relatively broad band located between the ventral part of the caudate nucleus and the cortex of the olfactory tubercle. Caudal to the olfactory tubercle, the population in the anterior part of the nucleus basalis merged with that in the horizontal part of the diagonal band from which it could be delimited because of its lower density (Fig. 1C). The population of nNOS positive neurons within the intermediate part of the nucleus basalis was separated in two subgroups corresponding to its dorsal and ventral subdivisions caused by the passage of the ansa peduncularis from the thalamus to the insula and claustrum (Fig. 1D–E). Caudal to the ansa peduncularis, labeled neurons were densely packed within the posterior part of the nucleus basalis which protrudes as a wedge between the putamen and the external medullary lamina of the globus pallidum up to the level of the tail of the nucleus caudatus (Figs. 1F and 2F). Many small nNOS positive cells appeared within the nucleus caudatus, the putamen and the claustrum (Fig. 1A–F). Though the neurons in the caudate nucleus were distributed across its whole extent, the core of the nucleus always contained less labeled cells than its cortical portion along the internal capsule and the lateral ventricle. The labeled neurons in the putamen appeared uniformly distributed whereas in the claustrum they were predominately concentrated in the ventral part of the nucleus. In contrast, hardly any labeled cell was seen within the limits of the globus pallidum apart from a few that sporadically appeared interspersed in its external medullary lamina or between the fibers of the internal capsule. These cells were large and rounded in shape more similar to the labeled neurons in the adjacent nucleus basalis than to the cells found in the other divisions of the corpus striatum. Scattered small nNOS positive neurons were also found in the amygdala, the deepest layer of the olfactory tubercle and within the small islands of Calleja. However, no labeled neurons were seen within the main island of Calleja that is located between the diagonal band and the nucleus accumbens (Fig. 1B). Outside the nuclei of the basal telencephalon, labeled cells were found as well in the cortex and in several diencephalic structures including the paraventricular hypothalamic nucleus, the lateral hypothalamic area and the zona incerta but were not mapped in the present work. These neurons were small, most frequently fusiform, with a relatively big nucleus located in the center of the perikarya.
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The NADPH-d staining confirmed the existence of two types of nitrergic cells that could be clearly differentiated (Fig. 3A–D). The first type consisted of the relatively large, rounded or triangular neurons with short prolongations that constituted the majority of those found in the diagonal band and the nucleus basalis (Fig. 3A–C). These large neurons showed a moderate to low NADPH-d activity and used to appear lightly stained with this technique. The second type consisted of the smaller perikarya found in the nuclei of the corpus striatum and the olfactory tubercle as well as intermingled with the larger neurons in the diagonal band and the nucleus basalis (Fig. 3B– D). This second type stained intensely for NADPH-d and varied from multipolar to fusiform showing extensive dendritic trees. By other side, the distribution of NADPH-d labeled cells was similar to the obtained with nNOS immunohistochemistry and no major anatomical differences were noted. Nonetheless, the number of nNOS labeled neurons in the diagonal band and nucleus basalis was always appreciably higher than the number of NADPH-d stained cells in the same areas of the adjacent sections (compare Figs. 2B and 3A). The double staining of nNOS immunoreactivity and NADPH-d in the same tissue section gave similar results (data not shown). The immense majority of the small perikarya, if not all, appeared doubled labeled showing a blackish coloration that could be clearly differentiated from either the brown color showed by the nNOS immunoreactive cells or the bright-blue color of the NADPH-d positive cells seen in the respective single labeled adjacent sections. Many of the large perikarya appeared as well double-labeled. Usually, they were of a mixed brown-bluish color but some times the two colors were segregated in different zones of a particular cytoplasm. Additionally, a significant number of large perikarya in the diagonal band and nucleus basalis appeared to be single labeled for nNOS immunoreactivity and a few large neurons, scattered among the fibers of the internal capsule, were single-labeled for NADPH-d. 4. Discussion The anatomical distribution of the nitrergic neurons in the basal forebrain of rodents, cats and primates has been studied by immunohistochemistry, in situ hybridization or the visualization of the NADPH-d activity (Ellison et al., 1987; Mizukawa et al., 1989; Vincent and Kimura, 1992; Egberongbe et al., 1994; Rodrigo et al., 1994; Hashikawa et al., 1994; Satoh et al., 1995; Iwase et al., 1998; Yew et al., 1999; Downen et al., 1999). A common feature to all these species is the presence of numerous NOS containing neurons throughout the caudate nucleus, accumbens and putamen as well as in the olfactory tubercle and in several nuclei of the amygdala. In this regard, the dog does not appear to be an exception since we have found nNOS positive cells in all these areas. Furthermore, the
Fig. 1. (A)–(F) Schematic drawings of coronal sections depicting the distribution of nNOS (left side) and ChAT (right side) immunoreactive neurons in the basal forebrain of the dog. Each dot represents a neuron in its precise localization. AC, anterior commissure; AMg, amygdala; ASt, amygdalo-striatal transition area; CL, claustrum; DBh, diagonal band, horizontal limb; DBv, diagonal band, vertical limb; IC, internal capsule; FX, fornix; GP, globus pallidum; IC, island of Calleja; ICm, island of Calleja major; LV, lateral ventricle; NBa, nucleus basalis anterior; NBid, nucleus basalis intermediodorsal; NBiv, nucleus basalis intermedioventral; NBp, nucleus basalis posterior; NC, caudate nucleus; OPt, optic tract; OT, olfactory tubercle; PT, putamen.
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Fig. 2. nNOS immunoreactive neurons in the basal forebrain. In all photographs dorsal is to the top and medial to the right. (A) vertical limb of the diagonal band at the level of Fig. 1B. Note that labeled neurons are lacking in the Island of Calleja major which contains a very dense, dust-like positive labeling. (B) Horizontal limb of the diagonal band at the level of Fig. 1C. (C) Nucleus basalis anterior at the level of Fig. 1B. (D) Nucleus basalis intermediodorsal at the level of Fig. 1E. (E) Nucleus basalis intermedioventral at the level of Fig. 1E. (F) Nucleus basalis posterior at the level of Fig. 1F. Note that the labeled cells are concentrated between the putamen and the external medullary lamina of the globus pallidum (eml). Arrowheads in (A)–(F) point to small, less abundant perikarya. Scale bar in (F) represents 200 mm for all photographs.
morphology of the labeled neurons in these nuclei of the dog was comparable to that reported in other species. They appeared uniformly small and when visualized by means of NADPH-d histochemistry showed an intense staining of their perikarya and extensive dendritic trees. In addition, a population composed of large and small nNOS neurons were seen in other basal forebrain areas such as the medial septum, the diagonal band and the nucleus basalis. Both, large and small nitrergic neurons have been described in the diagonal band and nucleus basalis of the rat, cats and primates, including humans (Ellison et al., 1987; Mizukawa et al., 1989; Vincent and Kimura, 1992; Hashikawa et al., 1994; Downen et al., 1999). However, these previous reports point to the possible existence of several notable species-specific differences. Thus, it has been reported that in the rat dense clusters of large intensely stained NADPH-d positive neurons are present in the medial septum and the vertical and horizontal limb of the diagonal band whereas they are lacking in the region of the nucleus basalis (Vincent and Kimura, 1992). On the other hand, humans, baboon and monkey present numerous NOS positive neurons in the horizontal limb of the diagonal band and in the anterior part of the nucleus basalis but very few in the medial septum,
vertical limb of the diagonal band, and the intermediate and posterior divisions of the nucleus basalis (Geula et al., 1993; Egberongbe et al., 1994; Satoh et al., 1995). As seen from our results, the distribution of nNOS immunoreactive neurons in the dog’s forebrain appears to be more extensive and uniform across these nuclei than in rodents and primates since we have found numerous labeled cells in the vertical and horizontal limbs of the diagonal band and throughout the rostrocaudal extension of the nucleus basalis. Other authors have described the presence of NADPH-d neurons in the same areas of the cat forebrain, which suggests that this pattern could be stable throughout the carnivores (Mizukawa et al., 1989). However, it could not be completely excluded that some of the reported discrepancies between species were related with procedural differences in the processing of the tissue or in the methodology utilized for the visualization of the NOS containing neurons. In the present work we have used either nNOS immunohistochemistry or NADPH-d staining and no major anatomical differences were noted. However, the number of nNOS labeled neurons in the diagonal band and nucleus basalis was always appreciably higher than the number of NADPH-d stained cells in the same areas of the adjacent sections. Furthermore, when
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the two staining procedures were combined in the same tissue section, a significant number of the large neurons in the diagonal band and nucleus basalis appeared single labeled for nNOS immunoreactivity. Similarly, the presence of nNOS positive-NADPH-d negative neurons has been described in the hippocampus of rats and humans and in the basal forebrain of monkeys (Dinerman et al., 1994; Satoh et al., 1995; Doyle and Slater, 1997). Most probably, these observations are related with the processing of the samples since the conservation of NADPH-d activity in the brain is known to be highly dependent on the degree of fixation of the tissue (Dinerman et al., 1994; Vincent, 1995). In any case, our results show that NADPH-d histochemistry could significantly underestimate the number of
nNOS containing neurons in the basal forebrain, particularly when it is applied to not-perfused tissue obtained from necropsies. On the other hand, the single NADPH-d labeled neurons that we have seen scattered among the fibers of the internal capsule could be expressing a different isoform of NOS, not recognized by our anti-nNOS antibody, as has been reported in healthy humans and AD patients (Dinerman et al., 1994; Luth et al., 2002). The relation between the nitrergic and the magnocellular cholinergic neurons of the basal forebrain is an issue of considerable interest. In the dog, as in other species this system has been divided in four groups of neurons called ChAT-1 to ChAT-4 (Mesulam et al., 1983). The ChAT-1 group is localized
Fig. 3. NADPH-d positive neurons in the basal forebrain. In all photographs dorsal is to the top and medial to the right. (A) Large neurons in the horizontal limb of the diagonal band showed at the same magnification of Fig. 2B which correspond to the same region in an adjacent section stained for nNOS immunoreactivity. Note that the number of NADPH-d stained cells is clearly lesser than the number of nNOS immunoreactive neurons in Fig. 2B. (B) Enlargement of the framed area in (A) at the same magnification that (C) and (D). Arrowheads point to small perikarya. (C) Nucleus basalis anterior at the level of Fig. 1B. Double arrowheads point to large perikarya. (D) Nucleus caudatus at the level of Fig. 1C. No large labeled neurons were found in this nucleus. Scale bar in (D) represents 100 mm for (A) and 50 mm for (B)–(D).
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Fig. 3. (Continued ).
in the medial septum; the ChAT-2 and ChAT-3 are in the vertical and horizontal limbs of the diagonal band, respectively; the ChAT-4 is within the nucleus basalis usually subdivided in anterior, intermediodorsal, intermedioventral and posterior parts (Kimura et al., 1981; Mesulam et al., 1983, 1984; Satoh and Fibiger, 1985; Vincent and Reiner, 1987; Oh et al., 1992; Bruckner et al., 1992; Lauterborn et al., 1993; Rieck et al., 1995; St. Jacques et al., 1996; Ichikawa et al., 1997; Kasashima et al., 1998; Oda and Nakanishi, 2000). Double labeling studies in the rat have reported that a high percent of ChAT positive cells co-express NADPH-d in the diagonal band, but this proportion decreases in the anterior part of the nucleus basalis and became virtually zero toward its intermediate and caudal parts (Brauer et al., 1991; Pasqualotto and Vincent, 1991; Kitchener and Diamond, 1993; Geula et al., 1993; Sugaya and McKinney, 1994). Similar double labeling experiments in primates including humans, have shown that very few if any neurons of the basal magnocellular cholinergic system co-
express NADPH-d (Ellison et al., 1987; Geula et al., 1993). To our knowledge, such double-labeling studies have not been carried out in the carnivores. However, the comparison of the anatomical distribution of the NADPH-d positive and ChAT immunoreactive neurons in the basal forebrain of the cat reveals an extensive anatomical overlapping of these two neurotransmitters systems (Vincent and Reiner, 1987; Mizukawa et al., 1989). Agreeing with this, we have found in the dog significant numbers of large nNOS labeled cells in all the nuclei occupied by the magnocellular cholinergic groups as described by St. Jacques et al. (1996). This suggests that the carnivores could present a higher degree of co-expression of ChAT and nNOS throughout these areas, particularly in the neurons of the intermediate and posterior part of the nucleus basalis. Further experiments are underway in our laboratory to specifically asses this issue in the dog. It has been proposed that an over-expression of NO by neurons and neuroglia in the basal forebrain could contribute to
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the degeneration of the cholinergic magnocellular neurons that occurs in AD (Benzing and Mufson, 1995; Smith et al., 1997; Law et al., 2001). Indeed, augmented expression of the neuronal and inducible isoforms of NOS has been demonstrated in the brain of normal adults and AD patients (Benzing and Mufson, 1995; Yew et al., 1999; Luth et al., 2001, 2002). Contrasting with this, other experiments have shown that the expression of NADPH-d is linked to a protective effect of these neurons against ischemia, neurotoxicity and various neurodegenerative processes (Ferrante et al., 1985; Koh and Choi, 1988; Hyman et al., 1992; Behrens et al., 1996). Taken together, these findings could help to explain the particular vulnerability of the human basal forebrain cholinergic neurons in AD since most of them are NADPH-d negative and, at least within the vertical limb of the diagonal band and the anterior part of the nucleus basalis, co-exist with nitrergic cells. However, the protective effect of NADPH-d would differ from one cholinergic group to another as a consequence of the variable distribution of the nitrergic neurons. In addition, it should be considered that other major species-specific differences in the cytochemical phenotype of the basal forebrain cholinergic neurons could modify their response to aging or their vulnerability to neurodegenerative processes from one species to another (Geula et al., 1993). Thus, we must be cautious when using the results obtained in rodents to infer the response to aging of this neurotransmitter system in humans or carnivores. During the last decade, the dog has acquired an increasing interest for the study of brain aging. Numerous reports suggest that the canine could be a useful model to contrast relevant aspects of the cholinergic hypothesis of human brain aging and AD (Cummings et al., 1996b; Head et al., 2000; Colle et al., 2000; Head and Torp, 2002; Araujo et al., 2005). However, the response to normal or pathological aging of the different neuronal populations in the basal forebrain of the dog is almost completely unknown and more data are necessary to adequately validate the canine model for human cognitive aging and dementia. In summary, this work represents the first study on the distribution of nNOS immunoreactive cells in the basal forebrain of the dog. Small, intensely stained cells were distributed within the caudatus, accumbens, putamen, olfactory tubercle and amygdaloidal body following a pattern that seems to be well preserved through different species. In addition, a population of mixed large and small nNOS positive cells was found in the medial septum, diagonal band and nucleus basalis overlapping the distribution of the magnocellular cholinergic system of the basal forebrain. Regarding these nuclei, our results are in agreement with the distribution of NADPH-d neurons in the cat but differ notably from those reported in rodents and primates. These differences could translate into a different response of the basal forebrain magnocellular neurons to normal or pathological aging in the carnivores. Acknowledgements This work has been financed by the Spanish Ministerio de Ciencia y Tecnologı´a and FEDER (project no. SAF2003-
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