A comparative cluster analysis of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry in the brains of amphibians

A Comparative Cluster Analysis of NicotinamideAdenine Dinucleotide Phosphate (NADPH)-diaphoraseHistochemistry in the Brains of Amphibians

Claudia Pinelli,1 Rakesh K. Rastogi,2 Anna Scandurra,1 Arun G. Jadhao,3 Massimo Aria,4

and Biagio D’Aniello2*1Department of Environmental, Biological, and Pharmaceutical Sciences and Technologies, Second University of Naples, 81100

Caserta, Italy2Department of Biology, University of Naples Federico II, 80126 Naples, Italy3Department of Zoology, RTM Nagpur University, Nagpur, 440033 (M.S.), India4Department of Economics and Statistical Science, University of Naples Federico II, 80126 Naples, Italy

ABSTRACTNicotinamide adenine dinucleotide phosphate–diapho-

rase (NADPH-d) is a key enzyme in the synthesis of the

gaseous neurotransmitter nitric oxide. We compare the

distribution of NADPH-d in the brain of four species of

hylid frogs. NADPH-d–positive fibers are present

throughout much of the brain, whereas stained cell

groups are distributed in well-defined regions. Whereas

most brain areas consistently show positive neurons in

all species, in some areas species-specific differences

occur. We analyzed our data and those available for

other amphibian species to build a matrix on NADPH-d

brain distribution for a multivariate analysis. Brain dis-

similarities were quantified by using the Jaccard index

in a hierarchical clustering procedure. The whole brain

dendrogram was compared with that of its main subdi-

visions by applying the Fowlkes–Mallows index for den-

drogram similarity, followed by bootstrap replications

and a permutation test. Despite the differences in the

distribution map of the NADPH-d system among spe-

cies, cluster analysis of data from the whole brain and

hindbrain faithfully reflected the evolutionary history

(framework) of amphibians. Dendrograms from the sec-

ondary prosencephalon, diencephalon, mesencephalon,

and isthmus showed some deviation from the main

scheme. Thus, the present analysis supports the major

evolutionary stability of the hindbrain. We provide evi-

dence that the NADPH-d system in main brain subdivi-

sions should be cautiously approached for comparative

purposes because specific adaptations of a single spe-

cies could occur and may affect the NADPH-d distribu-

tion pattern in a brain subdivision. The minor

differences in staining pattern of particular subdivisions

apparently do not affect the general patterns of staining

across species. J. Comp. Neurol. 000:000–000, 2014.

VC 2014 Wiley Periodicals, Inc.

INDEXING TERMS: NADPH-diaphorase; Amphibians; Nitric oxide; Brain evolution; Cluster analysis

One of the most widely distributed brain molecules is

the unconventional diffusible chemical messenger nitric

oxide (NO), which plays multiple diverse roles in both

vertebrates and invertebrates (for review, see Toda and

Ayajiki, 2006; Knott and Bossy-Wetzel, 2009). Within the

central nervous system, NO has been recognized as a

neurotransmitter and might modulate synaptic functions

(Park et al., 1998a, b; Renter�ıa and Constantine-Paton,

1999; Steinbusch et al., 2000; Prast and Philippu, 2001;

Esplugues, 2002). Furthermore, NO has been implicated

in olfaction, food and liquid intake, the modulation of

nociception, and the release of other neurotransmitters

(Ernst et al., 1999; Villani et al., 2001; Moreno et al.,

2002a, b). Moreover, this molecule has also been associ-

ated with certain neuronal disorders (Bruhwlyler et al.,

1993; Prast and Philippu, 2001; Gotti et al., 2004; Guix

et al., 2005).

NO is derived from L-arginine through a reaction that

requires nicotinamide adenine dinucleotide phosphate

(NADPH), O2, and mediation of the constitutive neuro-

nal isoform of nitric oxide synthase (nNOS). NADPH-

*CORRESPONDENCE TO: B. D’Aniello, Department of Biology, Universityof Naples Federico II, 80126 Naples, Italy. E-mail: [email protected]

Received December 13, 2013; Revised February 8, 2014;Accepted February 11, 2014.DOI 10.1002/cne.23561Published online February 18, 2014 in Wiley Online Library(wileyonlinelibrary.com)VC 2014 Wiley Periodicals, Inc.

The Journal of Comparative Neurology | Research in Systems Neuroscience 00:00–00 (2013) 1

RESEARCH ARTICLE

diaphorase (d) reduces chromogens, such as tetrazo-

lium salts, to insoluble dark formazan reaction products

in histochemical reactions (Thomas and Pears, 1961),

which allows NADPH-d histochemistry to be used to

localize this enzyme (Dawson et al., 1991; Hope et al.,

1991) in the nervous system.

Among amphibians, NADPH-d patterns have been

studied in the nervous systems of some urodeles

(Artero et al., 1995; Gonz�alez et al., 1996; Porteros

et al., 1996), a caecilian (Gonz�alez et al., 2002), and

several anurans (Br€uning and Mayer, 1996, 2001;

Mu~noz et al., 1996, 2000; Porteros et al., 1996; Pitzer

and Wirtshafter, 1997; Prasada Rao et al., 1997; L�az�ar

and Losonczy, 1999; Huynh and Boyd, 2007). Although

in some of these studies, the entire brain NADPH-d sys-

tem remains unavailable, the ongoing general observa-

tion is that such a substrate is present in all brain

subdivisions.

These data revealed that the NADPH-d brain peri-

karya pattern does not entirely overlap in any two of

the amphibian species, showing that this neuroanatomi-

cal system is variable. Indeed, comparison of phyloge-

netically related species (Pyron and Wiens, 2011), i.e.,

Rana (Pelophylax) esculenta (esculentus) and R. (P.)

perezi, considered as sibling species (Casola et al.,

2004), revealed that there are nine NADPH-d–positive

nuclei in the R. perezi brain, which were not reported in

the R. esculenta brain, and conversely, eight NADPH-d–

positive nuclei in the latter species were not present in

the brain of the former (compare Mu~noz et al., 1996

and L�az�ar and Losonczy, 1999). However, these obser-

vations are not surprising, because the brain could

respond to environmental variations through rapid

adaptive mechanisms, whereby different types of brain

organization could be observed in closely related spe-

cies within a subgroup of vertebrates, with respect to

Abbreviations

A anterior thalamic nucleusAC anterior commissureAD anterodorsal tegmental nucleusAMY amygdalaAMYl lateral amygdalaAMYm medial amygdalaAOB accessory olfactory bulbAQ aqueduct of SylviusAV anteroventral tegmental nucleusC central thalamic nucleusCB cerebellar nucleusCBL cerebellumCG central grayCGT thalamic geniculate bodyCOT commissural nucleus of the torus semicircularisDBB nucleus of the diagonal band of BrocaDCN dorsal column nucleusDG dorsal grayDHyp dorsal hypothalamusDP dorsal palliumDTh dorsal thalamusEa anterior entopeduncular nucleusFS fasciculus solitariusGR granular layer of cerebellumHB habenulaHR hindbrain reticular formationig internal granular layerIII third ventricleIN interpeduncular nucleusIR infundibular recessIV fourth ventricleIXm motor nucleus of glossopharyngeal nerveLa lateral thalamic nucleusLAM laminar nucleus of the torus semicircularisLC locus coeruleusLFB lateral forebrain bundleLP lateral palliumLpv lateral thalamic nucleus posteroventral divisionLV lateral ventricleMOB main olfactory bulbMOL molecular layer of cerebellumMP medial palliumMT magnocellular nucleus of the torus semicircularisNAC nucleus accumbensnB nucleus of BellonciNFLM nucleus of the medial longitudinal fasciculusNI nucleus isthmiNPC nucleus of the posterior commissureNPv nucleus of the periventricular organOB olfactory bulbsOC optic chiasma

ON optic nerveOT optic tectumOV optic ventricleP posterior thalamic nucleusPalC pallial commissurePC posterior commissurePD posterodorsal tegmental nucleusPOA preoptic areaPOAp posterior preoptic areaPON preoptic nucleusPONa anterior preoptic nucleusPOR preoptic recessPR principal nucleus of the torus semicircularisPRM nucleus profundus of mesencephaliPTG pretectal grayPTn pretectal nucleusPTN pretrigeminal nucleusPTRG pretoral grayPV posteroventral tegmental nucleusPVO nucleus of periventricular organR raphe nucleusSCN suprachiasmatic nucleusSl lateral septumSm medial septumSO superior oliveSol nucleus of the solitary tractSP Purkinje layer (cerebellum)STR striatumTC tectal commissureTEG tegmentumTEGd dorsal tegmentumTEGv ventral tegmentumTEL telencephalonTP posterior tubercleTR trigeminal nucleusTrVds descending trigeminal tractVds descending trigeminal nucleusVG ventral grayVHyp ventral hypothalamusVII facial nucleusVIII octaval nucleusVIIIv ventral octaval nucleusVisc nucleus visceralis secundariusVL ventrolateral thalamic nucleusVLs superficial ventral nucleusVM ventromedial thalamic nucleusVpr principal sensory trigeminal nucleusVTh ventral thalamusX motor nucleus of the vagusXII nucleus of hypoglossal nerve

C. Pinelli et al.

2 The Journal of Comparative Neurology |Research in Systems Neuroscience

different life histories (Northcutt, 2002; Kolm et al.,

2009), or even in the same species. In this regard, one

of the most common differences in brain anatomy con-

cerns sexual dimorphism (Morris et al., 2004); in some

cases, up to four different types of preoptic nucleus

morphologies have been described in the same species

(Jadhao et al., 2001). Of course this strong adaptive

brain plasticity can make brain comparisons difficult for

evolutionary purposes, because some adaptations can

originate in a species as a response to a contingent

environmental request, and thus do not reflect the real

evolutionary history.

In this study we aimed to explore whether a statisti-

cal approach could help to overcome this problem, in

conjunction with an investigation of a widely distributed

substance within the brain in order to present a more

extensive comparison of all brain areas. The wide

NADPH-d distribution in different brain areas, together

with its extensive variability, offers a valuable tool for

this purpose.

To this end we first examined the brains of four

hylids (Anura, Hylidae) to survey comparative NADPH-d

neuronal distribution. Then we utilized the existing

amphibian database to build a matrix of the presence/

absence of NADPH-d brain cell distribution for a multi-

variate statistical analysis. In this way, we obtained a

graphical representation based on brain NADPH-d dis-

similarities for a comparison with that deriving from

molecular phylogenetic studies. This analysis was per-

formed for the whole brain, as well as for its main sub-

divisions, following the current prosomeric model

(Puelles and Rubenstein, 2003). Despite some contro-

versy, the morphological studies match the molecular

data with regard to phylogenetic affinities (Wiens,

2004). Therefore, if the NADPH-d brain system has not

undergone extensive modifications due to different

selective pressures, we expected that a cluster analysis

of the presence and distribution pattern of NADPH-d in

the brain (and its main subdivisions) would agree with

the results of molecular studies. Otherwise the dendro-

grams obtained would not reflect the actual evolution-

ary affinities of amphibians.

MATERIALS AND METHODS

AnimalsThe following hylid species were studied: the Cuban

tree frog, Hyla (Osteopilus) septentrionalis, native to the

Bahamas, Cayman Islands, and Cuba, but introduced

elsewhere; the common tree frog, H. arborea, distrib-

uted in most parts of Europe; the Assam tree frog, H.

annectans, found in southwestern and central China,

northeast India, northern Myanmar, and Viet Nam; and

the green tree frog, Litoria caerulea, a widespread spe-

cies in Australia.

All samples (one female and four male H. septentrio-

nalis and H. annectans, two female and two male

H. arborea and L. caerulea) were purchased from a

commercial dealer. All animals were obtained in a non-

reproductive state and were acclimatized to the same

conditions (thermal, photoperiodic, and food) for 4

weeks, with food ad libitum. The study protocol was

performed in accordance with legislation on the care

and use of animals in research.

The animals were anesthetized through immersion in

0.03% MS-222 (tricaine methanesulfonate; Sigma, St.

Louis, MO) and intraventricularly perfused with 0.1 M

phosphate-buffered saline (PBS; pH 7.4), followed by

perfusion with 4% paraformaldehyde solution in PBS.

The entire procedure was performed in approximately

20 minutes. Subsequently, the brains were quickly

removed and immersed in the same fixative for 4 hours

at 4�C. Before freezing, the brains were cryoprotected

in 30% sucrose solution in 0.1 M PBS overnight and

subsequently placed in embedding medium (Tissue-Tek;

Sakura, Torrance, CA) for cryostat sectioning. Serial

transverse sections (20 lm) were mounted on polylysi-

nated glass slides and stored at 220�C.

NADPH-d histochemistryThe sections were rinsed in 0.1 M PBS (pH 7.4).

NADPH-d histochemistry was performed using the origi-

nal method (Scherer-Singler et al., 1983) with modifica-

tions. The sections were incubated in 100 ml PBS

containing 0.2% Triton X-100, 0.1% NADPH, reduced-

Na4 salt (Serva, Heidelberg, Germany), and 50 mg nitro

blue tetrazolium chloride (NBT; Serva) for 2 hours at

37�C. Subsequently, the sections were rinsed in PBS,

dehydrated, and coverslipped in Eukitt mounting media

(Sigma).

Microscopy and photodocumentationDigital photomicrographs were captured with a

Canon digital camera on a DMBR Leica photomicro-

scope. The images were slightly adjusted for brightness

and contrast by using Adobe Photoshop. The photomon-

tages and lettering were constructed by using Corel-

Draw software. The images were drawn with a camera

lucida.

NomenclatureFor the identification and nomenclature of various

brain areas and nuclei, we used the terminology

adopted from Nieuwenhuys et al. (1998), integrated

with several additional sources, including Frontera

(1952), Kemali and Braitenberg (1969), Northcutt and

NADPH-Diaphorase in Amphibian Brains

The Journal of Comparative Neurology | Research in Systems Neuroscience 3

Kicliter (1980), Wada et al. (1980), Neary (1990), Butler

and Hodos (1996), Mu~noz et al. (1996), Pinelli et al.

(1999), L�az�ar and Losonczy (1999), Huynh and Boyd

(2007), and Morona and Gonz�alez (2008, 2009). The

localization of various brain areas and nuclei was

framed within the newly defined territories in the telen-

cephalon (Mar�ın et al., 1998; Moreno and Gonz�alez,

2006; Endepols et al., 2006; Morona and Gonz�alez

2008, 2009) and the segmental model proposed for the

caudal prosencephalon (Puelles and Rubinstein, 2003)

adapted for anurans and urodeles.

Data analysisThe data for the presence/absence of brain NADPH-

d were organized according to a matrix (Table 1), which

was constructed by inserting a row for each of the

areas/nuclei of the brain where NADPH-d–positive cells

have been observed in at least one of the amphibian

species studied, and using data from previous studies

(Br€uning and Meyer, 1996; Gonz�alez et al., 1996, 2002;

Mu~noz et al., 1996; L�az�ar and Losonczy, 1999; Huynh

and Boyd, 2007), with the exception of R. pipiens, for

which only partial data are available (Pitzer and Wirtsh-

after, 1997).

Some NADPH-d areas described in Apoda and Cau-

data, such as the dorsal thalamic and tegmental nuclei,

comprise multiple entities in the anuran group, whereas

in other amphibian groups, these areas are organized

as single structures (Neary and Northcutt, 1983).

Furthermore, some studies did not precisely indicate

the nuclei, but rather only identified the main areas of

the brain where NADPH-d–positive neurons were

observed. To overcome these problems and to avoid

losing information, we utilized a matrix with additional

lines, specifying the areas consisting of multiple nuclei,

generically described as the “dorsal thalamus” and

“mesencephalic tegmentum.” Hence, these brain areas

are included as either a specific brain area and or an

aggregate.

Thus, 76 rows were obtained for the whole brain, of

which 40 rows corresponded to the forebrain (22 for

the secondary prosencephalon and 18 for the

diencephalon) and 36 rows to the brainstem (9 for the

mesencephalon, 3 for the isthmus, and 24 for the

hindbrain).

The presence of NADPH-d is indicated with “1” and

the absence of NADPH-d is indicated with “0.” To

explain dissimilarity between the areas/nuclei of the

brain, we used a hierarchical cluster analysis and

obtained dendrograms based on these measurements.

We were interested in comparing species by the pres-

ence of NADPH-d, because the co-absence for our goal

does not mean similarity. To this end, we chose the

Jaccard algorithm (Jaccard, 1908), also known as the

Jaccard similarity coefficient, which assigns a weight of

0 to each binary comparison showing the absence of

NADPH-d. So, not taking into account “0–0” negative

matches, the dissimilarity between two operational tax-

onomic units is not influenced by other variables not

included in the analysis (i.e., other molecules), and its

value is independent of the number of variables stud-

ied. Using these data sets, we quantified and graphi-

cally represented the level of dissimilarity of NADPH-d

brain distribution, considering the whole brain and its

main subdivisions. All data, which were analyzed by

using MATLAB (Mathworks, Union, NJ), can be freely

downloaded from the MATLAB File Exchange website

(Aria, 2014).

To compare the dendrogram obtained for the whole

brain with those originating from its main subdivisions,

we used the Fowlkes–Mallows (FM) index (1983; Aria,

2014), which measures the similarity between two par-

titions (clusters obtained after a clustering algorithm); it

can also be interpreted as the proportion of observa-

tions that are common (in the same cluster) in the two

hierarchical partitions (Meila, 2007). The FM index

range in the interval [0–1] and higher is the value

greater is the similarity between the clusters. To obtain

a more accurate estimation of the FM index, it was cal-

culated as the average value of a bootstrap resampling

procedure with 1,000 replications, so that the data

were modeled by resampling with replication of the

original sample (Efron and Tibshirani, 1993). In this

way, the estimation of the unknown parameter is

obtained as the expected value measured on the 1,000

bootstrapped samples. The statistical significance of

bootstrap estimations of the FM index was evaluated

by a permutation test (randomization test, rerandomiza-

tion test, and exact test). Because only 10 species

were available for testing, we were forced to limit our

comparison to partitions composed of two, three, and

four clusters, as further partitioning would take into

account few informative groups.

RESULTS

NADPH-d neuroanatomical distributionThe overall neuroanatomical pattern of NADPH-d in

the brains of the four hylids studied is illustrated in the

representative rostrocaudal progressive transverse sec-

tions shown in Figure 1. The NADPH-d activity was

rather diffuse, revealing the widespread distribution of

cells and fibers in all segments of the brain. The posi-

tive blue reaction was easily identified, although the

intensity, morphology, and size of the stained cell could

vary.

C. Pinelli et al.


TABLE 1.

Matrix of NADPH-d Presence (1) and Absence (0) in Amphibian Brain1

Brain area 1 2 3 4 5 6 7 8 9 10

ForebrainSecondary prosencephalon

MOB 0 1 1 0 1 1 1 0 1 1AOB 0 0 0 0 1 0 0 0 1 1DP 0 1 1 1 1 1 1 1 1 1MP 0 1 1 0 1 1 1 1 1 1LP 1 1 1 1 1 1 1 1 1 1STR 1 1 1 1 1 1 1 1 1 1Sm 1 1 1 0 1 1 1 1 1 1Sl 1 1 1 1 1 0 1 1 0 1DBB 1 1 1 0 1 1 1 0 0 0NAC 1 1 1 1 1 1 1 1 0 0AMY 1 1 1 1 1 1 1 1 1 1POA 1 1 1 1 1 1 1 0 1 1PON 1 1 1 1 1 1 1 1 1 1Ea 0 0 0 0 0 0 0 1 0 0AC 1 0 1 1 0 0 0 0 0 0POAp 0 0 0 0 0 0 0 0 1 0SCN 1 0 0 0 1 1 1 0 0 1DHyp 1 1 1 1 1 0 0 0 0 0VHyp 1 1 1 1 1 1 1 1 1 1NPv 0 0 0 0 1 0 1 0 0 0OC 0 1 0 0 0 0 0 0 0 0PVO 0 0 0 0 0 0 1 0 0 0

DiencephalonHB 0 0 0 0 1 0 0 0 0 1DTh 1 1 1 1 1 1 1 1 0 1A 0 0 0 0 1 1 0 1 0 0C 1 1 1 1 0 1 1 0 0 0P 0 0 0 0 0 0 0 1 0 0La 1 1 1 1 1 1 1 1 0 0VM 1 0 0 1 0 0 0 1 0 0VL 1 1 1 1 0 0 0 1 0 0nB 0 0 0 0 1 1 1 0 0 0VLs 0 1 1 0 1 0 0 0 0 0CGT 0 1 0 0 1 1 1 0 0 0TP 1 1 1 1 1 1 1 1 0 1Lpv 0 0 1 0 0 0 0 0 0 0PTn 0 0 0 0 0 0 0 0 0 1PTG 1 1 0 1 1 1 1 1 1 0PTRG 0 1 1 1 0 1 1 0 1 0NPC 0 0 0 0 1 0 0 0 0 0NFLM 0 0 0 0 1 1 1 0 0 0

BrainstemMesencephalon

OT 1 1 0 1 1 1 1 1 1 1LAM 1 1 1 0 1 1 1 0 0 0PR 1 1 1 1 1 1 1 1 0 0MT 1 1 1 1 1 1 1 1 0 0COT 0 0 0 0 1 1 1 0 0 0TEG 1 1 1 1 1 1 1 1 1 1AD 1 1 1 1 1 1 1 1 1 0AV 1 1 1 1 1 1 0 1 1 0PRM 1 0 0 1 1 1 1 0 0 0

IsthmusPD 1 1 1 1 1 1 0 1 1 0PV 1 1 1 1 1 1 0 1 1 0NI 0 0 0 0 1 0 0 1 1 1

HindbrainMOL 1 1 1 1 1 1 1 0 0 0P 0 0 1 0 0 0 0 1 0 0GR 1 0 1 0 1 1 1 0 0 0



In the four samples belonging to the same species,

we did not record appreciable differences in either the

distribution pattern or the neuron and fiber densities.

Secondary prosencephalonH. septentrionalisIn the rostral brain regions, the olfactory and the vom-

eronasal nerves, their encephalic targets, and the glo-

merular layers of the main and accessory olfactory

bulbs were intensely stained (Figs. 1Aa,b, 2A,B). Nota-

bly, some stained fibers of the olfactory tract were

observed running ventromedially into the medial septal

area (Fig. 1Ab,c).

The rostralmost NADPH-d–positive cell bodies were

dispersed in the diagonal band of Broca and nucleus

accumbens (Fig. 1Ad,e). The first discrete brain nucleus

of NADPH-d–positive cells was present in the lateral

septal nucleus (Fig. 3C–E). In the same sections, a few

stained cells were observed in the medial septal

nucleus, lateral pallium, and ventral striatum, and most

cells were located near the ventricle (Fig. 1Ad). At the

level of the anterior preoptic area, we observed round,

darkly stained cells intermingled with unstained cells in

the periventricular layer of the anterior preoptic nucleus

(Fig. 4D). Other stained cells were observed in the ante-

rior preoptic area (Fig. 1Ae,f), and a few were present

near the lateral forebrain bundle in the amygdala pars

lateralis, underneath the pallial commissure in the

amygdala pars medialis (Fig. 1Ae,f) and in the anterior

commissure (Fig. 1Af). In the chiasmatic region, stained

cells were present in the suprachiasmatic nucleus (Fig.

1Ag). At the level of the infundibular ventricle, many

densely packed and darkly stained NADPH-d–positive

neurons were observed in the ventral hypothalamus,

near the infundibular recess of the third ventricle (Figs.

1Ah,i, 4L). A few stained cells were also observed in

the dorsal hypothalamus.

Stained NADPH-d fibers were visible throughout the

pallium, septum, and striatum, with a dense network in

the proximity of the neurons of the septal area and

striatum (Fig. 1Ac–e). At the preoptic level, widespread

fiber staining of a different caliber typified the ventral

region of the brain, with the greatest intensity near the

lateral longitudinal fascicle region. In the chiasmatic

region, a dense network of thick fibers surrounded the

stained cells of the suprachiasmatic nucleus, a few of

which decussated through the optic chiasm (Fig. 1Ag).

A denser fiber network was observed immediately pos-

terior to the optic chiasm, at the level of the infundibu-

lar ventricle (Fig. 1Ah,i).

H. arboreaThe glomerular layers of the main and accessory olfactory

bulbs were not stained, whereas diffuse fiber staining

was observed in the medial neuropil from the accessory

olfactory bulb to the periventricular layer (Figs. 1Ba,b,

2C,D). Small stained cells were located in the internal

granular layer of the main olfactory bulb, close to the ros-

tral extent of the lateral ventricles (Fig. 1Bb).

All pallial divisions were characterized by scattered

cells (Fig. 1Bb–g) concentrated in the more posterior

TABLE 1. Continued

Brain area 1 2 3 4 5 6 7 8 9 10

CB 1 1 1 1 0 1 1 0 1 0IN 1 0 0 0 0 0 0 0 0 0Vpr 0 1 0 1 1 1 1 1 0 0Vds 1 1 1 0 1 1 1 1 0 0TR 0 0 0 0 0 0 0 0 1 0PTN 0 0 0 0 0 0 1 1 1 0VII 0 0 0 0 0 1 0 0 0 0VIII 0 1 0 0 1 1 1 0 0 0IXm 0 1 0 0 0 0 0 0 0 0X 0 0 0 0 0 0 1 0 0 0XII 0 1 0 0 0 1 0 0 0 0Sol 0 1 1 1 1 1 1 1 1 1DG 0 0 0 0 0 0 0 0 0 1VG 0 0 0 0 0 0 0 0 0 1CG 1 1 1 1 0 1 1 1 0 0SO 0 1 0 0 0 0 1 0 0 0LC 0 0 0 0 0 0 1 0 0 0DCN 0 0 0 0 1 1 1 0 0 1R 0 0 0 0 1 1 1 0 1 1Visc 0 0 0 0 0 0 0 1 0 0HR 1 1 1 1 1 1 0 1 1 1

1For brain areas, see abbreviations list. In the first line: 1, Hyla septentrionalis; 2, H. arborea; 3, H. annectans; 4, Litoria caerulea; 5, Rana perezi

(from Mu~noz et al., 1996); 6, R. esculenta (from L�az�ar and Losonczy, 1999); 7, R. catesbeiana (from Huynh and Boyd, 2007); 8, Xenopus laevis

(from Br€uning and Mayer, 1996); 9, Pleurodeles waltl (from Gonz�alez et al., 1996); 10, Dermophis mexicanus (from Gonz�alez et al., 2002).

C. Pinelli et al.


Figure 1. (Parts I and II). Distribution of NADPH-d–positive cell bodies and fibers in the brain of four anuran amphibians. A–D: Camera

lucida drawings of rostrocaudal progressive transverse sections of the brain of H. septentrionalis (A), H. arborea (B), H. annectans (C), and

L. caerulea (D) at the levels indicated by lowercase letters in the schematic dorsal view of the frog brain at the lower left. NADPH-d–

positive cell bodies are indicated as large dots, and the fibers are indicated as dashes and fine dots. The number of dots corresponds to

the densities of the NADPH-d–positive elements, as described in the Results section. Empty dots in the olfactory bulbs indicate the glo-

meruli. For abbreviations, see list. Scale bar 5 100 lm in A–D.



Figure 1. continued

C. Pinelli et al.


regions of the lateral pallium (Fig. 3A). In the lateral

and dorsal pallium, darkly stained cells were present in

the neuropil, from the periventricular layer to the exter-

nal edge. Stained cells were rarely observed in the

medial pallium, medial and lateral septum, diagonal

band of Broca, and nucleus accumbens (Fig. 1Bd,e).

The striatum was rich with positively stained cells either

dorsally or ventrally (Figs.1Bd, 3B). All striatal cells

were predominantly distributed throughout the periven-

tricular region, with a few positive cells lining the epen-

dymal layer. NADPH-d–positive cells were observed in

the amygdala pars lateralis (Fig. 1Be,f), with a few cells

present in the amygdala pars medialis, (Fig. 1Bf). In the

periventricular region, only a few faintly stained cells

were observed in the anterior preoptic nucleus and in

the preoptic area (Figs. 1Be,f, 4C). Weakly stained cells

were present behind the optic chiasm in the rostrodor-

sal and ventrocaudal lateral part of the infundibulum

(Fig. 1Bi).

Stained fibers were present in all pallial divisions and

were generally more abundant in the ventral and medial

areas, particularly in the medial and lateral septum and

the striatal region. The anterior preoptic area was char-

acterized by a dense network of thin fibers, which were

less abundant posteriorly. Many of these fibers were

observed surrounding and scattered throughout the lat-

eral forebrain bundle and decussating into the pallial

and anterior commissure. Many stained fibers were

also observed to decussate at the level of the optic

chiasm, where some stained cells were also observed

(Fig. 1Bh). Long, thick fibers from the optic chiasm

were observed radiating toward the lateral forebrain

bundle and in the lateral geniculate complex.

H. annectansIn the olfactory bulbs, whereas the glomerular layer of

the main and accessory olfactory bulbs were only par-

tially stained, intense fiber staining was observed in the

neuropil medial to the accessory olfactory bulb (Figs.

1Ca,b, 2E,F). The most rostrally located NADPH-d–

positive cell bodies were observed in the internal granu-

lar layer of the olfactory bulbs (Fig. 1Cb).

NADPH-d–positive cells were present in all pallial

subdivisions (Fig. 1Cb–f). In particular, a number of

cells were present in the dorsal and lateral pallium but

Figure 2. NADPH-d positivity in the olfactory bulbs. A,B: H. sep-

tentrionalis. Intense staining of the glomerular layer of the main

(A) and the accessory olfactory bulbs (B). C,D: H. arborea. The

glomerular layer of the main (C) and the accessory olfactory

bulbs (D) was not stained. Note diffuse staining medial to the

accessory olfactory bulb. E,F: H. annectans. The glomerular layer

of the main (E) and the accessory olfactory bulbs (F) was only

partially stained. Note the intense staining medial to the acces-

sory olfactory bulb. G,H: L. caerulea. The glomerular layer of the

main (E) and the accessory olfactory bulbs (F) was intensely

stained. Note discrete staining medial to the accessory olfactory

bulb. For abbreviations, see list. Scale bar 5 100 lm in A–H.



were rarely observed in the medial pallium. A few cells

were also observed in the medial and lateral septum

(Fig. 1Ce,f). The striatum was rich in stained cells, pre-

dominantly located in the periventricularly, with den-

drites pointing lateroventrally (Fig. 1Cd). Other

telencephalic cells were observed in the diagonal band

of Broca and in the nucleus accumbens (Fig. 1Cc,d). In

the sections at the level of the preoptic area, intensely

stained cells were present in the amygdala, under the

anterior commissure (Figs. 1Ce,f, 4B), and a few faintly

stained cells could be observed in the periventricular

region of the anterior preoptic nucleus and scattered

into the anterior preoptic area (Fig. 1Ce). In the hypo-

thalamus, behind the optic chiasm, numerous round,

darkly stained cells were present in the ventral hypo-

thalamus, in a periventricular position (Fig. 1Ci–m),

whereas only a few cells were observed in the dorsal

hypothalamus (Fig. 1Cl,m).

A dense network of thin, stained fibers was present

in all telencephalic divisions, generally more abundant

in the ventral and medial areas. Notably, an aggregate

of fibers was present in the striatal region and the

ventrolateral wall of the hemispheres, at the boundary

between the striatum and lateral pallium (Fig. 1Cc,d).

Figure 3. NADPH-d–positive elements in the telencephalon. A: NADPH-d–positive neurons (arrows) in the lateral pallium (LP) of H. arborea.

B: NADPH-d–positive neurons (arrows) in the striatum (STR) of H. arborea. C: NADPH-d–positive neurons in the lateral septum (Sl) of H.

septentrionalis. D: Enlargement of the boxed area in C, showing the NADPH-d–positive cells (arrows) of the lateral septum located dorsally.

E: Enlargement of the boxed area in C, showing the NADPH-d–positive cells (arrows) located ventrally, leaning against the ventricular wall.

Scale bar 5 100 lm in A–E.

C. Pinelli et al.

10 The Journal of Comparative Neurology | Research in Systems Neuroscience

Figure 4. NADPH-d–positive elements in the diencephalon. A: Asymmetrical NADPH-d staining of the dorsal habenular nuclei of H. arborea.

B: NADPH-d–positive neurons (arrows) in the amygdala pars lateralis (AMYl) of H. annectans. C: A solitary NADPH-d–positive neuron

(arrow) in the preoptic area (POA) of H. arborea. D: Stained NADPH-d–positive cells in the periventricular cell layer of the anterior preoptic

nucleus (PONa) intermingled with the unstained cells of H. septentrionalis. E: A dense network of NADPH-d–positive fibers (arrowheads) of

various thicknesses, decussating into the optic chiasm (OC) of L. caerulea. F: NADPH-d–positive cells and fibers at the level of the lateral

geniculate complex (CGT) of H. annectans. G: Dense terminal network of NADPH-d–positive fibers in the dorsal thalamus (DTh) of H.

annectans. H: NADPH-d–positive cells (arrows) in the anterior division of the lateral thalamic nucleus of H. arborea. I: NADPH-d–positive

cells (arrows) in the ventral part of the lateral thalamic nucleus of H. arborea. – Many round, NADPH-d–positive densely packed neurons

in the ventral hypothalamus (VHyp) of H. septentrionalis, around the infundibular recess (IR). Scale bar 5 100 lm in A–I,L.



The fibers were particularly abundant at the level of

the amygdala pars lateralis, dorsally and laterally to the

lateral forebrain bundle and in the anterior preoptic

area. A few fibers were observed decussating through-

out the pallial and anterior commissure (Fig. 1Cf). The

hypothalamus showed major fiber concentrations, with

many fibers decussating through the optic chiasm.

L. caeruleaBoth the glomerular layers of the main and accessory

olfactory bulb were intensely stained (Fig. 2G,H). Dis-

crete staining was also visible in the neuropil medial to

the accessory olfactory bulb (Fig. 2H).

Few NADPH-d–positive cells were present in the dor-

sal and lateral pallium (Fig. 1Dd). In the septum, only

the lateral region contained stained cells, with a major

concentration of cells in the ventral region and the peri-

ventricular cell layer (Fig. 1Dd). Scattered positive cells

were also located in both the dorsal and ventral stria-

tum, predominantly located periventricularly, and a few

cells were located in the nucleus accumbens (Fig. 1Dd).

At the level of the preoptic area, some cells were

observed in the amygdala, concentrated in its pars lat-

eralis (Fig. 1De,f). Some cells were present in the ante-

rior commissure and the anterior preoptic area. Faintly

stained cells characterized the periventricular layer of

the preoptic nucleus around the rostral preoptic recess

(Fig. 1Df,g). In the infundibular nuclei, a few cells were

present both dorsally and ventrally (Fig. 1Di,l).

The pallium, septum, and striatum were characterized

by a diffuse network of weakly stained fibers, not well

discriminated in the pallial division, but more abundant

in the ventral and medial areas, with a major concentra-

tion in the ventrolateral wall of the hemispheres at the

boundary between the striatum and lateral pallium (Fig.

1Dd,e). The anterior preoptic area was characterized by

a dense network of thin fibers, particularly abundant

ventrally in the neuropil around the preoptic recess.

Thicker fibers were observed decussating into the optic

chiasm (Fig. 4E).

DiencephalonH. septentrionalisIn the thalamus, darkly stained NADPH-d–positive cells

were scattered throughout the ventromedial and ventro-

lateral thalamic nuclei, and many neurons were present

in the central and lateral thalamic nuclei (Fig. 1Ag,h).

NADPH-d–positive cells were also observed in the pos-

terior tubercle, and a few scattered cells were present

in the pretectal gray matter (Fig. 1Ai).

H. arboreaThe diencephalon showed diffuse positivity. Weak asym-

metrical staining was observed only in the dorsal habe-

nular nuclei (Fig. 4A). Stained cells were present in the

lateral geniculate complex (Fig. 1Bg) and in the central,

lateral, and ventrolateral nuclei of the thalamus (Figs.

1Bh,i, 4H). In the ventral region of the lateral thalamic

nucleus, the cells had a pyramidal body and conspicu-

ous vertically oriented dendrites (Fig. 4I). Some NADPH-

d–positive cells were also observed in the superficial

ventral nucleus (Fig. 1Bi) and in the posterior tubercle

(Fig. 1Bl). Distinct intensely stained cells were spread

throughout the pretectal and pretoral gray (Fig. 1Bl).

The entire diencephalon contained a densely stained

neuropil (Fig. 1Bh,i). The dorsal and central thalamic

neuropil were crowded with tiny stained fibers,

arranged in parallel bands, whereas fibers of various

thicknesses and lengths were observed in the lateral

and ventral regions. Furthermore, a concentration of

stained fibers was present at the level of the eminentia

thalami and the olfactohabenularis anterior tract.

H. annectansIn the thalamus, a few stained cells appeared in the lat-

eral and central nuclei and the superficial ventral

nucleus. However, the major cellular density was seen in

the thalamic ventrolateral nucleus and in the lateral tha-

lamic nucleus posteroventral division (Fig. 1Cg,h).

Stained cells were also observed in the posterior tuber-

cle and into the pretoral gray. The diencephalon showed

a weak fiber staining into the dorsal habenular nuclei,

decussating into the habenular commissure. The entire

thalamus was characterized by a dense and diffuse ter-

minal network of thin, stained fibers. In the central tha-

lamic neuropil, the fibers were arranged in parallel bands

(Fig. 4G), whereas in the lateral and ventral regions,

fibers of various thicknesses and lengths were observed.

Furthermore, a conspicuous aggregate of stained fibers

was observed above the lateral forebrain bundle in the

lateral geniculate complex (Figs. 1Ch, 4F).

L. caeruleaIn the thalamus, NADPH-d activity was observed in the

central, lateral, and ventral nuclei of the thalamus (Fig.

1Dh). More caudally, a few NADPH-d–positive cells

were observed in the posterior tubercle and fusiform,

and moderately stained cells spread into the pretectal

and pretoral gray (Fig. 1Di). The habenular nuclei were

weakly and asymmetrically stained; some stained fibers

were observed in the habenular commissure. Most tha-

lamic subdivisions contained thin, diffuse NADPH-d–

positive fibers, with the densest distribution in the ven-

tral portion. In the anterior and ventromedial thalamic

nuclei, the fibers were typically arranged in parallel

bands. Thicker fibers were observed in the more lateral

and ventral portions of the neuropil. Numerous fibers

C. Pinelli et al.


were observed crossing into the posterior and tectal

commissure.

MesencephalonH. septentrionalisNADPH-d–positive cells were rarely observed in the

optic tectum located in the more external layer, and a

few weakly stained fibers were arranged into different

layers (Fig. 5A). Numerous NADPH-d–positive cells were

present either dorsally or ventrally in the anterior and

posterior tegmentum (Fig. 1Ai–n). Moderately stained

cells, located below the optic lobes, were observed in

the torus semicircularis in the laminar nucleus, the

nucleus principalis of the torus semicircularis, and the

Figure 5. NADPH-d–positive elements in the transverse sections of the hylid optic tectum. The ependyma is shown at the bottom. A: H. sep-

tentrionalis. Weakly stained fibers were arranged into the different layers (arrowheads). B: H. arborea. Several NADPH-d–positive cells (arrows)

were preferentially distributed throughout layers 4 and 6. C: H. annectans. Conspicuous aggregates of stained fibers were ordinately arranged

throughout layers 3, 5, 7, and 9; a few fibers were also present in the superficial neuropil. D: L. caerulea. A few weakly stained cells (arrows)

were predominantly located in layers 4 and 6, and the fibers were ordinately arranged in all layers. Scale bar 5 100 lm in A–D.



anterodorsal and anteroventral tegmental nuclei, and

darkly stained cells characterized the toral magnocellu-

lar nucleus (Fig. 1Ai,l). Rare well-stained cells were also

observed in the nucleus profundus mesencephali (Fig.

1Al). NADPH-d–stained fibers were abundant through-

out the entire tegmentum.

H. arboreaIn the optic tectum, several NADPH-d–positive cells were

preferentially distributed in layers 4 and 6 (Figs. 1Bm, 5B)

and alternated with stained fibers arranged predominantly

in layers 3, 5, and 7 (Fig. 5B). Many stained cells were

observed in the anterodorsal (Fig. 6C) and anteroventral

(Fig. 6E,F) tegmental nuclei and in the nucleus principalis,

magnocellularis (Fig. 6C,D) and laminaris of the torus

semicircularis (Fig. 1Bm,n). Moderately stained cells were

observed in the anterior portion of the anteroventral teg-

mental nucleus and the medial portion of the anterodorsal

tegmental nucleus (Fig. 6C) compared with the other

regions. The cell size and typology of these mesencephalic

nuclei were extremely variable. Small or medium-sized

cells were predominantly observed in the nucleus lamina-

ris of the torus semicircularis, medial region of the antero-

dorsal tegmental nucleus (Fig. 6C) and anteroventral

tegmental nuclei. In contrast, large multipolar cells charac-

terized the nucleus magnocellularis (Fig. 6C,D) and princi-

palis of the torus semicircularis, the lateral part of the

anterodorsal tegmental nucleus (Fig. 6C). Stained fibers

were particularly abundant, extending throughout almost

all subdivisions in the mesencephalon. A dense and dif-

fuse network of thin fibers was present in the neuropil

among the stained cell groups and near the ventricle, and

thick, intensely stained bundles were observed laterally

and ventrally. Some fibers were observed crossing the

dorsal tectal and posterior commissure.

H. annectansConspicuous aggregates of stained fibers were ordi-

nately arranged in the optic tectum, predominantly in

layers 3, 5, 7, and 9 (Fig. 5C). Abundant positive cells

were present in the nucleus principalis of the torus

semicircularis and the nucleus magnocellularis (Fig.

1Cm). Fewer cells were present in the nucleus laminaris

of the torus semicircularis, in the anterodorsal and

anteroventral tegmental nuclei (Fig. 1Cm–o). Almost all

subdivisions of the mesencephalon were characterized

by the presence of a dense background of thin, stained

fibers. As in the diencephalon, in the central body of

the tegmentum, the fibers were arranged in parallel

bands, whereas thicker and longer fibers were observed

in the lateral and ventral areas. Many of these fibers

also decussated into the tectal and posterior commis-

sure (Fig. 6A).

L. caeruleaThe optic tectum had some weakly stained cells, pre-

dominantly located in layers 4 and 6, and fibers were

present in all layers (Figs. 1Dl, 5D). An intense enzy-

matic reaction was observed in the principal and mag-

nocellular nuclei of the torus semicircularis and in the

anterodorsal tegmental nucleus. Moderate staining was

revealed in the anteroventral tegmental nucleus (Fig.

6B) and nucleus profundus. Stained fibers were particu-

larly abundant in the mesencephalon, and they com-

pletely filled in almost all subdivisions with a dense and

diffuse network of thin fibers, whereas thicker fibers

were observed near the stained cells and in the lateral

and ventral neuropil.

IsthmusH. septentrionalisStained cells were observed medially and ventrally in

the posterodorsal and posteroventral tegmental nuclei,

extending caudally and disposed lateroventrally to the

fourth ventricle (Fig. 1Am,n). A dense plexus of fibers

emerged from these nuclei, which encircled and pene-

trated the isthmic nucleus. Other stained cells were

observed in the interpeduncular nucleus (Fig. 1Am).

H. arboreaMany stained cells were observed in posterodorsal (Fig.

6E,F) and posteroventral tegmental nuclei. Small or

medium-sized cells were predominantly observed in the

posteroventral tegmental nuclei. In contrast, large multi-

polar cells characterized the posterodorsal tegmental

nucleus (Fig. 6E). The stained cells were particularly

abundant, were intensely stained, and occurred medial

and ventral to the isthmic nucleus, with most dendrites

pointing ventrally and ventrolaterally toward the reticu-

lar formation (Figs. 1Bn, 6E,F). The isthmic nucleus

itself was not stained; however, because a few thick

axons originating from the posterodorsal tegmental cell

group traversed the body, this region appeared to be

striped, with intense enzymatic activity in the surround-

ing cells and fibers (Fig. 6E).

H. annectansWhereas fewer cells were present in the posteroventral

tegmental nuclei, a conspicuous group of large and

darkly stained NADPH-d–positive cells was present in

the posterodorsal tegmental nucleus (Fig. 1Cl–o),

located medial and ventral to the isthmic nucleus and

crossed by thick axons.

L. caeruleaWhereas weak staining characterized the posteroventral

tegmental nucleus (Fig. 1Dl–n), the most conspicuous

C. Pinelli et al.


Figure 6. NADPH-d–positive elements in the mesencephalon. A: H. annectans. Many NADPH-d–positive fibers (arrowheads) decussating

into the tectal (TC) and posterior commissure (PC) of the optic tectum (OT). B: L. caerulea. Many NADPH-d–positive neurons and fibers at

the level of the anteroventral tegmental nucleus (AV). C: H. arborea. Many NADPH-d–positive neurons (white arrows) and fibers in the

anterodorsal tegmental nucleus (AD) and in the magnocellular nucleus of the torus semicircularis (MT). D: H. arborea. Enlargement of the

boxed area in C, showing NADPH-d–positive cells (arrows) and fibers in the magnocellular nucleus of the torus semicircularis (MT). E: H.

arborea. A conspicuous group of darkly stained NADPH-d–positive cells in the posterodorsal tegmental nucleus (PD) located medially and

ventrally to the isthmic nucleus (NI), crossed by thick axons originating from the adjacent cell group. F: H. arborea. A dense network of

NADPH-d–positive fibers in the ventral isthmic region. G: H. arborea. Thick and darkly stained NADPH-d–positive fibers (arrowheads) below

the cerebellum. H: H. annectans. A thick stained NADPH-d–positive fiber (arrowheads) in the granular layer of the cerebellum (GR). Scale

bar 5 100 lm in A–H.



group of large and strongly stained cells was observed

in the posterodorsal tegmental nucleus, located medial

to the isthmic nucleus (Fig. 1Dn). Most of the dendrites

originating from these cells pointed ventrally and ven-

trolaterally in the reticular formation, and only a few

cells were directed dorsally to the isthmic nucleus.

HindbrainH. septentrionalisIn the cerebellum, faintly stained cells were observed in

both the granular and molecular layer, whereas Purkinje

cells were not stained. Thick, intensely stained fibers

were observed running lateral and medial to the region

above the fourth ventricle in the granular and Purkinje

cell layers (Figs. 1Ao, 7A). In the basal rhombence-

phaon, NADPH-d activity was present in weakly stained

cells of the cerebellar nucleus (Fig. 1Ao), in a few cells

of the descending trigeminal nucleus and the central

gray and hindbrain reticular formation (Fig. 1Ap,q).

H. arboreaIn the cerebellum, many darkly stained cells of the

molecular layer were NADPH-d–positive (Figs. 1Bo, 7B).

Thick, darkly stained fibers were observed in the granular

and Purkinje cell layers, whereas cell staining was rarely

observed in the molecular layer (Figs. 6G, 7B). In the

caudal brainstem, many NADPH-d–labeled cells of differ-

ent morphologies and staining intensities were observed

in the cerebellar nucleus, sensory and descending tri-

geminal nucleus, motor nucleus of the glossopharyngeal

nerve, ventral octaval nucleus, oliva superioris, motor

nucleus of hypoglossal nerve, and nucleus of the solitary

tract. Moreover, scattered positively stained cells were

present ventromedially throughout the central gray

region and in the reticular formation (Figs. 1Bo–q, 8A,B).

All NADPH-d–positive cells were associated with an intri-

cate network of stained fibers that could easily be traced

for long distances. Whereas almost the entire neuropil

was densely innervated with thin fibers, in the reticular

formation there was a profusion of thicker fibers running

medially and laterally, and these fibers frequently decus-

sated at different levels.

H. annectansIn the cerebellum, intensely stained cell bodies were

present in the granular layers, whereas in the molecular

and Purkinje cell layers the cells were faintly stained

(Figs. 1Cp,q, 7C). Thick, darkly stained fibers were

observed among granular (Fig. 6H) and Purkinje cells,

whereas these cells were rarely observed in the molec-

ular layer (Fig. 7C). In the basal rhombencephalon,

weakly NADPH-d–positive cells were revealed in the

cerebellar nucleus (Fig. 1Cq), whereas well-stained cells

were present in the descending trigeminal nucleus,

scattered in the central gray, in the reticular formation,

and ventrolateral to the solitary tract (Fig. 1Cr,s). Posi-

tive thin NADPH-d fibers were scattered throughout all

brainstem divisions, whereas thick and abundant fibers

appeared ventromedially and laterally (Fig. 8C).

L. caeruleaIn the cerebellar division, faintly NADPH-d–positive cells

were present in the molecular layer, whereas thick, darkly

stained fibers were observed in the molecular and Pur-

kinje cell layers (Figs. 1Do, 7D). In the basal rhombence-

phalon, round, weakly stained cells were seen in the

central gray, principal sensory trigeminal nucleus, with

some cells scattered in the reticular formation and many

well-stained cells surrounding the solitary tract (Figs.

1Do–q, 8D). Positive thin NADPH-d fibers characterized

most of the rhombencephalic neuropil, whereas thick

fibers were more abundant ventromedially and laterally.

Cluster analysisIn terms of the distribution of NADPH-d in the brain of

all amphibians studied so far, the degree of dissimilarity

between different samples, calculated by using MATLAB

software, is shown in Figure 9. The results obtained from

the statistical analysis of the matrix associated with the

whole brain (Fig. 9A) revealed dissimilarity ranging

between 26% and 64%. The lowest percentage of dissimi-

larity occurred between R. perezi and R. esculenta, which

in turn had 30% dissimilarity with R. catesbeiana as an

outgroup. A 27% dissimilarity was observed between the

cluster of H. septentrionalis and L. caerulea and also

between H. arborea and H. annectans. The highest per-

centage of dissimilarity between Hylidae was 34%. Xeno-

pus laevis, with 49% dissimilarity, is represented as an

outgroup of Ranidae and Hylidae, which are sister groups

(Neobatrachia). The cluster comprising the newt Pleuro-

deles waltl and the Mexican burrowing caecilian Dermo-

phis mexicanus, with a 53% dissimilarity, differed from

the anuran group by 64%.

Secondary prosencephalon (Fig. 9B)Compared with the results obtained using whole brain

data, the secondary prosencephalon alone showed a

high degree of dissimilarity ranging between 6% and

44%. The lowest degree of brain dissimilarity was

observed between H. arborea and H. annectans. R. cat-

esbeiana and R. perezi had a 17% dissimilarity, with R.

esculenta as an outgroup (22% dissimilarity). The ranids

differed by 25% from the cluster H. arborea and H.

annectans. P. waltl and D. mexicanus, with 22% dissimi-

larity between them, had a 35% dissimilarity from all

the species listed above. The outgroup of all these

C. Pinelli et al.


species was X. laevis (42% dissimilarity). Finally, H. sep-

tentrionalis and L. caerulea, which together clustered

with 28% brain dissimilarity, appeared to be outside all

the other samples studied, with 44% dissimilarity.

Comparison of the dendrogram of the secondary

prosencephalon with that of the whole brain by the FM

index gave low values of similarity, and the permutation

test showed no significant values (Table 2).

Diencephalon (Fig. 9C)Analysis of the diencephalon revealed a large range of

dissimilarity, between 10% and 85%. The closest pattern

Figure 7. Transverse sections of the hylid cerebellum. A: H. septentrionalis. Thick, intensely stained NADPH-d–positive fibers were observed

in the granular cell layer (GR) (thick black arrow) and in the Purkinje cell layer (SP) (thick white arrow). The insets show faintly stained cells

(arrows) in the molecular cell layer (MOL) and GR. Note that the cells of the SP were not stained. B: H. arborea. Thick, darkly NADPH-d–

stained fibers were observed in the GR (thick black arrow) and SP (thick white arrow), up to the fourth ventricle. The inset shows many

darkly stained NADPH-d–positive cells (arrows) in the MOL. C: H. annectans. Thick, darkly NADPH-d–stained fibers were observed in the GR

(thick black arrow) and SP (thick white arrow). The inset shows well-stained NADPH-d cell bodies (white arrows head) in the GR and faintly

stained cells in the MOL (black arrowheads) and SP (arrows). D: L. caerulea. Thick, darkly NADPH-d–stained fibers were observed in the GR

and SP. The inset shows faintly NADPH-d–positive cells (arrows) in the MOL. Scale bar 5 50 lm in D (applies to A–D).



was for the cluster of R. esculenta and R. catesbeiana,

followed by H. septentrionalis and L. caerulea, with

13%, and H. arborea and H. annectans, with 30%. R.

perezi was a sister species of the other two ranids,

with 43% dissimilarity. X. laevis was situated in

between hylids as a sister species of H. septentrionalis

and L. caerulea, with 37% dissimilarity, and 48% with

the cluster of H. arborea and H. annectans. The hylids

(including X. laevis) differed from ranids by 58%. D.

mexicanus was represented in the dendrogram as an

outgroup of Anura, with 80% dissimilarity, and P. waltl

had 85% dissimilarity as outgroup of all the other spe-

cies listed.

Comparison of the dendrogram of the diencephalon

with that of the whole brain by the FM index gave high

similarity values, and the permutation test showed

strongly significant values (Table 2).

Mesencephalon (Fig. 9D)The percentage of mesencephalon dissimilarity ranged

between 0% and 64%. The cluster of R. perezi and R.

esculenta showed no dissimilarity, having as an out-

group R. catesbeiana, with 12% dissimilarity. H. arborea

and H. septentrionalis clustered with 13% dissimilarity.

Another cluster was L. caerulea and X. laevis, with 15%

dissimilarity, which in turn had an 18% dissimilarity with

the cluster of H. arborea and H. septentrionalis. These

four species had a 25% dissimilarity with ranids. H.

annectans appeared as an external species of anurans,

with 31% dissimilarity. P. waltl and D. mexicanus clus-

tered with 50% dissimilarity and were an external group

of anurans with 64% dissimilarity.

Comparison of the dendrogram of the mesencepha-

lon with that of the whole brain by the FM index gave

high values of similarity, and the permutation test

showed moderately significant values for partitions with

two and three clusters, but no significant values for

four clusters (Table 2).

Isthmus (Fig. 9E)The dendrogram obtained from the isthmus was totally

anomalous. Comparison with that of the whole brain by

the FM index gave low values of similarity, and the per-

mutation test showed no significant values (Table 2).

Hindbrain (Fig. 9F)The hindbrain showed a dissimilarity ranging between

31% and 77%. R. perezi and R. esculenta clustered with

the lower dissimilarity, with R. catesbeiana (dissimilarity

44%) as a sister species. H. septentrionalis and

H. annectans clustered with 33% dissimilarity, and

H. arborea and L. caerulea with 46%. These last two

clusters differed by 54%. Ranids and hylids had 58%

dissimilarity. X. laevis, with 66% dissimilarity, is repre-

sented as an outgroup of Neobatrachia. The cluster

comprising the newt P. waltl and D. mexicanus, with

Figure 8. NADPH-d–positive elements in the rhombencephalon. A:

H. arborea. Corresponds to a level similar to “p” in Figure 1. The

inset is an enlargement of the boxed area. CG, central gray; HR,

reticular formation; IV, fourth ventricle; IXm, motor nucleus of glos-

sopharyngeal nerve; SO, oliva superioris; Vds, decending trigeminal

nucleus; VIIIv, ventral octaval nucleus. B: H. arborea. Positive cells

(arrows) at the level of the motor nucleus of the hypoglossal nerve

(XII). C: H. annectans. Positive fibers in the hindbrain reticular for-

mation (HR). Note the decussation of thick axons. D: L. caerulea.

NADPH-d–positive cells (arrows) in the nucleus of the solitary tract

(Sol) and fibers in the descending trigeminal tract (TrVds). Scale

bar 5 100 lm in A–D; 50 lm inset of A.

C. Pinelli et al.


66% dissimilarity, differed from the anuran group by

77%.

Comparison of the dendrogram of the hindbrain with

that of the whole brain by the FM index gave high val-

ues of similarity, and the permutation test showed mod-

erately significant values for partitions with two and

three clusters and strongly significant values for four

clusters (Table 2).

DISCUSSION

The histochemical method used in this work facili-

tated the identification of several areas of the brain

where NADPH-d is expressed. In general, NADPH-d was

widely distributed throughout the brain subdivisions.

Whereas positive neurons were found in most brain

areas or discrete nuclei, the fibers were more or less

densely scattered throughout the brain.

Figure 9. – Dissimilarities in NADPH-d distribution in amphibian brains. Dendrograms showing the dissimilarity degree in the NADPH-d dis-

tribution of the whole amphibian brain and its main subdivisions obtained by using the Jaccard index in a hierarchical clustering procedure.

1, Hyla septentrionalis; 2, H. arborea; 3, H. annectans; 4, Litoria caerulea; 5, Rana perezi; 6, R. esculenta; 7, R. catesbeiana; 8, Xenopus lae-

vis; 9, Pleurodeles waltl; 10, Dermophis mexicanus.



A clear-cut comparative analysis of the present

results and those reported in other amphibian species

is complicated by the fact that in several studies, the

authors used a slightly different nomenclature for the

same brain areas or provided general indications.

Comparative analysisSecondary prosencephalonIn the frog species studied to date, a certain degree of

variability in the distribution of NADPH-d in the olfac-

tory bulbs has emerged. Our data showed that positive

neurons are present in the main olfactory bulb of H.

arborea and H. annectans, a feature shared with a uro-

dele and an apodan amphibian (Gonz�alez et al., 1996,

2002) and other species of anurans (Mu~noz et al.,

1996; L�az�ar and Losonczy, 1999; Huynh and Boyd,

2007), except for R. pipiens (Pitzer and Wirtshafter,

1997) and X. laevis (Br€uning and Mayer, 1996). Con-

versely, positive neurons in the accessory olfactory bulb

were not observed in any of the hylid species studied,

but these cells were reported in R. pipiens, P. waltl, and

D. mexicanus (Mu~noz et al., 1996; Pitzer and Wirtsh-

after, 1997; Gonz�alez et al., 1996, 2002).

A considerable variability in NADPH-d reactivity in

olfactory components was also observed in the fibers,

for which NADPH-d positivity was detected in almost all

the anuran species studied and in P. waltl (Gonz�alez

et al., 1996). However, in some frog species, such as

H. arborea, both the main and accessory olfactory bulbs

were completely negative; in other cases, such as Pipa

sp., only the part of the olfactory bulb pertaining to the

lateral diverticulum was very positive, and a few fibers

were described in the main olfactory bulb associated

with the medial diverticulum, whereas no fibers were

observed in the accessory olfactory bulb (Meyer et al.,

1997). Olfactory NADPH-d–positive fibers were not

reported in the main and accessory olfactory bulbs in

D. mexicanus (Gonz�alez et al., 2002).

In all species studied, other areas of the secondary

prosencephalon, such as the lateral pallium, the stria-

tum, and the amygdala, showed consistently positive

NADPH-d neuronal expression, and a high percentage

of homology was observed in other areas, such as the

dorsal and medial pallium, the medial and lateral sep-

tum, the diagonal band of Broca, the nucleus accum-

bens, the preoptic area, and the preoptic nucleus.

Occasionally, some species showed unique features,

as in the case of NADPH-d neurons of the entopedun-

cular nucleus, described only in X. laevis, R. esculenta,

and R. pipiens (Br€uning and Mayer, 1996; Pitzer and

Wirtshafter, 1997; L�az�ar and Losonczy, 1999). Interest-

ingly, NADPH-d neurons in the anterior commissure of

the hylids (except for H. arborea) were not observed in

any other species of amphibian studied so far. Another

peculiarity is the presence of NADPH-d neurons in the

olfactory tubercle of R. pipiens (Pitzer and Wirtshafter,

1997). However, this brain nucleus is quite controver-

sial: Hoffmann (1973) and Kicliter and Ebbesson (1976)

included this region in the diagonal band of Broca,

whereas R€othig (1912), Scalia et al. (1968), and North-

cutt and Royce (1975) considered this region as part of

the nucleus of the medial septum.

All amphibian species studied so far were reported

to contain NADPH-d–positive areas in the ventral hypo-

thalamus, whereas NADPH-d positivity in the dorsal

TABLE 2.

Statistical Significance of Bootstrap Estimations of the Fowlkes–Mallows (FM) Index Evaluated by a Permutation Test1

Brain area

Nbs. of

clusters Mean SE CI 95% P value

Secondary prosencephalon 2 0.6159 0.1483 0.4377–0.7710 0.3843 0.6055 0.1484 0.4252–0.7461 0.2164 0.5987 0.1710 0.3819–0.7770 0.076

Diencephalon 2 0.9010 0.1455 0.5690–0.9826 <0.0013

3 0.9308 0.1278 0.6142–1.0000 0.0023

4 0.7963 0.1240 0.4910–0.8660 <0.0013

Mesencephalon 2 0.8238 0.1526 0.5690–0.9826 0.0162

3 0.8329 0.1649 0.5791–1.0000 0.0142

4 0.5234 0.1370 0.3629–0.7235 0.128Isthmus 2 0.5493 0.0697 0.4377–0.6339 0.736

3 0.5289 0.0853 0.4009–0.6447 0.3454 0.4525 0.2017 0.3086–0.6979 0.234

Hindbrain 2 0.8412 0.1646 0.5639–0.9826 0.0302

3 0.8782 0.1680 0.5252–1.0000 0.0262

4 0.8482 0.1912 0.5819–0.9770 0.0103

1P values were obtained by permutation test with 1,000 replications. CI, confidence interval; SE, standard error.2Moderately significant, 0.01 < P � 0.05.3Strongly significant, P � 0.01.

C. Pinelli et al.


hypothalamus was a more specific feature of hylids and

R. perezi (Mu~noz et al., 1996). Although the dorsal

hypothalamus was not explicitly mentioned by Mu~noz

et al. (1996), we were able to detect this region by

using their proposed schemes.

Some particular features are the presence of NADPH-

d–positive neurons in the periventricular organ of R. cat-

esbeiana (Huynh and Boyd, 2007) and R. perezi (Mu~noz

et al., 1996), and in the optic chiasm of H. arborea.

DiencephalonAll anuran species studied so far have been reported to

contain NADPH-d–positive perikarya in the posterior

tubercle and the lateral thalamic nucleus. The posterior

tubercle was also positive in D. mexicanus (Gonz�alez

et al., 2002). Some areas of the diencephalon were

more specific features of hylids, such as the NADPH-d

neuronal distribution in the ventrolateral thalamic

nucleus. Furthermore, the central thalamic nucleus was

NADPH-d–positive in R. esculenta (L�az�ar and Losonczy,

1999), and R. catesbeiana (Huynh and Boyd, 2007) and

in hylids. In any case, NADPH-d–positive cells were

detected in these areas and also in non-anuran amphib-

ians. Conversely, diencephalic areas, such as the genic-

ulate body (except for H. arborea) and the thalamic

nucleus of Bellonci, were also common features of Rani-

dae, which were not shared by Hylidae.

Some particular features are the presence of

NADPH-d–positive neurons in the posteroventral subdi-

vision of the lateral thalamic nucleus of H. annectans

and R. perezi (Mu~noz et al., 1996) and in the habenulae

of R. perezi (Mu~noz et al., 1996). This latter feature was

shared with D. mexicanus (Gonz�alez et al., 2002).

The NADPH-d neurons in the fasciculus longitudinalis

medialis and the tectal commissure were only reported

in ranids (Mu~noz et al., 1996; L�az�ar and Losonczy,

1999; Huynh and Boyd, 2007) as a characteristic of the

family.

Notably, the presence of NADPH-d in the nucleus of the

posterior commissure of R. perezi (Mu~noz et al., 1996)

and the pretectal nucleus of D. mexicanus (Gonz�alez et al.,

2002) was not reported in any other species studied.

MesencephalonThe presence of NADPH-d positivity in the midbrain

showed homogeneity in the anterodorsal and anteroven-

tral tegmental nuclei; with the exception of R. catesbei-

ana (Huynh and Boyd, 2007) and D. mexicanus

(Gonz�alez et al., 2002), this feature has been noted in

almost all amphibians. Another area of the brain in

which NADPH-d–positive neurons were largely spread

among different amphibian subgroups was the optic

lobe, except for H. annectans. The areas widely shared

among anurans include the laminar, principal, and mag-

nocellular nuclei of the torus semicircularis. A feature

present only in ranids was the presence of NADPH-d–

positive neurons in the commissural nucleus of the

torus semicircularis.

IsthmusThe presence of NADPH-d positivity in the isthmus

showed homogeneity in the posterodorsal and postero-

ventral nuclei, with the exception of R. catesbeiana

(Huynh and Boyd, 2007) and D. mexicanus (Gonz�alez

et al., 2002). The nucleus isthmi was negative for

NADPH-d in all four hylid species, whereas positivity

was reported in this brain area in R. perezi (Mu~noz

et al., 1996) and X. laevis (Br€uning and Mayer, 1996),

as well as in both non-anuran amphibians.

HindbrainThe hindbrain showed the greatest variability: no con-

sistently NADPH-d–positive area was observed for any

of the amphibians studied thus far. A high percentage

of similarity in amphibians was recorded in the nucleus

of the solitary tract, with the exception of H. septentrio-

nalis, and in the reticular formation, except for R. cates-

beiana (Huynh and Boyd, 2007). Other constantly

positive, highly shared nuclei included the descending

trigeminal nucleus, with the exception of L. caerulea, R.

catesbeiana (Huynh and Boyd, 2007), and D. mexicanus

(Gonz�alez et al., 2002), and the central gray, except for

R. perezi (Mu~noz et al., 1996), D. mexicanus (Gonz�alez

et al., 2002), and P. waltl (Gonz�alez et al., 1996).

The unique features of the Ranidae among anurans

included NADPH-d activity in the dorsal column nucleus

and the raphe nucleus. The raphe nucleus was also

positive in P. waltl (Gonz�alez et al., 1996) and D. mexi-

canus (Gonz�alez et al., 2002).

Many peculiarities were revealed for the hindbrain,

including at least four areas that were specific charac-

teristics of the NADPH-d neuronal distribution of single

species. Among anurans, the nucleus of the nervi facia-

lis was noted to contain NADPH-d–positive neurons

only in R. esculenta (L�az�ar and Losonczy, 1999), the

nucleus visceralis secundarius only in X. laevis (Br€uning

and Mayer, 1996), the dorsal and ventral gray only in

D. mexicanus (Gonz�alez et al., 2002), and the interpe-

duncular nucleus only in H. septentrionalis.

Cluster analysisIn the present study, we used a statistical approach

to quantify the degree of dissimilarities in brain NADPH-

d distribution among different species, taking into

account both the entire brain and its main subdivisions.

This analysis was made possible because of the great



number of brain areas/nuclei showing NADPH-d–

positive cells.

There is some dispute on the use of the data to

obtain morphological or molecular phylogenetic trees

(Wiens, 2004). However, assuming that the phyloge-

netic trees of amphibians obtained on the basis of

molecular data are correct, our data for the entire brain

based on a simple morphological character are appa-

rently consistent with the molecular data. In particular,

the dendrogram obtained here by hierarchical clustering

using the Jaccard index was similar to those achieved

in molecular biological studies on amphibians (Ford and

Cannatella, 1993; Hay et al., 1995; Biju and Bossuyt,

2003; Roelants and Bossuyt, 2005; Zhang et al., 2005;

Pyron and Wiens, 2011; Pyron, 2011), by grouping Rani-

dae and Hylidae (Neobatrachia) (and having as an

external group X. laevis [Pipidae]) and combining Gym-

nophiona and Caudata together in the Procera clade, as

supported by some studies (Feller and Hedges, 1998;

Zhang et al., 2005; San Mauro et al., 2005; Marjanovic

and Laurin, 2007). However, our dendrogram contrasts

with other studies in which a sister group relationship

between Anura and Caudata was proposed (Frost et al.,

2006; Pyron, 2011; Roelants et al., 2007; San Mauro,

2010; Wiens, 2011; Zhang et al., 2005).

In acceptance of the reliability of the dissimilarity den-

drogram obtained for NADPH-d distribution for the whole

brain in reflecting the evolution of amphibians, among

the main brain subdivisions only the hindbrain agrees

quite well. Indeed, the dendrogram obtained from this

area is significantly similar to that extrapolated from the

whole brain. In contrast, the secondary prosencephalon

and isthmus dendrograms do not reflect the amphibian

phylogenetic relationships, and comparison with that of

the whole brain did not support a significant similarity.

The diencephalon and the mesencephalon only partially

reflected the evolutionary relationship among amphib-

ians, with X. laevis in an anomalous position (i.e., in

between hylid species). These results might indicate that

the diencephalic and mesencephalic NADPH-d system of

X. laevis has undergone an adaptation converging toward

the hylids. Furthermore, the apodan D. mexicanus had a

diencephalic NADPH-d system that was closer to that of

the anurans with respect to P. waltl. Nevertheless, com-

parison of the diencephalic dendrogram with that of the

whole brain revealed a significant similarity; at the same

time, values for the mesencephalon were only partially

significant.

CONCLUSIONS

In the present study, we mapped the distribution of

NADPH-d–positive cells in the brains of four species of

treefrogs.

A total of 76 NADPH-d–positive brain areas of

amphibians studied to date were found. Some of these

areas, such as the anterior commissure in H. septentrio-

nalis, H. annectans, and L. caerulea, the optic chiasm

and the motor nucleus of the glossopharyngeal nerve in

H. arborea, and the interpeduncular nucleus of H. sep-

tentrionalis, have not previously been described. Among

the total number of NADPH-d–positive areas, only five

regions were consistently positive in all species studied,

representing 6.6% of the whole data set.

Although no individual differences in the distribution

of NADPH-d were found in samples from the same spe-

cies, comparison between different species revealed

appreciable diversity. Nevertheless, our data, when stat-

istically analyzed and combined with those obtained

from previous studies, faithfully reflected the evolution-

ary history of amphibians when we considered the

whole brain and the hindbrain; for the other brain areas,

our dendrograms did not match those from phyloge-

netic history. Natural selection could potentially act

selectively on brain areas, leading to specific character-

istics, a possibility that would confirm a previous study

showing that selection acts independently on different

brain regions in cichlid fish (Pollen et al., 2007;

Gonzalez-Voyer et al., 2009). In this context, and in

terms of NADPH-d brain distribution in amphibians, our

data support the concept of a major evolutionary stabil-

ity of the hindbrain, which is consistent with other hind-

brain studies in vertebrates. For example, the medulla

is more conservative because it has changed little/very

little in vertebrate phylogeny (Finlay et al., 2001; North-

cutt, 2002; Gonzalez-Voyer et al., 2009).

Overall, in this study we provide evidence that, at

least for the NADPH-d brain system, the use of main

brain subdivisions should be approached carefully for

comparative purposes because specific adaptations

may occur in a single species, a phenomenon that

apparently has no effect on the whole brain, because it

is hidden by the great number of brain areas in which

NADPH-d is present.

CONFLICT OF INTEREST STATEMENT

The authors have no conflicts of interest.

ROLE OF AUTHORS

All authors had full access to all the data in the

study and take responsibility for the integrity of the

data and the accuracy of the data analysis. Study con-

cept and design: C.P., B.D., R.K.R. Acquisition of data:

C.P., A.S. Analysis and interpretation of data: C.P., B.D.,

A.G.J. Drafting of the manuscript: C.P., B.D. Critical

C. Pinelli et al.


revision of the manuscript for important intellectual

content: R.K.R. Statistical analysis: M.A.

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A comparative cluster analysis of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry in the brains of amphibians