Neurons co-localizing calretinin immunoreactivity and reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity in the hippocampus and dentate gyrus of the rat

Brain Research 744 Ž1997. 112–120

Research report

Neurons co-localizing calretinin immunoreactivity and reduced nicotinamide adenine dinucleotide phosphate diaphorase Ž NADPH-d. activity in the hippocampus and dentate gyrus of the rat M. Megıas D. Crespo ´ ) , R. Verduga, C. Fernandez-Viadero, ´ Department of Anatomy and Cell Biology, Faculty of Medicine, UniÕersity of Cantabria, 39011 Santander, Spain Accepted 27 August 1996

Abstract Co-localization of calretinin immunoreactivity and nicotinamide adenine dinucleotide phosphate diaphorase ŽNADPH-d. activity was studied in the rat hippocampus and dentate gyrus. Neurons co-expressing both markers ŽCRrNADPH-d. were observed throughout the hippocampus and dentate gyrus. However, they were more abundant in the stratum pyramidale and radiatum of CA3, stratum pyramidale of CA1, and in the juxtagranular zone of the hilus. The NADPH-d activity appeared in 37% of the calretinin immunoreactive neurons in CA3, 42% in CA1, and 36% in the dentate gyrus, whereas calretinin immunoreactivity occurred in 41% of the NADPH-d positive neurons in the hippocampus, and 16% in the dentate gyrus. The morphology and location of the double marked cells could not be used as a characteristic of the co-localizing neurons. The heavily stained NADPH-d neurons occurring mainly in CA1 do not show calretinin immunoreactivity. NADPH-d fiber swellings could be observed in close apposition to calretinin immunoreactive neurons and dendrites, suggesting synaptic contacts. It has been reported that calretinin immunoreactivity and NADPH-d activity co-localize infrequently in other areas such as the neocortex, striatum, hypothalamus and tegmental nucleus. The relatively high proportion of double marked cells found in the hippocampus and dentate gyrus could be indicative of the importance of the CRrNADPH-d interneurons in the circuitries of these areas. Keywords: Hippocampus; Calretinin; Nicotinamide adenine dinucleotide phosphate diaphorase; Nitric oxide; Co-localization

1. Introduction Co-localization studies of different neuronal markers, such as neurotransmitters, enzymes, cytosolic proteins, receptors and neuronal surface molecules, have revealed a great deal of neuronal heterogeneity, even in the same neural area, and a diversity of functions performed by these neurons w9,21x. Several neuronal markers have been studied by immunohistochemistry in the mammal hippocampus and dentate gyrus. One of the most widely distributed is the neurotransmitter GABA, which has been used to characterize a population of non-pyramidal neurons w3x. This large GABAergic population has been divided into several subpopulations depending upon the presence or ausence of immunoreactivity for some neuropeptides such as somatostatin andror neuropeptide Y ŽNPY. w15,25,28,43x, cholecystokinin ŽCCK. w26,43x, and vasoactive intestinal polypeptide ŽVIP. w26x. Furthermore, some

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GABAergic neurons may co-express the E–F hand type calcium-binding proteins, calbindin w19x, parvalbumin w27x, and calretinin w31,34x. Calretinin has been widely used as a neuronal marker w37,39,49x. The calretinin immunoreactive neurons have been well described in the hippocampus and dentate gyrus w17,31,34,35,41x, where they have been grouped into two subpopulations: one co-localizes with GABA and the other does not Žspinous neurons in the stratum lucidum of hippocampal CA3 and in the hilus of the dentate gyrus. w17x. Calretinin immunoreactivity does not coexist either with the other E–F hand type calcium-binding proteins, parvalbumin and calbindin, or with neuropeptides w30,31,36,38,41x, except VIP w38x. It has been suggested that the expression of calcium-binding proteins by cells has effects on their electrophysiological characteristics, excitotoxicity resistance or neuronal plasticity w4,7,20x. Nicotinamide adenine dinucleotide phosphate-diaphorase ŽNADPH-d. histochemical activity is another neuronal marker Žsee references in w42x. present in specific neuronal populations in the nervous system w33,47x. Re-

0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 0 7 5 - X

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cently, a great deal of attention has been given to NADPH-d activity since it has been shown to be a marker for neurons that also express nitric oxide synthase ŽNADPH-drNOS. w6,22x. Nitric oxide has been considered a novel neurotransmitter w5x. In the hippocampus and dentate gyrus NADPH-d histochemistry defines a specific subpopulation of GABAergic neurons that always express NOS ŽGABArNADPH-drNOS. w44x. Furthermore, studies in the rat w11x and mouse w8x hippocampus have demonstrated that GABArNADPH-drNOS neurons do not express immunoreactivity to either parvalbumin or somatostatin. In

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the dentate gyrus there is co-localization of GABArNADPH-drNOS and somatostatin w8,11x. The co-existence of NADPH-drNOS with GABA and somatostatinrNPY has been reported in some cortical areas w30,45x, and in the striatum w29,46,48x. Co-localization of calretinin and NADPH-drNOS has been studied in the neocortex w12,16,30x, striatum w29x, hypothalamus w2x and tegmental nuclei w10x. In all these areas there is a conspicuous segregation of these markers. The fact that the two markers do not usually co-localize could suggest that their functions may be mutually exclu-

Fig. 1. Co-localization of calretinin immunoreactivity and NADPH-d activity in the rat dentate gyrus. The brown nucleus is indicative of CR immunoreactivity. A: CRrNADPH-d neuron Žlarge arrowhead. located in the hilar juxtagranular zone. This neuron has one dendrite extending parallel to and another crossing the granular layer. A nearby NADPH-d positive neuron is present Žsmall arrowhead.. B: in the molecular layer CRrNADPH-d-neurons Žlarge arrow. are scarce, while calretinin immunoreactive Žsmall arrow. or NADPH-d positive-neurons Žarrowhead. are more abundant. C: the usual location of CRrNADPH-d neurons Žsmall arrowhead. is in the hilar juxtagranular zone, where NADPH-d positive Žlarge arrowhead. and calretinin Žarrow. neurons are also more frequent. D: appositions between NADPH-d fiber swellings Žarrows. and a calretinin immunopositive soma in the molecular layer. gl, granular layer; h, hilus, ml, molecular layer. Scale bar in A and D: 25 mm; in B and C: 30 mm.

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sive for certain cellular behaviors. The purpose of this study was to analyze the occurrence, distribution and morphological features of those neurons that co-express calretinin and NADPH-d activity in the hippocampus and dentate gyrus of the rat.

under light microscopy. The best results were obtained between 1–3 h of incubation. Since NBT turns into a visible blue product Žformazan., a set of sections were promptly removed from the developer solution to test the reaction pattern of the neurons that rapidly displayed intense reactivity. This was necessary in order to detect the product of the following immunohistochemical staining performed on the same section Žsee results.. Furthermore, as the brown 3,3X-diaminobenzidine tetrahydrochloride ŽDAB. insoluble product turns dark with histochemistry, immunocytochemistry was always performed second. This allows histochemical Žblue. and immunohistochemical Žbrown. reaction products to be readily distinguished.

2. Materials and Methods Adult Sprague–Dawley rats were obtained from Charles River ŽBarcelona.. Animals were anesthetized with 2.5% tribromoethanol and perfused transcardially with normal saline Ž0.9% NaCl., followed by a fixative solution containing 4% paraformaldehyde, 0.2% glutaraldehyde in phosphate-buffered saline ŽPBS 0.1 M pH 7.3.. The brains were removed, postfixed for 16–18 h in fresh fixative solution without glutaraldehyde at 48C, cryoprotected by immersion for 12 h in 30% sucrose in PBS, and 50 mm thick sections were obtained in a freezing microtome.

2.2. Light microscopic immunocytochemistry The peroxidase-antiperoxidase ŽPAP. method was performed after the NADPH-staining. Sections were thoroughly rinsed in PBS and then preincubated for 1 h at room temperature in a blocking solution containing 1% BSA, 0.3% Triton X-100 and 0.1% sodium azide in PBS. Sections were incubated with the primary antiserum for 18–20 h at 258C, a polyclonal antibody Ž1:3000. raised in rabitt against calretinin ŽM. Celio., washed several times in PBS, and then introduced in the secondary antiserum for 1 h at 258C Žanti-rabbit IgG 1:50.. Both primary and secondary antisera were diluted in PBS containing 1% BSA, 0.3% Triton X-100 and 0.1% sodium azide. Sections were rinsed and placed for 1 h at 258C in rabbit PAP. After several washes in PBS they were incubated in a solution of 0.005% DAB in PBS for 15 min, and then 0.005% H 2 O 2

2.1. NADPH diaphorase histochemistry Sections were rinsed in PBS for 24 h at room temperature. Then, the general process described by Valtschanoff et al. w44x was followed. Briefly, tissue sections were incubated for 10 min in phosphate buffer ŽPB 0.1 M, pH 7.3., containing 0.25% Triton X-100, followed by incubation in a developer solution of 0.5 mgrml b-NADPH and 0.2 mgrml nitro blue tetrazolium ŽNBT.. Sections were maintained in this second incubation with gentle shaking at room temperature for 10 min. After this, they were kept at 378C, and the appropriate staining time pattern was checked

Table 1 Numbers and percentages of calretinin immunoreactive-, NADPH-d positive- and CRrNADPH-d-neurons in the differents fields of the hippocampus and dentate gyrus NADPH-d positive neurons

Dentate gyrus Hilus Granular layer Molecular layer Total CA3 S. oriens S. pyramidale S. lucidumq S. radiatum S. lacunosum-moleculare Total CA1 S. oriens S. pyramidale S. radiatum S. lacunosum-moleculare Total Total

Calretinin immunorreactive neurons

CRrNADPH-d positive neurons

% of NADPH-d positive neuronsco-expresssing co-expressing calretinin

% of calretinin neurons NADPH-d activity

306 39 140 485

179 12 25 216

71 4 3 78

23 10 2 16

40 33 12 36

29 100 173 62 364

64 170 157 15 406

10 62 75 3 150

34 62 43 4 41

15 36 47 20 37

66 162 162 23 413 1262

75 197 102 24 398 1020

29 90 47 3 169 397

44 55 30 13 41 31

38 45 46 12 42 39

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was added. Once rinsed several times in PBS, sections were mounted on gelatinized slides, dehydrated and coverslipped. 2.3. SemiquantitatiÕe analysis Sections showing satisfactory histochemical and immunochemical reactions were selected. A total of ten sections from five different rats Žtwo section per animal. were employed. Hippocampal and dentate gyrus NADPH-d andror calretinin reactive neurons were counted at midseptotemporal levels.

3. Results Three types of neurons were identified: neurons positive for calretinin, those positive for NADPH-d, and those positive for both calretinin and NADPH-d ŽCRrNADPHd.. Single and double stained cells were easy to differenti-

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ate, since NADPH-d positive cells displayed a blue cytoplasm with a light non-staining cell nucleus and calretinin immunoreactive cells showed a brown staining in both the cytoplasm and cell nucleus ŽFig. 1C.. Thus, the CRrNADPH-d neurons presented a very dark stained-cytoplasm and a brown cell nucleus ŽFig. 1C and Fig. 3D.. Although the immunolabeling appeared to be throught the entire thickness of the sections, it is possible that some deep calretinin neurons could not be detected and then the number of double marked cells could be an understimation. The percentage of calretinin neurons co-localizing NADPH-d activity was 36% in the dentate gyrus, 37% in CA3, and 42% in CA1 whereas the percentage for NADPH-d neurons co-expressing calretinin immunoreactivity was only 16% in the dentate gyrus and 41% in CA1 and CA3. More detailed data are given in Table 1. The morphology of the NADPH-d positive, calretinin immunoreactive and CRrNADPH-d neurons varied considerably such that their features could not be used as a criterion for classification.

Fig. 2. CRrNADPH-d neurons in the hilus. A: a hilar CRrNADPH-d neuron Žarrowhead. is located deep in the hilus where its dendrites remain confined. B: the dendrites from neurons in the juxtagranular zone are seen to cross the granular layer. This is not exclusively related to the presence of calretinin immunoreactivity or NADPH-d activity, since CRrNADPH-d Žlarge arrowhead. and NADPH-d Žsmall arrowhead. positive neurons send out their dendrites in the same way. gl, granular layer; h, hilus; ml, molecular layer. Scale bar: 40 mm in A and 50 mm in B.

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3.1. Dentate gyrus Neurons co-localizing calretinin and NADPH-d were located mainly in the hilar juxtagranular region of the dorsal blade of the granular layer ŽFig. 1A,C.. A few were also present in deeper hilar zones ŽFig. 2A.. CRrNADPH-d neurons in the granular and molecular layers were very scarce ŽFig. 1B.. Therefore, the highest percentage of co-localizing neurons was observed in the hilar zone Žsee Table 1..

Morphologically, the hilar CRrNADPH-d neurons often had an oval to round soma with their dendrites extending parallel to or crossing the granular blade ŽFig. 1A.. Other double marked cells showed pyramid-like somata with a dendrite crossing the granular layer ŽFig. 2B.. This pyramidal type was more frequently observed in NADPH-d positive cells than in calretinin immunoreactive cells. CRrNADPH-d neurons located in deeper hilar zones had their dendrites confined to the nearby area without crossing the granular blade ŽFig. 2B.. Some spiny dendrites in

Fig. 3. CRrNADPH-d neurons in the hippocampus. A: CRrNADPH-d neurons Žlarge arrowheads. located in the stratum radiatum and lucidum of CA3. The staining of calretinin neuron cytoplasms shows different intensities Žarrows.. However, NADPH-d positive neurons can be readily distinguished Žsmall arrowhead.. B: neurons located in the CA1 stratum pyramidale. CRrNADPH-d Žsmall arrowhead. and NADPH-d positive neurons Žlarge arrowhead. can display very similar morphology. Thus, this is not a distinct characteristic of CRrNADPH-d neurons. C: CRrNADPH-d neurons Žlarge arrowhead. occur mainly in, or near, the CA1 stratum pyramidale. Small arrowheads indicate NADPH-d positive neurons and arrows show calretinin immunoreactive neurons. D: NADPH-d positive fiber swellings Žarrowheads. can be seen in apposition to CRrNADPH-d neurons located in CA1 stratum pyramidale Žpyr.. sl, stratum lacunosum; so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum. Scale bar in A and C: 80 mm, in B 30 mm and in D 25 mm.

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the hilus were positive to NADPH-d, but co-localization with calretinin immunoreactivity could not be discerned in these structures. In the molecular layer, CRrNADPH-d neurons appeared scattered and the dendrites arising from neurons in the juxtagranular zone were frequent. In this layer, the most striking finding was the observation of direct appositions of calretinin dendrites and somata with NAPDH-d positive fiber swellings ŽFig. 1D.. On the other hand, these appositions were seldom observed in the hilus. 3.2. Hippocampus CRrNADPH-d neurons occurred mainly in the pyramidal layer and stratum radiatum, both of the CA3 and CA1 hippocampal subfields ŽTable 1.. They presented variable size and morphology ŽFig. 3A,C. and their dendrites appeared to be distributed throughout the hippocampal strata ŽFig. 3C.. In the stratum lucidum of the CA3, doublelabeled neurons displayed smooth dendritic trees, and the few spiny dendrites observed were only calretinin positive. Some had their dendrites extending from the pyramidal layer across the stratum radiatum to the stratum lacunosum-moleculare. The most frequent type of CRrNADPH-d neurons in CA1 were cells with a soma located in the juxtapyramidal zone from which an apical dendrite arose. This type of morphology was not specific for doublelabeled neurons, since both calretinin immunoreactive and

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NADPH-d positive neurons showed a similar morphology ŽFig. 3B.. Between the stratum radiatum and stratum lacunosum moleculare there were numerous calretinin and NADPH-d neurons, where the markers co-localized in only a few neurons. In the transitional area of CA1 and the subiculum, very large, heavily stained NADPH-d neurons were present ŽFig. 4A., but seldom appeared in the hilus, CA3, or CA1. In the promptly removed sections from the developer solution, these neurons showed their nuclei free of staining for calretinin ŽFig. 4B.. The NADPH-d fibers were mainly concentrated in the stratum lacunosum-moleculare. However, they could also be observed in other hippocampal strata in close apposition to calretinin immunopositive somata, as described for the dentate gyrus. A portion of these cells occasionally stained with both calretinin and NADPH-d ŽFig. 3D..

4. Discussion The main result of the present study is the existence of a neuronal population in the hippocampal formation co-expressing NADPH-d activity and calretinin immunoreactivity Žsee Table 1.. The relatively hight percentaje of co-localization is in contrast with other telencephalic areas, where these markers are co-expressed infrecuently w12,16,29x. In the hippocampal formation NADPH-d activity is

Fig. 4. Large, heavily stained NADPH-d neurons in CA1. These cells are mainly located in the CA1-subiculum area. A: two NADPH-d positive neurons show golgi-like staining Žarrowheads.. B: a weak NADPH-d staining of this type of neurons leaves the nucleus visible, where no calretinin immunoreactivity is present Žlarge arrowhead.. Darken nuclei can be observed in the calretinin immunoreactive neurons Žsmalls arrowhead.. so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum. Scale bar: 50 mm in A and 75 mm in B.

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present in NOS immunoreactive neurons w44x. Hence, we may state that the CRrNADPH-d neurons reported here are CRrNADPH-drNOS neurons. CRrNADPH-drNOS neurons in the hippocampus and dentate gyrus are a neuronal type which has been infrequently reported in other encephalic areas w2,10,12,29,30x. The co-localization of NADPH-d activity with other neuronal markers varies according to the zone studied. For instance, NADPH-d co-localizes with somatostatin and NPY in the neocortex, striatum and dentate gyrus w8,11,46,48x, but does not do so in the hippocampus w8,11x. Our observations indicate that in the hippocampus there are two main types of NADPH-d neurons. A first type corresponds to those large, heavily stained NADPH-d neurons, which are GABAergic and somatostatin positive w8x Žour unpublished observations., but calretinin immunonegative. Neurons with these features are present in the neocortex and striatum w46,48x. A second group consists of those small, weakly stained neurons that show immunoreactivity for GABA w44x and occasionally for calretinin, but not for somatostatin w11x. In the dentate gyrus, most of the NADPH-drNOS neurons are GABAergic and somatostatin positive, but stain weakly for NADPH-d w11x. Calretinin neurons can also be divided into two subpopulations: one type containing GABA ŽCRrGABA. and a nonGABAergic type Žspiny neurons of the hilus and stratum lucidum. w17,31x. Our results show that in the CR neurons there is a subpopulation with NADPH-d activity. In the hippocampus, NADPH-drNOS positivity always co-localizes with GABA w44x, therefore we can rule out NADPH-d activity in the spiny calretinin non-GABAergic neurons of the stratum lucidum of the CA3. However, in the hilar region, spiny NADPH-d dendrites were observed and a possible co-localizing of calretinin and NADPH-d in the spiny hilar neurons can not be completely excluded. Another point of interest was the occurrence of appositions between NADPH-d fibers and calretinin dendrites and somata in the hippocampus and dentate gyrus. Swellings of nerve fibers in direct apposition to another element have been considered as indicative of synaptic contacts w18x. The distribution pattern of NADPH-d positive fibers does not coincide with the extrahippocampal afferent projections described previously. Calretinin extracortical afferents have been demonstrated from the medial septum-diagonal band of Broca Žwhere NADPH-d positive neurons are present and project to the hippocampus and dentate gyrus w24x. and raphe nuclei w1x. It is possible, therefore that NADPH-d positive fibers arise both from those areas projecting to the hippocampus and dentate gyrus and from hippocampal and dentate NADPH-d interneurons. The role of the NO and calcium binding proteins in neuronal physiology is not clear. It has been suggested that the presence of NOS in the neurons of the dentate gyrus co-expressing somatostatin makes these cells especially susceptible to degeneration in seizures w11x. However, this

could not be extended to the hippocampal somatostatin neurons w32x. The susceptibility to seizures of calretinin neurons is not uniform, and in ischemic models there is a greater decrease in the number of spiny versus sparsely spiny calretinin neurons in the dentate gyrus and hippocampus w13x. Therefore, a direct correlation between degeneration vulnerability and the presence of calretinin or NOS is not apparent. It might be interesting to investigate whether the delay in degeneration of calretinin neurons after kainic acid injections w13,14x is related to the presence of NADPH-drNOS. In the light of the present results relating to the hippocampus and the dentate gyrus, the heterogeneity of GABAergic interneurons has to be extended with the addition of a subpopulation containing calretinin and NADPH-d. Neurons with specific markers in the striatum, neocortex and hilar regions have been found to exhibit different electrophysiological properties w23,40x. Thus, we suggest that the CRrNADPH-d interneurons could play a distintic role in comparison to CR and NADPH-d cells in the circuitries of the hippocampus and dentate gyrus.

Acknowledgements The authors thank Prof. I. Williams for the stylistic review of the manuscript. This work has been supported by a grant from the Comision ´ Mixta CASYC–University of Cantabria.

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