ORIGINAL RESEARCH ARTICLE
published: 23 April 2014
doi: 10.3389/fnana.2014.00024
Comparative neuronal morphology of the cerebellar cortex
in afrotherians, carnivores, cetartiodactyls, and primates
Bob Jacobs1*, Nicholas L. Johnson1, Devin Wahl1, Matthew Schall1, Busisiwe C. Maseko2,
Albert Lewandowski3, Mary A. Raghanti4, Bridget Wicinski5, Camilla Butti5, William D. Hopkins6,
Mads F. Bertelsen7, Timothy Walsh8, John R. Roberts8, Roger L. Reep9, Patrick R. Hof5,
Chet C. Sherwood10 and Paul R. Manger2
1 Laboratory of Quantitative Neuromorphology, Psychology, Colorado College, Colorado Springs, CO, USA
2 Faculty of Health Sciences, School of Anatomical Sciences, University of the Witwatersrand, Johannesburg, South Africa
3 Cleveland Metroparks Zoo, Cleveland, OH, USA
4 Department of Anthropology, Kent State University, Kent, OH, USA
5 Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
6 Division of Developmental and Cognitive Neuroscience, Yerkes National Primate Research Center, Atlanta, GA, USA
7 Center for Zoo and Wild Animal Health, Copenhagen Zoo, Frederiksberg, Denmark
8 Smithsonian National Zoological Park, Washington, DC, USA
9 Department of Physiological Sciences, University of Florida, Gainesville, FL, USA
10 Department of Anthropology, The George Washington University, Washington, DC, USA
Edited by:
Suzana Herculano-Houzel,
Universidade Federal do Rio de
Janeiro, Brazil
Reviewed by:
James M. Bower, University of
Texas Health Science Center San
Antonio, USA
Suzana Herculano-Houzel,
Universidade Federal do Rio de
Janeiro, Brazil
Andrew Iwaniuk, University of
Lethbridge, Canada
*Correspondence:
Bob Jacobs, Laboratory of
Quantitative Neuromorphology,
Psychology, Colorado College, 14 E.
Cache La Poudre, Colorado Springs,
CO 80903, USA
e-mail: bjacobs@
coloradocollege.edu
Although the basic morphological characteristics of neurons in the cerebellar cortex
have been documented in several species, virtually nothing is known about the
quantitative morphological characteristics of these neurons across different taxa. To
that end, the present study investigated cerebellar neuronal morphology among eight
different, large-brained mammalian species comprising a broad phylogenetic range:
afrotherians (African elephant, Florida manatee), carnivores (Siberian tiger, clouded
leopard), cetartiodactyls (humpback whale, giraffe) and primates (human, common
chimpanzee). Specifically, several neuron types (e.g., stellate, basket, Lugaro, Golgi,
and granule neurons; N = 317) of the cerebellar cortex were stained with a modified
rapid Golgi technique and quantified on a computer-assisted microscopy system. There
was a 64-fold variation in brain mass across species in our sample (from clouded
leopard to the elephant) and a 103-fold variation in cerebellar volume. Most dendritic
measures tended to increase with cerebellar volume. The cerebellar cortex in these
species exhibited the trilaminate pattern common to all mammals. Morphologically,
neuron types in the cerebellar cortex were generally consistent with those described
in primates (Fox et al., 1967) and rodents (Palay and Chan-Palay, 1974), although there
was substantial quantitative variation across species. In particular, Lugaro neurons in the
elephant appeared to be disproportionately larger than those in other species. To explore
potential quantitative differences in dendritic measures across species, MARSplines
analyses were used to evaluate whether species could be differentiated from each other
based on dendritic characteristics alone. Results of these analyses indicated that there
were significant differences among all species in dendritic measures.
Keywords: dendrite, morphometry, Golgi method, brain evolution, cerebellum
INTRODUCTION
In terms of gross anatomy, the cerebellum appears to have a com-
mon plan in all mammals (Bolk, 1906; Breathnach, 1955; Larsell,
1970; Sultan and Braitenberg, 1993), although absolute and rel-
ative size can vary considerably (Marino et al., 2000; Maseko
et al., 2012b). Histologically, cerebellar cortex exhibits a generally
trilaminate architecture, which is similar in birds and mammals
(Ramón y Cajal, 1909, 1911; Iwaniuk et al., 2006; Sultan and
Glickstein, 2007). Whereas limited aspects of cerebellar neuron
morphology have been described in some vertebrate species (e.g.,
mormyrid electric fish: Han et al., 2006; teleost fish: Murakami
and Morita, 1987; alligator: Nicholson and Llinas, 1971; cat:
Melik-Musyan and Fanardzhyan, 2004; duck: O’Leary et al., 1968;
dolphin: Adanina, 1965; rhesus monkey: Fox et al., 1967; Rakic,
1972; human: Braak and Braak, 1983), the most detailed research
has focused on rodents. In particular, Palay and Chan-Palay
(1974) provided a comprehensive examination of the cerebellar
cortex of the rat, documenting organizational features, neuronal
morphology, and ultrastructure at the electron microscopic level.
Recently, we expanded the scope of such investigations with an
examination of neuronal morphology in the cerebellar cortex
of the African elephant (Maseko et al., 2012a). The current,
rapid Golgi study is part of a larger project to document neu-
ronal morphology of both the cerebral neocortex (Jacobs et al.,
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NEUROANATOMY
Jacobs et al. Neuronal morphology in cerebellar cortex
2011) and the cerebellar cortex in large brained mammals not
previously examined. Such comparative investigations may help
discern which aspects of neuronal morphology are general to all
vertebrates, and which are specific to particular species (Meek
et al., 2008). To this end, we examine cortical neuronal mor-
phology in the cerebella of eight different mammalian species
comprising four diverse taxa: afrotherians (African elephant,
Florida manatee), carnivores (Siberian tiger, clouded leopard),
cetartiodactyls (humpback whale, giraffe), and primates (human,
common chimpanzee).
Although there are many representative freehand and cam-
era lucida drawings of cerebellar cortex neurons (Ramón y Cajal,
1909, 1911; Chan-Palay and Palay, 1970, 1972; Palay and Chan-
Palay, 1974; Braak and Braak, 1983; Bishop, 1993; Lainé and
Axelrad, 1996), very few cerebellar neurons have been digitally
reconstructed relative to those in the neocortex and hippocam-
pus (Halavi et al., 2012). In fact, it is revealing that, of the
10,004 digital reconstructions currently in the online repository
at Neuromorpho.org, only 24 are cerebellar neurons (as opposed
to 5405 cerebral cortex neurons). In terms of digital reconstruc-
tions, the Purkinje neuron has been traced much more than
other cerebellar neurons, perhaps because of its central role as the
sole output neuron for the cerebellar cortex in tetrapods (Marr,
1969; Dean et al., 2010). The most complete Purkinje cell trac-
ings are typically the result of injection techniques (e.g., Lucifer
yellow: Sawada et al., 2010; biocytin: Roth and Häuser, 2001),
and immunohistochemistry (Wu et al., 2010) with confocal laser
microscropy, although the number of reconstructions usually
remains small (<30). An even more limited number of Purkinje
neuron reconstructions have been obtained using horseradish
peroxidase and Golgi-Cox impregnations with light microscopy
(Calvet and Calvet, 1984; Rapp et al., 1994; Milatovic et al., 2010).
There appear to be no digital reconstructions of Purkinje neurons
based on rapid Golgi stains. Finally, apart from a small num-
ber of traced molecular layer interneurons (N = 26; Sultan and
Bower, 1998), there are few complete digital reconstructions of
other neuronal types in cerebellar cortex.
In terms of comparative neuromorphology, research has gen-
erally focused on qualitative descriptions of Purkinje neurons. For
example, there are well-documented morphological differences
between tetrapods and teleosts such as the mormyrids, which
have Purkinje neuron dendrites with a distinct palisade pattern
(Meek and Nieuwenhuys, 1991). Quantitatively, however, there
is very little comparative morphological information on cerebel-
lar cortical neurons. To this end, the present study documents
the morphological attributes of several types of cerebellar neu-
rons. Following descriptions in rodents (Palay and Chan-Palay,
1974) and other mammals (rhesus monkey: Fox et al., 1967; cat:
Larsell and Jansen, 1972; human: Braak and Braak, 1983), the
superficial molecular layer contains the two-dimensional den-
dritic arrays of Purkinje neurons. These Purkinje neurons are
described only qualitatively in the present study because rapid
Golgi impregnations under light microscopy make complete
and accurate tracings of their dense, distal dendritic segments
extremely problematic, if not impossible. Also in the molecular
layer are inhibitory interneurons, classically divided into (1) the
relatively small stellate neurons in the outer two thirds of the
layer, which are characterized by contorted, frequently dividing
dendritic trees that radiate in multiple directions and by axons
that are generally oriented horizontally; and (2) the somewhat
deeper basket neurons, characterized by extensive, sea-fan shaped
dendritic arbors and horizontally oriented axons that terminate
in multiple pericellular baskets around the somata of Purkinje
neurons. Although we follow the classical terminology for these
interneurons in the present paper, it should be noted that both
developmental research (Rakic, 1972) and empirical investiga-
tions (Sultan and Bower, 1998; Leto et al., 2006; Schilling et al.,
2009) support early speculation (Ramón y Cajal, 1909, 1911) that
these molecular inhibitory interneurons may actually be a uni-
form cell type whose ultimate morphology is determined by local
cues at particular depths of the molecular layer.
Under the molecular layer, the Purkinje cell layer contains the
large somata of Purkinje neurons, arranged in a single row, pro-
viding a clear demarcation between the other two layers. The
deep granule cell layer contains the somata of two relatively large
interneurons: (1) located immediately beneath the Purkinje cell
layer, the Lugaro neurons (Golgi, 1874; Lugaro, 1894) are charac-
terized by triangular or elongated fusiform shaped somata from
which relatively long, thick, unbranched dendrites originate, typ-
ically extending in an arc under the Purkinje cell layer; and (2)
somewhat deeper in the granule cell layer, the Golgi neurons
(Golgi, 1874) are characterized by round somata with multi-
ple dendrites radiating in all directions. Finally, throughout the
granule cell layer are the very densely packed granule neurons,
characterized by small, round somata extending several short, rel-
atively unbranched dendrites characterized by gnarled, claw-like
terminations.
The goals of the present comparative study were three-fold: (1)
provide a qualitative description of neuronal morphology in the
cerebellar cortex across the eight species examined; (2) provide
quantitative data on the dendritic characteristics of these neurons;
and (3) examine potential species differences in the dendritic
measures of the traced neurons.
MATERIALS AND METHODS
SPECIMENS
Tissue was obtained from eight species in the following phy-
logenetic groups: afrotherians (African elephant, Florida mana-
tee), carnivores (Siberian tiger, clouded leopard), cetartiodactyls
(humpback whale, giraffe), and primates (human, common
chimpanzee). For captive animals (Siberian tiger, clouded leop-
ard, chimpanzee), observations prior to death revealed no obvi-
ous behavioral abnormalities or deficits. Similar observations
were not possible for animals in their natural habitat (African
elephant, giraffe, humpback whale, Florida manatee, human). In
post-mortem examinations, the brains of all animals exhibited
no obvious abnormalities in terms of gross neuroanatomy. For
five species (African elephant, Siberian tiger, clouded leopard,
humpback whale, giraffe), cerebellar volume for at least one of
the animals was obtained through magnetic resonance imaging
(Maseko et al., 2012b). For the other species (Florida mana-
tee, human, chimpanzee), direct measurement was not obtained
because we did not have the opportunity for MRI scanning, nor
was destructive dissection of the cerebellum an option. Instead,
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Jacobs et al. Neuronal morphology in cerebellar cortex
we had to rely on species mean values from the published liter-
ature. The present study was approved by the Colorado College
Institutional Review Board (#011311-1) and the University of the
Witwatersrand Animal Ethics Committee (2008/36/1).
African elephant (Loxodonta africana)
Cerebellar tissue from two 20 to 30-year-old, solitarymale African
elephants scheduled for population management culling was
obtained after they were euthanized as described in Manger et al.
(2009). In situ perfusion-fixation of the brains was conducted
by removal of the head, flushing of the head with cold saline,
and intra-carotid perfusion with 4% paraformaldehyde in 0.1M
phosphate buffer (autolysis time, AT, averaged = 135min). The
brains were then removed from the skull, placed in the same cold
fixative and stored in 4% paraformaldehyde in 0.1M phosphate
buffer for 72 h. One brain had a mass of 5145 g and a cerebel-
lar volume of 946ml; the other brain had a mass of 4835 g and a
cerebellar volume of 902ml (Maseko et al., 2012b). Small tissue
blocks containing the cerebellar regions of interest were stored in
0.1% sodium azide in 0.1M phosphate buffer saline at 4◦C for 8
months before Golgi staining.
Florida manatee (Trichechus manatus latirostris)
Following a watercraft collision in Florida, a sub-adult female
manatee was euthanized. The head was perfused (by Roger L.
Reep) via bilateral cannulation of the internal carotids, with 20 l
phosphate buffer followed by 10 l of 4% paraformaldehyde. The
brain (brain mass = 316 g; estimated cerebellar volume = 44ml;
Reep and O’Shea, 1990) was removed (AT = 6 h) and stored in
a cold 2% paraformadehyde solution for ∼2 days. One cerebel-
lar tissue block was removed and stored in cold (2◦C) phosphate
buffer solution for 3 additional days before Golgi staining.
Siberian tiger (Panthera tigris altaica)
One 12-year-old female from the Copenhagen Zoo in Denmark
was euthanized. In situ perfusion-fixation (by Mads F. Bertelsen)
of the brain (AT < 30min) followed the same protocol as in
the elephant (brain mass = 258 g; cerebellar volume = 37ml).
Cerebellar tissue blocks were stored in 0.01% sodium azide in
0.1M phosphate buffer saline at 4◦C for 6 months before Golgi
staining.
Clouded leopard (Neofelis nebulosa)
Two adult female clouded leopards were euthanized for medical
reasons: a 20-year old from the Smithsonian National Zoological
Park in Washington, DC., and a 28-year old from the Cleveland
Metroparks Zoo (AT < 30min for both animals). The brains
were immersion fixed in 10% formalin for 10 (20-year old) and
34 days (28-year old). Brain mass was 82 g for the 20-year old
and 73 g for the 28-year old; cerebellar volume was an average of
8.6ml for both animals. Subsequently, the brains were stored in
0.1% sodium azide in 0.1M phosphate buffer saline at 4◦C prior
to Golgi staining (5 months for the 20-year old; 3 years for the
28-year old).
Humpback whale (Megaptera novaeangliae)
A ∼2 year-old male humpback whale, 9.45m in length, was
stranded in East Hampton, Long Island, New York in April, 2010.
A necropsy was performed (by Patrick R. Hof, Bridget Wicinski,
and Camilla Butti) immediately after death. The brain (brain
mass = 3606 g; cerebellar volume = 695ml) was removed (AT
= 8 h) and immersion-fixed in 4% paraformaldehyde for 2 years
prior to Golgi staining.
Giraffe (Giraffa camelopardalis)
The brains of three solitary, free ranging, sub-adult (∼2–3 years of
age) male giraffes were obtained and processed in the same man-
ner as the elephant (Dell et al., 2012). Brain masses—cerebellar
volumes for these three animals were 610 g—83ml, 527 g—69ml,
and 480 g—67ml. Cerebellar blocks were stored in 0.1% sodium
azide in 0.1M phosphate buffer for 4 months prior to Golgi
staining.
Human (Homo sapiens)
Human tissue was provided by Dr. R. Bux of the El Paso County
coroner’s office in Colorado Springs. Tissue blocks were removed
from the cerebellum of a neurologically normal, 54-year-old male
who had died of acute myocardial infarction (brain mass =
1435 g; estimated cerebellar volume = 139ml; Smaers et al., 2011;
Maseko et al., 2012b). Tissue was immersion fixed in 10% for-
malin and stored at 2◦C for ∼1 week before Golgi staining
(AT = 5 h).
Common chimpanzee (Pan troglodytes)
Two adult chimpanzees were obtained from the Yerkes National
Primate Center: a 23-year-old female who died under anesthe-
sia, and a 39-year-old male euthanized due to congestive heart
failure. Brains were immersion fixed in 10% formalin (13 days
for the 23-year old; 4 months for the 39 year old; AT < 1 h).
Subsequently, brains were stored in 0.1% sodium azide in 0.1M
phosphate buffer saline at 4◦C prior to Golgi staining (4 years for
the 23-year old; 2 years for the 39-year old). Brain mass was 408 g
for the 23-year old and 392 g for the 39-year old; cerebellar vol-
ume was estimated to be an average of 43ml for both animals
(Smaers et al., 2011; Maseko et al., 2012b).
TISSUE SELECTION
In five of the species (Florida manatee, Siberian tiger, hump-
back whale, human, and chimpanzee), tissue blocks (3–5mm
thick) were removed from the dorsal posterior aspect of the pos-
terior lobe and from the dorsal anterior aspect of the anterior
lobe of the left cerebellar hemisphere (Figure 1). In the remain-
ing three species (African elephant, giraffe, and clouded leopard),
the same regions were sampled from the right cerebellar hemi-
sphere. Tissue was coded to prevent experimenter bias, stained via
a modified rapid Golgi technique (Scheibel and Scheibel, 1978),
and sectioned serially perpendicular to the long axis of the folia at
120μm with a vibratome (Leica VT1000S, Leica Microsystems,
Inc.). Because of the small number of neurons traced in each
species, neurons from anterior and posterior cerebellar lobes were
combined for all subsequent analyses.
NEURON SELECTION AND QUANTIFICATION
Neurons were selected for tracing based on established criteria
(Roitman et al., 2002; Anderson et al., 2009; Jacobs et al., 2011; Lu
et al., 2013), which required an isolated, darkly stained soma near
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Jacobs et al. Neuronal morphology in cerebellar cortex
FIGURE 1 | Dorsal views of the brains from the eight species in the
current study illustrating the relative location from which tissue blocks
were selected from the cerebellum for staining. Represented from top
to bottom are: afrotherians (African elephant, Florida manatee), carnivores
(Siberian tiger, clouded leopard), cetartiodactyls (humpback whale, giraffe),
and primates (human, common chimpanzee). For the two primates, the
dorsal portion of the cerebrum has been removed to reveal the cerebellum.
Note scale bar is different for each species.
the center of the 120μm section, with as fully impregnated, unob-
scured, and complete dendritic arbors as possible (i.e., no beading
or interruptions). In the tracing process, dendritic branches
were not followed into adjacent sections. Although serial section
reconstructions of dendrites are possible with some histological
techniques (e.g., intracellular injections), accurate reconstruc-
tions are problematic in Golgi stained material, where multiple
neural elements overlap in the same section. As such, only por-
tions captured in the 120μm-thick section could be compared
in the present study, resulting in an overall underestimation of
dendritic values insofar as neurons with longer dendrites are
disproportionately cut in the sectioning process. Prior to quan-
tification, Golgi-stained sections were examined to determine
neuronal types. The neurons of interest included the molecu-
lar layer interneurons (e.g., stellate, basket), as well as Lugaro,
Golgi, and granule cells. As noted above, we kept the classical
distinction between stellate and basket neurons for the molecular
layer interneurons, although it has been argued that they actually
constitute the same neuronal population (Sultan and Bower,
1998). Additionally, no distinction was made between superficial
and deeper stellate neurons, or between large and small Golgi
neurons (Palay and Chan-Palay, 1974). Purkinje neurons were
photomicrographed but not traced due to the complexity of their
dendritic plexus. Candelabrum (Lainé and Axelrad, 1994), unipo-
lar brush (Altman and Bayer, 1977), and synarmotic (Landau,
1933; Flace et al., 2004) neurons were not observed in the current
preparations. Certain morphological characteristics of neurons
traced in the elephant have previously been reported (Maseko
et al., 2012a), but are included here with a more in-depth anal-
ysis. Golgi impregnation was inconsistent across species, both in
terms of overall numbers of neurons and the types of neurons
that stained. Consequently, neurons traced in different individ-
uals within a species (i.e., elephant, clouded leopard, giraffe,
chimpanzee) were combined without consideration of individual
differences.
Quantification was performed under a Planachromatic 60x
oil objective (N.A. 1.4), except for elephant neurons, which
were quantified under a Planachromat 40x dry objective (N.A.
0.70). Based on prior research, this difference in microscope
objectives was not expected to significantly affect dendritic
measures (Anderson et al., 2010). Neurons were traced along x-,
y-, z-coordinates using a Neurolucida system (MBF Bioscience,
Williston, VT) interfaced with an Olympus BH-2 microscope
equipped with a Ludl XY motorized stage (Ludl Electronics,
Hawthorne, NY) and a Heidenhain z-axis encoder (Schaumburg,
IL). A MicroFire Digital CCD 2-megapixel camera (Optronics,
Goleta, CA) mounted on a trinocular head (model 1-L0229,
Olympus, Center Valley, PA) displayed images on a 1920 × 1200
resolution Dell E248WFP 24-inch LCD monitor. Somata were
traced first at their widest point in the 2-dimensional plane to
provide an estimate of the cross-sectional soma area, a measure
that appears highly correlated with soma volume (Ulfhake,
1984). Subsequently, dendrites were traced somatofugally in their
entirety, recording dendritic diameter. Dendritic arbors with
unclear, sectioned, broken, ambiguous, or obscured terminations
were identified as incomplete endings. Of the 13,698 dendritic
segments quantified, 45% were intermediate segments. With
regard to terminal segments, 58% were complete, and 42% were
incomplete, which is a higher completion ratio than obtained in
neocortex with the same methodology (Jacobs et al., 1997, 2001).
Neurons with sectioned segments were not differentially analyzed
because elimination of neurons with incomplete segments would
have biased the sample toward smaller neurons (Schadé and
Caveness, 1968; Uylings et al., 1986).
Neurons were traced by three investigators (Busisiwe C.
Maseko, Nicholas L. Johnson, Devin Wahl). Intrarater reliabil-
ity was determined by having each rater trace the same soma
and dendritic segment 10 times. The average coefficient of varia-
tion for soma size (2.5%) and total dendritic length (TDL, 2.8%)
indicated little variation in the tracings. Intrarater reliability was
further tested with a split plot design (α = 0.05), which indicated
no significant difference between the first five tracings and the last
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Jacobs et al. Neuronal morphology in cerebellar cortex
five tracings. Interrater reliability was determined through com-
parison of 10 dendritic system tracings with the same tracings
completed by the primary investigator (Bob Jacobs). Interclass
correlations across soma size and TDL averaged 0.99 and 0.99,
respectively. An analysis of variance (ANOVA; α = 0.05) failed
to indicate significant differences among the tracers for the three
measures. Additionally, the primary investigator reexamined all
completed tracings under the microscope to ensure accuracy.
CELL DESCRIPTIONS AND DEPENDENT DENDRITIC MEASURES
Neurons were classified according to somatodendritic morpho-
logical characteristics, closely following well-established descrip-
tive criteria (Ramón y Cajal, 1909, 1911; Rakic, 1972; Palay
and Chan-Palay, 1974; Braak and Braak, 1983; Melik-Musyan
and Fanardzhyan, 2004). Quantitatively, soma size (i.e., surface
area,μm2) and depth from the pial surface (μm) were measured.
Dendritic branches extending from the soma were characterized
centrifugally (Bok, 1959; Uylings et al., 1986), and quantified with
four previously established measures (Jacobs et al., 2011): den-
dritic volume (Vol, μm3; the total volume of all dendrites); total
dendritic length (TDL, μm; the summed length of all dendritic
segments); mean segment length (MSL, μm; the average length
of each dendritic segment); and dendritic segment count (DSC;
the number of dendritic segments). One additional measure
from Maseko et al. (2012a) was examined: dendritic tortuos-
ity (Tor), a measure of the relative straightness-twistedness of
a dendrite. The tortuosity index was calculated by dividing the
total length of dendritic segments from the origin point on the
soma to the end point by the length of a vector: an index of 1
equals a straight line; an index greater than 1 means the path of
the dendrites is more complex than a straight line (Foster and
Peterson, 1986;Wen et al., 2009). Finally, dendritic branching pat-
terns were analyzed using a Sholl analysis (Sholl, 1953), which
quantified dendritic intersections at 20-μm intervals radiating
somatofugally.
INFERENTIAL STATISTICAL ANALYSES OF INTERSPECIES DIFFERENCES
Measures (Vol, TDL, MSL, DSC, Tor) for every dendritic seg-
ment, along with soma size and depth, were aggregated for
each neuron (SPSS release 20.0.0). A total of 317 neurons were
traced in 12 members of 8 species, with the following break-
down: African elephant (n = 20; 18 in one animal, 2 in the other),
Florida manatee (n = 25), Siberian tiger (n = 33), clouded leop-
ard (n = 32; 21 in the 20-year old; 11 in the 28-year old),
humpback whale (n = 47), giraffe (n = 56; 21, 21, and 14 in
each of the three animals), chimpanzee (n = 86; 51 in the 23-
year old; 35 in the 39-year old), human (n = 28). As indicated in
Table 1, neuron types were unevenly distributed among species
and some neuron types did not stain in some species (i.e.,
granule neurons in afrotherians, and Golgi neurons in the mana-
tee).
For inferential analyses, we initially constructed a table of
dendritic measures for all neuron types to explore differences
among species (Table 2). Given that cerebellar volume increases
with the size of the brain (Smaers et al., 2011; Maseko et al.,
2012b), the table was used to evaluate if the same is true for den-
dritic measures. Confidence intervals of 95% were constructed
Table 1 | Number of tracings for each neuronal type within each
species.
Species Neuron type
Stellate Basket Lugaro Golgi Granule Total
African elephant 5 5 5 5 0 20
Florida manatee 6 8 1 0 0 15
Siberian tiger 5 8 3 7 10 33
Clouded leopard 16 5 4 1 6 32
Humpback whale 9 11 11 6 10 47
Giraffe 17 13 4 7 15 56
Human 7 6 5 5 5 28
Common chimpanzee 20 16 17 17 16 86
Total 85 72 50 48 62 317
for each measure. Because elephants had the largest cerebellar
volume (averaging 924ml) and clouded leopards the smallest
(averaging 8.6ml), these two species were used as the references
against which the confidence intervals for the other species were
compared. Unfortunately, because only one Golgi neuron stained
in the clouded leopard, we could not construct confidence inter-
vals for that species and neuron; instead, only the elephant was
used as a reference for Golgi neurons. Similarly, the elephant had
no stained granule neurons, so only the clouded leopard was used
for comparison. In examining the table columns for each type of
dendritic measure, any species-specific value outside the reference
indicates a difference between the two distributions. The goal was
to evaluate how much, or how little, the reference distributions
for elephant and clouded leopard overlapped with the distribu-
tions of the dendritic values for the other species. By comparing
the amount of overlap between the distributions, a much better
idea of the comparative distributions was provided than if just F-
tests based on means had been presented. Significant differences
were highlighted in bold for comparison to the elephant and red
for comparisons with the clouded leopard. Because of the usage of
95% confidence intervals, any two distributions that did not over-
lap were different at p ≤ 0.05. For example, in Table 2, the TDL
lower (176.5) and upper confidence intervals (2735.2) for Golgi
neurons in the chimpanzee are in bold text, indicating that the
Golgi TDL values in the chimpanzee (1455.9 ± 639.7μm) are sig-
nificantly [F(1315) = 720.62, p ≤ 0.05] different from those in the
reference animal, namely the elephant (Golgi TDL = 5664.2 ±
878.9μm). Although Table 2 provides detailed information, it
does not specify whether any of the species can be identified from
just dendritic characteristics. Because accurate differentiation of
species may require combinations of multiple dendritic measures,
a more comprehensive analysis was necessary.
There are six potential analytic obstacles to overcome in the
present, or any, quantitative neuromorphological analysis. First,
the brains for each species were fixed and preserved differ-
ently, which contributed to “noise,” or error variability in mea-
surements both within the same species and between different
species. Second, differential stain impregnation introduced mea-
surement error at the dendritic level. Third, some neuron types
did not stain in some animals. Fourth, the standard assumption
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Jacobs et al. Neuronal morphology in cerebellar cortex
Table 2 | Dendritic measures and soma size for each neuronal type across species.a
Speciesa Cell type Vol TDL
Mean SD L95% U95% Mean SD L95% U95%
Clouded leopard Stellate 691.9 222.2 247.4 1136.4 1367.2 489.8 387.6 2346.7
Basket 1463.4 660.4 142.7 2784.2 2156.5 890.2 376.1 3937.0
Lugaro 1664.6 694.7 275.3 3054.0 899.1 531.1 −163.1 1961.4
Golgi 1693.7 − − − 1528.3 − − −
Granule 691.9 222.2 247.4 1136.4 140.6 69.1 2.5 278.7
Siberian tiger Stellate 790.0 188.2 413.6 1166.4 1463.7 401.2 661.3 2266.0
Basket 1081.2 585.5 −89.8 2252.1 1284.7 544.2 196.2 2373.2
Lugaro 3252.9 1550.6 151.8 6354.0 1286.3 153.7 978.9 1593.7
Golgi 5018.1 3215.0 −1411.9 11, 448.1 3076.0 744.9 1586.3 4565.7
Granule 129.9 35.6 58.7 201.1 303.7 57.0 189.8 417.6
Florida manatee Stellate 1836.2 302.7 1230.8 2441.6 1969.8 490.4 989.0 2950.6
Basket 2202.3 399.3 1403.7 3000.9 1993.1 421.8 1149.4 2836.8
Lugaro 2401.6 − − − 1651.9 − − −
Golgi − − − − − − − −
Granule − − − − − − − −
Common chimpanzee Stellate 524.5 304.1 −83.6 1132.7 2029.6 1170.8 −312.0 4371.2
Basket 954.0 494.5 −35.0 1943.0 2408.3 1235.3 −62.4 4879.0
Lugaro 1453.8 858.5 −263.2 3170.8 952.3 479.3 −6.2 1910.9
Golgi 1287.0 631.2 24.7 2549.3 1455.9 639.7 176.5 2735.2
Granule 46.0 23.0 0.0 91.9 126.1 37.7 50.7 201.4
Giraffe Stellate 1725.8 616.2 493.5 2958.2 2254.1 733.9 786.3 3721.9
Basket 1905.1 714.1 476.8 3333.4 2314.5 764.6 785.2 3843.7
Lugaro 3222.4 1887.7 −553.0 6997.7 2136.8 1449.0 −761.2 5034.8
Golgi 6333.3 2150.1 2033.0 10, 633.6 2907.3 1425.6 56.1 5758.5
Granule 241.4 58.6 124.2 358.5 275.4 116.0 43.4 507.4
Human Stellate 1977.5 601.3 774.8 3180.2 3583.7 1016.9 1550.0 5617.4
Basket 2163.5 699.1 765.3 3561.7 3374.5 1083.8 1206.9 5542.1
Lugaro 3812.9 1887.3 38.2 7587.5 2467.2 599.3 1268.5 3665.8
Golgi 5942.7 3530.7 −1118.7 13, 004.0 5098.0 2866.9 −635.9 10, 831.9
Granule 117.5 48.8 20.0 215.1 166.3 44.3 77.7 254.9
Humpback whale Stellate 781.8 249.1 283.6 1280.0 1785.9 520.9 744.1 2827.7
Basket 1641.4 513.0 615.5 2667.4 3018.0 839.3 1339.5 4696.5
Lugaro 3902.2 2716.4 −1530.6 9335.0 1121.3 708.5 −295.6 2538.2
Golgi 2358.4 962.9 432.6 4284.1 1759.6 435.7 888.1 2631.0
Granule 78.0 29.5 19.0 136.9 180.9 58.0 64.9 296.8
African elephant Stellate 7681.2 2844.1 1993.0 13, 369.3 5501.0 1561.2 2378.5 8623.4
Basket 12, 890.4 4406.2 4078.0 21,702.8 6212.4 2501.7 1209.0 11, 215.7
Lugaro 37, 395.8 17, 331.0 2733.8 72, 057.7 3019.1 884.0 1251.2 4787.1
Golgi 16, 637.0 3951.5 8734.1 24, 540.0 5664.2 878.9 3906.4 7422.0
Granule − − − − − − − −
(Continued)
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Jacobs et al. Neuronal morphology in cerebellar cortex
Table 2 | Continued
Speciesa Cell type MSL DSC
Mean SD L95% U95% Mean SD L95% U95%
Clouded leopard Stellate 29.3 4.5 20.4 38.3 47.5 18.3 10.9 84.1
Basket 40.4 3.2 34.1 46.8 52.8 20.8 11.2 94.4
Lugaro 45.1 3.6 37.9 52.3 20.3 12.7 −5.1 45.6
Golgi 50.9 − − − 30.0 − − −
Granule 13.0 5.3 2.4 23.6 11.5 6.0 −0.5 23.5
Siberian tiger Stellate 43.1 8.3 26.5 59.7 36.0 14.9 6.2 65.8
Basket 40.4 14.7 10.9 69.9 32.6 9.5 13.6 51.6
Lugaro 109.2 82.1 −55.1 273.4 16.3 9.1 −1.8 34.5
Golgi 38.0 12.3 13.4 62.7 86.0 29.7 26.7 145.3
Granule 11.0 3.1 4.7 17.3 29.8 11.3 7.2 52.4
Florida manatee Stellate 62.1 9.3 43.5 80.6 31.5 3.6 24.4 38.6
Basket 67.2 14.9 37.3 97.1 30.5 7.4 15.8 45.2
Lugaro 57.0 − − − 29.0 − − −
Golgi − − − − − − − −
Granule − − − − − − − −
Common chimpanzee Stellate 33.2 9.1 15.0 51.4 58.5 23.4 11.6 105.3
Basket 39.3 7.9 23.5 55.0 62.8 34.0 −5.2 130.9
Lugaro 57.3 20.5 16.4 98.2 18.0 8.8 0.4 35.6
Golgi 46.8 12.9 21.1 72.5 30.9 9.9 11.0 50.7
Granule 10.5 2.4 5.7 15.2 12.6 4.4 3.8 21.4
Giraffe Stellate 52.2 10.6 31.0 73.3 45.0 17.4 10.3 79.7
Basket 65.4 25.2 14.9 115.9 37.4 10.7 16.0 58.8
Lugaro 41.4 11.6 18.1 64.7 55.0 42.4 −29.8 139.8
Golgi 45.8 9.6 26.5 65.0 64.4 31.3 1.8 127.1
Granule 12.5 4.3 4.0 21.1 23.5 9.4 4.7 42.2
Human Stellate 53.1 10.4 32.4 73.8 67.1 12.1 42.9 91.4
Basket 49.8 6.3 37.2 62.5 69.3 27.1 15.1 123.5
Lugaro 85.4 30.2 24.9 145.9 32.0 11.9 8.2 55.8
Golgi 67.5 11.7 44.1 90.9 73.4 32.5 8.4 138.4
Granule 14.0 3.4 7.2 20.7 12.0 2.4 7.1 16.9
Humpback whale Stellate 43.8 9.5 24.9 62.7 41.1 9.8 21.6 60.7
Basket 52.5 11.0 30.5 74.4 59.1 20.7 17.7 100.4
Lugaro 58.3 25.8 6.6 109.9 21.0 13.3 −5.6 47.6
Golgi 42.2 12.0 18.3 66.1 43.8 14.5 14.9 72.7
Granule 10.1 2.6 4.9 15.3 18.4 6.2 6.1 30.7
African elephant Stellate 49.0 5.6 37.8 60.1 114.4 40.2 33.9 194.9
Basket 64.8 10.6 43.6 86.0 96.2 33.6 29.1 163.3
Lugaro 70.6 14.1 42.3 98.9 44.2 17.1 10.0 78.4
Golgi 40.4 7.1 26.3 54.5 143.2 32.9 77.4 209.0
Granule − − − − − − − −
(Continued)
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Jacobs et al. Neuronal morphology in cerebellar cortex
Table 2 | Continued
Speciesa Cell type Tor Soma size
Mean SD L95% U95% Mean SD L95% U95%
Clouded leopard Stellate 1.12 0.05 1.02 1.21 118.1 18.7 80.7 155.5
Basket 1.11 0.03 1.05 1.16 143.8 11.8 120.2 167.4
Lugaro 1.11 0.03 1.05 1.17 359.3 112.6 134.1 584.6
Golgi 1.16 − − − 242.0 − − −
Granule 1.29 0.14 1.01 1.57 50.7 6.7 37.3 64.2
Siberian tiger Stellate 1.15 0.05 1.05 1.24 125.2 13.2 98.7 151.7
Basket 1.12 0.05 1.03 1.22 148.2 21.9 104.4 192.0
Lugaro 1.11 0.03 1.05 1.16 384.9 140.0 104.8 665.0
Golgi 1.11 0.02 1.07 1.14 480.1 94.1 291.8 668.3
Granule 1.19 0.07 1.06 1.32 63.8 10.6 42.5 85.1
Florida manatee Stellate 1.17 0.07 1.04 1.31 170.3 16.6 137.1 203.4
Basket 1.17 0.06 1.06 1.29 203.8 21.9 160.0 247.7
Lugaro 1.20 – – – 270.0 – – –
Golgi – – – – – – – –
Granule – – – – – – – –
Common chimpanzee Stellate 1.13 0.03 1.07 1.20 86.3 23.0 40.3 132.2
Basket 1.11 0.02 1.06 1.15 114.6 29.5 55.5 173.7
Lugaro 1.13 0.04 1.05 1.20 271.8 63.8 144.1 399.4
Golgi 1.13 0.02 1.09 1.17 285.6 92.5 100.5 470.7
Granule 1.26 0.17 0.92 1.61 52.9 5.4 42.1 63.7
Giraffe Stellate 1.16 0.03 1.10 1.23 116.5 25.4 65.8 167.2
Basket 1.16 0.04 1.07 1.25 153.1 44.2 64.6 241.6
Lugaro 1.15 0.03 1.08 1.22 271.6 108.7 54.1 489.1
Golgi 1.16 0.05 1.06 1.26 538.2 131.5 275.3 801.2
Granule 1.38 0.21 0.96 1.80 68.0 11.3 45.3 90.7
Human Stellate 1.17 0.03 1.12 1.22 87.0 23.8 39.3 134.7
Basket 1.16 0.03 1.10 1.23 113.3 20.5 72.2 154.3
Lugaro 1.12 0.03 1.07 1.17 315.5 153.0 9.5 621.5
Golgi 1.14 0.02 1.10 1.18 433.1 185.1 63.0 803.3
Granule 1.51 0.39 0.72 2.29 49.7 6.9 35.9 63.6
Humpback whale Stellate 1.10 0.04 1.03 1.18 101.8 27.3 47.2 156.4
Basket 1.10 0.02 1.06 1.14 150.6 52.6 45.4 255.9
Lugaro 1.12 0.05 1.03 1.22 541.1 336.2 −131.4 1213.5
Golgi 1.13 0.04 1.06 1.21 495.5 372.6 −249.7 1240.7
Granule 1.17 0.08 1.01 1.33 63.9 12.0 39.8 88.0
African elephant Stellate 1.29 0.14 1.01 1.57 50.7 6.7 37.3 64.2
Basket 1.15 0.05 1.06 1.24 443.8 189.7 64.3 823.2
Lugaro 1.13 0.05 1.03 1.23 1353.7 394.7 564.4 2143.1
Golgi 1.19 0.02 1.14 1.23 736.5 131.9 472.6 1000.3
Granule − − − − − − − −
aDependent measures are: Volume (Vol, µm3), Total Dendritic Length (TDL, µm), Mean Segment Length (MSL), Dendritic Segment Count (DSC), Tortuosity (Tor),
and Soma Size (µm2). Species are arranged from smallest (clouded leopard) to largest (African elephant) in terms of brain mass. L95% = lower 95% confidence
interval; U95% = upper 95% confidence interval. Bold numbers represent comparisons that were significantly different from the elephant values; red numbers
represent comparisons that were significantly different from the clouded leopard values. See text for further explanation.
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Jacobs et al. Neuronal morphology in cerebellar cortex
of any conventional statistical analysis that requires the use of
covariances (e.g., principal components, ordinary least squares
regression, and even Pearson correlations) is that error terms
are uncorrelated (Williams et al., 2013). This requirement is not
met when, between variables, there are relationships that are due
to multiple sources. Such is the case in neuroanatomy, where
animals have both a genetic and a socio-developmental back-
ground. The confluence of these phylogenetic and ontogenetic
factors shape the underlying neuronal morphology of an individ-
ual animal as well as the common characteristics of conspecifics.
Consequently, it is not possible to evaluate the null hypothesis
in the present study with a conventional statistical test (Williams
et al., 2013). Fifth, sample size constrains the ability to use statis-
tics to test differences between members of a species or between
species themselves. One common rule for designs with nested lev-
els within groups calls for a minimum of 30 units at each level
of measurement (Bell et al., 2008). A study of dendritic charac-
teristics between two species would require 30 specimens from
each, with a sample of enough neurons to generate 30 randomly
selected neurons, from which 30 randomly chosen dendritic trees
would be selected, and so on. It is highly unlikely that a researcher
would have access to 60 animals divided equally between two
species, or the ability to stain and trace a minimum of 1800 com-
plete dendritic trees. Sixth, inferential statistical methods such
as t-tests and ANOVAs, with their standard errors, test statis-
tics, and p-values require that study samples be randomly selected
(Friedman, 1991; Berk and Freedman, 2003). In comparative
neuromorphology research, there are no random samples where
each unit analyzed has an equal probability of being selected. All
samples in such studies are those of convenience, and may not
represent any definable population larger than itself (Freedman,
2004).
The present study therefore sought to employ an analytic
method that could provide solid evidence of the ability to dif-
ferentiate species from dendritic measures despite the variability
introduced by factors such as differential brain fixation and
neuronal staining. Moreover, the analyses had to make these
predictions despite violations of the uncorrelated error, random
sample, and sample size requirements, which invalidate the use of
any conventional statistical tests (e.g., F-tests or principal com-
ponents). Thus, we were limited to nonparametric techniques
that make no assumptions about distributional, correlational,
random sampling, or other requirements. The technique chosen
is referred to as MARS, MARSplines, or Multivariate Adaptive
Regression Splines (Statistica, release 12; StatSoft Inc, Austin, TX;
Friedman, 1991; Hastie et al., 2009). The MARSplines technique
is appropriate because it does not assume or impose any restric-
tions or conditions for the differentiation of species with dendritic
measures, and it can create useful models even in quite difficult
situations similar to those faced in quantitative neuromorphol-
ogy (http://sdn.statsoft.com/STATISTICAVisualBasic.aspx?page=
category&item=modules%3AStatistics%3ASTAMARSplines).
Further, examination of the mathematics of MARSplines demon-
strates that there are no distributional assumptions or sample size
requirements for the r2 statistic it generates. For these reasons,
the MARSplines analysis was employed in the present study to
explore potential species differences in dendritic measures.
Table 3 | Matrix of Spearman’s rho correlations between cerebellar
volume and dendritic measures (and soma size) across all neuron
types.a
Dependent
measure
Neuron type
Stellate Basket Lugaro Golgi Granule
Vol 0.574** 0.512** 0.554** 0.407** 0.233
0.507** 0.393** 0.384** 0.174 0.233
TDL 0.511** 0.520** 0.353* 0.343* 0.007
0.434** 0.422** 0.156 0.095 0.007
MSL 0.583** 0.456** 0.177 0.088 0.026
0.582** 0.406** 0.054 0.210 0.026
DSC 0.175 0.331** 0.349* 0.301* 0.026
0.048 0.223 0.189 0.051 0.026
Tor 0.242* 0.199 0.060 0.537** −0.021
0.180 0.153 0.094 0.377* −0.021
Soma size 0.032 0.209 0.385** 0.350* 0.177
−0.116 0.016 0.153 0.132 0.177
aCorrelations in black font are for all eight species (N = 317 neurons); correlations
in red font are for all species except the elephant (N = 297 neurons). Note the
reduction in the magnitude of all correlations when the elephant is removed from
the analysis. *p = 0.05; **p = 0.01.
RESULTS
OVERVIEW
In terms of gross anatomy across the sampled species, there was a
64-fold variation in brain mass (from an average of 78 g in the
leopards to an average of 4990 g in the elephants) and a 103-
fold variation in cerebellar volume (from an average of 9ml in
the leopards and an average of 924ml in the elephants). The
larger variation in cerebellar volume appears to be a result of
a disproportionately large cerebellum in the elephant (Maseko
et al., 2012b). A Spearman’s rho correlation between brain mass
and cerebellar volume revealed a strong positive relationship
[r(13) = 0.977, p = 0.01]. Cerebellar volume averaged 13.6 ±
3.1% of total brain mass, with the following breakdown: whale
(18.6%), elephant (18.5%), giraffe (13.6%), tiger (13.8%), man-
atee (13.4%), leopard (11.1%), chimpanzee (10.4%), and human
(9.3%). Dendritic measures and soma size also tended to increase
with cerebellar volume for most neuronal types, particularly in
terms of dendritic Vol and TDL (Table 3). However, removing
the elephant data resulted in a 32% decrease in the magnitude
of these correlations and a reduction in the number of significant
correlations from 17 to 8 (Table 3), suggesting that the elephant
measures were skewing overall results.
Histologically, there was considerable variation in the Golgi
stain across species. Each impregnation was nevertheless of suf-
ficient quality to allow for adequate quantification of selected
neurons (Figures 2–7). The expected trilaminate architecture of
cerebellar cortex was present in all species. The molecular layer,
similar to supragranular layers in the cerebral neocortex (Jacobs
et al., 1997, 2001), tended to stain better (i.e., exhibited a clearer
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Jacobs et al. Neuronal morphology in cerebellar cortex
FIGURE 2 | Photomicrographs of Golgi-stained stellate neurons. (A) Florida manatee; (B) chimpanzee (see also Figure 11Q); (C) human (see also
Figure 11D); (D) giraffe; and (E) African elephant (see also Figure 8C). Scale bars: 100μm.
background with less obstructed, more complete neurons) than
the deeper granule cell layer, which allowed more molecular than
granular layer inhibitory interneurons to be traced (Table 1). A
Spearman’s rho correlation indicated a significant (p = 0.01) pos-
itive relationship between soma size and all dendritic measures in
the total sample [Vol: r(317) = 0.826; TDL: r(317) = 0.497; MSL:
r(317) = 0.647; DSC: r(317) = 0.253] except Tor, which was neg-
ative [r(317) = −0.285]. In terms of morphology, traced neurons
tended to be similar across all species. When comparing across all
neuron types, molecular layer interneurons consistently fell in the
middle of all dendritic measures. The largest neurons traced were
the Lugaro neurons (dendritic Vol ranged from 1454μm3 in the
chimpanzee to 37,396μm3 in the elephant) and the Golgi neu-
rons (TDL ranged from 1456μm in the chimpanzee to 5664μm
in the elephant). Lugaro neurons also tended to have the longest
MSL values (ranging from 41μm in the giraffe to 109μm in
the tiger) whereas Golgi neurons tended to have the highest
DSC values (ranging from 30 in the leopard to 143 in the ele-
phant). Granule neurons exhibited the lowest values for every
measure except Tor, which obtained its highest value in granule
neurons (ranging from 1.17 in the humpback whale to 1.51 in the
human).
Sample tracings of neuronal types for each species are provided
in Figures 8–11. Mean values of selected dependent measures
(i.e., Vol, TDL, MSL, DSC) for each neuronal type across species
are presented in Figure 12. Although the graphs in Figure 12
illustrate mean values, only the ranges across species are used
in the text below for these dendritic measures because (1) there
is asymmetric variation in the dependent measures across and
within species and neuronal types, and (2) the extremely large
values exhibited by the elephant for most of the dendritic mea-
sures distort the overall means. The morphological characteristics
of these neuronal types are addressed in detail below, followed by
the results from interspecies comparisons of dendritic measures.
MOLECULAR LAYER
Stellate neurons (Figure 2) were the most superficial neurons
traced (Meansoma depth = 2.3 ± 108μm). Their round or ovoid
somata were smaller than all other neurons except granule
cells, with a 2.13-fold range in size across species (chim-
panzee = 86μm2 < human < whale < giraffe < leop-
ard < tiger < manatee < elephant = 183μm2). Sample trac-
ings of stellate neurons are provided for each species: African
elephant (Figures 8A–C), Florida manatee (Figures 8M–Q),
Siberian tiger (Figures 9A–C), clouded leopard (Figures 9S–V),
humpback whale (Figures 10F–H), giraffe (Figures 10R–T),
human (Figures 11D–F), and chimpanzee (Figures 11P–R).
Morphologically, stellate neurons exhibited twisting dendrites
that frequently approached the pial surface. Some appeared bipo-
lar (Figure 10S) whereas others had multiple dendrites radiating
in all directions (Figures 8A, 11D,P). Stellate neurons had 4.1 pri-
mary branches per neuron (ranging from 2.8 in the tiger to 5.6
in the elephant) with a dendritic plexus that generally appeared
more complex in the human and elephant than in other species.
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Jacobs et al. Neuronal morphology in cerebellar cortex
FIGURE 3 | Photomicrographs of Golgi-stained basket neurons. (A)
Humpback whale; (B) chimpanzee; (C) giraffe; and (D) African elephant. The
pericellular baskets encapsulating Purkinje cell bodies (unstained) are
represented in (E) Siberian tiger and (F) humpback whale. Scale bars:
100μm.
Quantitatively, dendritic measures varied considerably, although
they tended to be greater for the elephant, particularly for den-
dritic Vol and TDL (Figures 12A–D). Variation in each dendritic
measure for stellate neurons across species was as follows: Vol =
14.63-fold, TDL = 4.02-fold, MSL = 2.14-fold, DSC = 3.56-fold,
and Tor = 1.07-fold.
Basket neurons (Figure 3) were located in the lower third
of the molecular layer (Meansoma depth = 350 ± 134μm). Their
typically ovoid somata were larger than observed in stel-
late neurons, with a 3.86-fold difference in size across
species (human = 113μm2 < chimpanzee < leopard <
tiger < whale < giraffe < manatee < elephant = 444μm2).
Sample tracings of basket neurons are provided for each
species: African elephant (Figures 8D,E), Florida manatee
(Figures 8R–V), Siberian tiger (Figures 9I–K), clouded leop-
ard (Figures 9W–Y), humpback whale (Figures 10A–E), giraffe
(Figures 10U–X), human (Figures 11A–C), and chimpanzee
(Figures 11S–V). Morphologically, basket neurons were usu-
ally, but not always (Figures 8D, 11C), characterized by den-
dritic branches that extended laterally from the soma, travelling
horizontally a short distance before curving toward the pial sur-
face in a typical sea-fan shape (Figures 8E, 9X, 10D, 11S). Axons
were visible in some neurons, allowing them to be traced over
distances of several 100μm (Figures 8R, 11C,U). These axons
travelled transversely above the Purkinje cell layer and were
sometimes observed to terminate in multiple pericellular nests
(Figure 3F) with paintbrush tips (Figure 3E; Ramón y Cajal,
1909, 1911) around the somata of Purkinje cells. Basket neurons
had an average of 4.1 primary dendrites (ranging from 3.0 in
the whale to 6.1 in the manatee). As with stellate neurons, they
appeared more dendritically complex in the human and elephant
relative to the other species. Quantitatively, there was considerable
variation among species, with the elephant generally exhibiting
the largest dendritic values (Figures 12E–H). Ranges of variation
in each dendritic measure for basket neurons across species were
as follows: Vol = 13.51-fold, TDL = 4.83-fold, MSL = 1.72-fold,
DSC = 3.10-fold, and Tor = 1.07-fold.
PURKINJE CELL LAYER
Purkinje neurons were not quantified in the present sample
because their dendritic complexity precluded (accurate) tracings;
in fact, the distal dendritic plexuses in many of these neurons
were completely black under the 60x objective. Nevertheless, sam-
ple photomicrographs (Figure 4) illustrate large, piriform somata
from which the complex, prototypical two-dimensional dendritic
plexus ascended throughout the molecular layer. These appeared
morphologically similar across all species except the humpback
whale (Figure 4C), where tertiary dendritic branches tended to
ascend to the pial surface in straight, unbending manner. As such,
the main dendritic branches of the humpback Purkinje neuron
are much less convoluted than observed in the other species. This
morphological difference is particularly clear when comparing
the skeletal tracings of the humpback whale Purkinje neurons
(Figures 4F,G) to those of the giraffe (Figures 4H,I), the other
cetartiodactyl in the current study.
GRANULE CELL LAYER
Lugaro neurons (Figure 5) were usually located superficially in
the granule cell layer immediately below the Purkinje cell bod-
ies (Meansoma depth = 543 ± 179μm). Those Lugaro neurons
located between Purkinje cell and granule cell layers usually pos-
sessed fusiform somata (e.g., Figures 8K, 10K, 11Z,AA); those
deeper in the granule cell layer were more likely to possess tri-
angular somata. In terms of soma size, these were the largest
observed in the present sample, with a 5.01-fold difference in
size across species (manatee = 270μm2 < chimpanzee and
giraffe < human < leopard < tiger < whale < elephant =
1354μm2). There was an average of 3.9 primary dendrites per
neuron (ranging from 3.2 in the tiger to 5.3 in the leopard). Those
neurons with fusiform somata tended to be horizontally ori-
ented in the parasagittal plane with dendrites that branched little
and extended over several 100μm (Figures 8K, 9L, 10J,K, 11G).
Those with more triangular shaped somata were oriented in vari-
ous directions, including perpendicular to the Purkinje cell layer,
and were particularly common in the elephant (Figures 8J,K,
9N, 11Y). In contrast to other cerebellar neurons, which tended
to be relatively uniform in appearance, the Lugaro neurons were
more morphologically diverse, as indicated in the tracings for
each species: African elephant (Figures 8I–L), Florida mana-
tee (Figure 8W), Siberian tiger (Figures 9 L–N), clouded leop-
ard (Figures 9Z–AA), humpback whale (Figures 10I–L), giraffe
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Jacobs et al. Neuronal morphology in cerebellar cortex
FIGURE 4 | Photomicrographs of Golgi-stained Purkinje neurons. (A)
African elephant; (B) human; (C) humpback whale; (D) Florida manatee; and
(E) giraffe. Note the large similarity between the neurons of all species
except the humpback whale, where the Purkinje neuron appears to have
more vertically oriented tertiary branches. Sample tracings of the main
dendritic branches illustrating this morphological difference are provided for
the humpback whale (F,G), which differs substantially even from the other
cetartiodactyl in the study, the giraffe (H,I). Scale bars: 100μm.
(Figures 10Y–AA), human (Figures 11G–I), and chimpanzee
(Figure 11W–AA). Quantitatively, Lugaro neurons were dispro-
portionately larger in the elephant than in any of the other species,
particularly in terms of dendritic Vol (Figures 12I–L). Variation
in each dendritic measure for Lugaro neurons across species was
as follows: Vol = 25.72-fold, TDL = 3.36-fold, MSL = 2.66-fold,
DSC = 3.44-fold, and Tor = 1.08-fold.
Golgi neurons (Figure 6) were also usually located superfi-
cially in the granule cell layer, although some were much deeper
(Meansoma depth = 512 ± 175μm). They had irregular stellate,
triangular, or polygonal somata, with a 3.05-fold difference
in size across species (leopard = 242μm2 < chimpanzee <
human < tiger < whale < giraffe < elephant = 737μm2).
With an average of 6.5 primary dendrites per neuron (rang-
ing from 5.3 in the whale to 8.2 in the elephant), they exhib-
ited the highest number of primary dendrites in the present
sample. These dendrites radiated relatively thick branches in
all directions, forming a characteristic three-dimensional spher-
ical field, as illustrated in Neurolucida tracings: African ele-
phant (Figures 8F–H), Siberian tiger (Figures 9D–H), clouded
leopard (Figures 9BB,CC), humpback whale (Figures 10P,Q),
giraffe (Figures 10BB–EE), human (Figures 11J–L), chimpanzee
(Figures 11GG–JJ). Quantitatively, as with most other neurons
in the current sample, these achieved their greatest extent in the
elephant (Figures 12M–P). Variation in each dendritic measure
for Golgi neurons across species (except the manatee) was as fol-
lows: Vol = 12.94-fold, TDL = 3.89-fold, MSL = 1.76-fold, DSC
= 4.77-fold, and Tor = 1.07-fold.
Granule neurons (Figure 7) were, on average, the most deeply
located of all traced neurons (Meansoma depth = 615 ± 181μm),
and the smallest, with a 1.36-fold difference in size across
species (human = 50μm2 < leopard < chimpanzee < whale
and tiger < giraffe = 68μm2). These were characterized by
small, round cell bodies from which an average of 3.5 short
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Jacobs et al. Neuronal morphology in cerebellar cortex
FIGURE 5 | Photomicrographs of Golgi-stained Lugaro neurons. (A, see
also Figure 8J) and (C, see also Figure 8L) African elephant; in these two
neurons, note the bouquet shaped dendritic arbor in (A) and the more
solitary, unbranched dendritic arbor in (C), with both descending to the
underlying white matter. The (B) humpback whale (see also Figure 10L)
and (D) chimpanzee also have predominantly unbranched dendritic trees.
ML, molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer. Scale
bars: 100μm.
dendrites emerged (ranging from 3.0 in the human to 3.8
in the giraffe, whale, and tiger), each terminating in gnarled,
claw-like inflorescences. Axons, when visible, tended to ascend
immediately toward the molecular layer (Figures 9Q, 10FF,
11M,BB,CC). Across species, there was little qualitative variation
in granule neuron morphology: Siberian tiger (Figures 9O–R),
humpback whale (Figures 10M–O), giraffe (Figures 10FF–HH),
human (Figures 11M–O), and chimpanzee (Figures 11BB–FF).
Quantitatively, granule neurons had the lowest median values
of all dendritic measures except Tor, for which they exhibited
the highest values (Figures 12Q–T). Variations in each dendritic
measure for granule neurons across species (except the elephant
and manatee) were as follows: Vol = 5.34-fold, TDL = 2.41-fold,
MSL = 6.40-fold, DSC = 2.50-fold, and Tor = 1.29-fold.
SHOLL ANALYSES
Several limited observations can be made on the basis of the
Sholl analyses (Figure 13). First, the peak in the number of
intersections appeared to be around 100μm from the soma
FIGURE 6 | Photomicrographs of Golgi-stained Golgi neurons. (A) and
(B, see also Figure 8H) African elephant; (C) chimpanzee (see also
Figure 11GG); (D) Siberian tiger (see also Figure 9E); (E) giraffe (see also
Figure 10DD); and (F) clouded leopard (see also Figure 9BB). Scale bars:
100μm.
for most neuron types for all species. Second, Lugaro neurons
(Figures 13I–L) did not exhibit the same sharp peak in intersec-
tions as did other neurons; rather, they were relatively flat in their
dendritic envelope, with dendrites that extended great distances
from the soma, particularly in humans and elephants. Third,
the elephant profile (Figures 13A,E,M) appeared markedly differ-
ent (i.e., much higher peaks) from other species’ profiles. Forth,
granule neurons (Figures 13Q–S) had a much lower number of
intersections than did other neurons, and exhibited a peak around
25μm from the soma.
INFERENTIAL STATISTICAL ANALYSES ACROSS SPECIES
To examine species differences in dendritic measures, analy-
ses proceeded with a third order MARSplines differentiation of
species using TDL, MSL, DSC, Vol, Tor, and soma size. In brief,
the procedure tested the dendritic measures and soma size of each
neuron to assess if it could be identified as belonging to a par-
ticular species. Eight binary variables were created, one for each
species. The analysis proceeded by utilizing a MARSplines model
to test the hypothesis that species could be differentiated from
each other based on just dendritic measures and soma size. As an
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Jacobs et al. Neuronal morphology in cerebellar cortex
FIGURE 7 | Photomicrographs of Golgi-stained granule neurons. (A)
Siberian tiger; (B) humpback whale; (C) chimpanzee; (D) Siberian tiger; and
(E) giraffe. Scale bars: 100μm.
example, to test the hypothesis with giraffes, a new attribute called
giraffe was created, and it took a value 1 when the dendritic mea-
sures came from a giraffe neuron and a 0 when they did not. Eight
of these binary (1/0) attributes were created, one for each species.
Each of the 317 rows of neuronal data was coded with eight 1/0
attributes. For example, there was a row for a giraffe neuron with
the eight new species attributes arranged in alphabetical order
(chimpanzee, clouded leopard, elephant, giraffe, human, hump-
back whale, manatee, tiger); then, these attributes were coded 0,
0, 1, 0, 0, 0, 0, 0, respectively. Next, the MARSplines analysis was
used with the giraffe attribute as the dependent measure and the
dendritic measurements as the independent measures to test the
null hypothesis by comparing neuronal measures contributed by
the giraffe to those contributed by the other seven species. In
the present study, the null hypothesis was that there was no rela-
tionship between the dendritic measures and species, or, in other
words, that the dendritic measures for a given neuron could not
be assigned reliably to a given species. The null hypothesis was
rejected at either p < 0.10 on the F-test or a correct prediction
(≥0.90) about whether a neuron did or did not belong to a par-
ticular species. Results were represented as counts (i.e., number
of neurons that successfully differentiated a given species from all
the other species) and percentages of correct and incorrect species
assignments, an r2 statistic, and an F statistic. F statistics were
produced using each binary species attribute as the categorical
or class measure and the “is or isn’t” as the target species score
estimated from the MARSplines. The estimated scores were con-
tinuous, and the cut point for whether or not the estimated value
was the species under analysis or some other species was 0.5. This
cut point was used in calculating the percentage of correct predic-
tions. That is, if giraffes were being evaluated, the observed value
of the giraffe 1/0 attribute would have been 1 and if the estimated
value of giraffe was ≥0.5, we would conclude that the MARSpline
equation correctly differentiated giraffes from all other species in
that row of data.
The null hypothesis of no relationship between dendritic mea-
sures and species was rejected for all species because all F statis-
tics were significant at p ≤ 0.01: elephant [F(1, 253) = 8097.30,
r2 = 0.967], manatee [F(1, 267) = 334.33, r2 = 0.526], tiger
[F(1, 315) = 137.93, r2 = 0.265], leopard [F(1, 315) = 110.63, r2 =
0.260], whale [F(1, 315) = 373.76, r2 = 0.208], giraffe [F(1, 315) =
290.12, r2 = 0.448], human [F(1, 315) = 196.05, r2 = 0.349], and
chimpanzee [F(1, 315) = 720.62, r2 = 0.443]. Further, as noted in
the correct-incorrect confusion matrices (Table 4), the percent-
age of correct predictions for neuronal fit to a particular species
ranged from 85.5% in the chimpanzee to 99.6% in the elephant.
To elaborate, in the elephant, 19 of 20 neurons were correctly
identified as belonging to the elephant, and 235 of 235 were cor-
rectly identified as not belonging to the elephant (thus, 99.6%).
In the chimpanzee, 63 of 86 neurons were correctly identified as
belonging to the chimpanzee, and 208 of 231 were correctly iden-
tified as not belonging to the chimpanzee (thus, 85.5%). What
these results indicate is that dendritic measures and soma size
were accurate predictors of each species in the current sample
because these measures, taken together, allowed neurons to be
correctly identified as belonging to a particular species.
Further, the procedure provided the relative importance of
each attribute (i.e., dendritic Vol, TDL, soma size, etc.) in deter-
mining whether a neuron belonged or did not belong to a
particular species (Table 4). These measures indicated the num-
ber of times each attribute or predictor was used in the equations
testing the null hypothesis for each species. In this framework, for
example, an attribute with three appearances in the analysis for
a given species would be three times more important to the pre-
diction than an attribute with only one appearance. As noted in
Table 4, across all species, dendritic Vol, Tor, and TDL appeared to
be the overall most important (i.e., most utilized by the analysis)
measures for differentiating each species from the others, whereas
DSC was the least important. However, the combination of these
variables was unique for each species. For example, in the ele-
phant, TDL (17 appearances) and dendritic Vol (16 appearances)
were the most influential measures in species identification; in the
chimpanzee, however, soma size (9 appearances) and dendritic
Vol (6 appearances) were the most important predictors.
DISCUSSION
The present study contributes to a limited database of compara-
tive neuroanatomy (Manger et al., 2008) by examining cerebellar
neuronal morphology across a wide variety of large brainedmam-
mals both qualitatively and quantitatively. Although the current
sample exhibited a large range in cerebellar volume, the overall
volume fraction of the cerebellum (13.6 ± 3.1%) is consistent
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Jacobs et al. Neuronal morphology in cerebellar cortex
FIGURE 8 | Neurolucida tracings of neurons in the cerebellar cortex of the African elephant (top) and Florida manatee (bottom) indicating relative soma
depth from the pial surface (inµm). Stellate neurons (A–C; M–Q); basket neurons (D,E; R–V); Lugaro neurons (I–L; W); Golgi neurons (F–H). Scale bar: 100μm.
with that reported by Clark et al. (2001) across 9 mammalian
taxa, namely 13.5 ± 2.4%. There was considerable uniformity
across species in terms of histology insofar as the cerebellar
cortex followed the trilaminate pattern typical of birds and mam-
mals (Ramón y Cajal, 1909, 1911; Sultan and Glickstein, 2007).
In terms of morphology, each neuronal type within the cere-
bellar cortex was generally consistent across the eight species.
Quantitatively, however, there was substantial species variation
in dependent dendritic measures for each neuronal type, with
neurons in the elephant tending to be larger than those in other
species for most measures. Finally, inferential analyses detected
significant species differences in dendritic measures and soma
size.
METHODOLOGICAL CONSIDERATIONS
General constraints pertaining to Golgi-stained materials have
been extensively outlined elsewhere (Jacobs and Scheibel, 2002;
Jacobs et al., 2011). These include (1) characteristics of
incomplete impregnations (Williams et al., 1978; Braak and
Braak, 1985), (2) the effects of post-mortem delay and subopti-
mal fixation (de Ruiter, 1983; Jacobs and Scheibel, 1993; Jacobs
et al., 1993, 2001; Friedland et al., 2006), and (3) the relative mer-
its of the Golgi stain compared to other histological techniques
(Scheibel and Scheibel, 1978; Buell, 1982; Ohm and Diekmann,
1994; Jacobs et al., 1997). Another inherent limitation in Golgi
studies is the effect of section thickness on estimations of den-
dritic extent (Jacobs et al., 1997). Larger neurons (such as those
in the elephant) are more affected by sectioning, resulting in
an attenuation of dendritic measures. In the present study, this
means that actual differences among species are probably larger
than the data suggest. Thus, the present dendritic measurements
should be seen as representing relative rather than absolute val-
ues. To completely eliminate cut dendrites would require (1)
tissue sections ∼1000μm thick, which would make them com-
pletely opaque, or (2) tracing cut dendritic segments across serial
sections, a technique that is not accurate or feasible in a Golgi
study this extensive, where multiple, overlapping neural pro-
cesses appear in any given section of tissue. Finally, neurons in
the present study were classified based solely on somatodendritic
architecture and their relative location within the cerebellar cor-
tex, which is typical for Golgi impregnations. It was not possible
to further subcategorize neuron types based on axonal plexi dis-
tributions (Bishop, 1993; Lainé and Axelrad, 1996), lipofuscin
pigmentation (Braak and Braak, 1983), or immunohistochem-
istry and neurochemical phenotypes (Lainé and Axelrad, 2002;
Simat et al., 2007).
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Jacobs et al. Neuronal morphology in cerebellar cortex
FIGURE 9 | Neurolucida tracings of neurons in the cerebellar cortex of
the Siberian tiger (top) and clouded leopard (bottom) indicating relative
soma depth from the pial surface (in µm). Stellate neurons (A–C; S–V);
basket neurons (I–K; W–Y); Lugaro neurons (L–N; Z–AA); Golgi neurons
(D–H; BB,CC); granule neurons (O–R). Axons, when present, are indicated in
red. Scale bar: 100μm.
NEURONAL MORPHOLOGY: QUALITATIVE OBSERVATIONS
In general, neurons in the present sample were similar in mor-
phology to those described in primates (Fox et al., 1967; Rakic,
1972; Braak and Braak, 1983; Mavroudis et al., 2013) and rodents
(O’Leary et al., 1968; Chan-Palay and Palay, 1972; Palay and
Chan-Palay, 1974). For example, molecular layer interneurons
in the present sample had hemielipsoid dendritic systems that
greatly resembled those described by Rakic (1972), with deeper
neurons having an ascending dendritic plexus, intermediate neu-
rons having a dendritic plexus extending in all directions, and
superficial neurons having mostly descending dendritic branches.
The axonal ramifications observed in basket neurons were also
similar to those described in monkeys (Fox et al., 1967), cats
(Bishop, 1993), and rats (Palay and Chan-Palay, 1974). There
were, however, a few morphological observations of particu-
lar interest. First, the Lugaro neurons in most of the current
species resembled those described in the literature (Christ, 1985;
Melik-Musyan and Fanardzhyan, 1998, 2004). They also resem-
bled those identified by Braak and Braak (1983) in humans as
Type II neurons despite Braak and Braak stating that these types
of cells may be displaced basket neurons, a finding disputed
by others (Lainé and Axelrad, 1996). In contrast, many Lugaro
neurons in the elephant appeared distinctive because of their ver-
tical orientation and idiosyncratic dendritic arrangements. These
were unusual in the present sample, although they have been
briefly described in the cat (Sahin and Hockfield, 1990; Melik-
Musyan and Fanardzhyan, 1998) and the duck (O’Leary et al.,
1968). Second, most of the traced Golgi neurons resembled those
observed in the literature, and are consistent with Braak and
Braak’s (1983) Type I neuron designation. However, there are
some (e.g., Figures 8G, 11K) that resembled Braak and Braak’s
Type III description (see their Figure 7) insofar as they exhibited,
among other characteristics, a very dense dendritic arborization
that extended into the molecular layer.
Purkinje neurons were remarkably similar in their basic mor-
phology across most species examined, and were consistent with
those described in other species (rat: Roth and Häuser, 2001;
Sawada et al., 2010; guinea-pig: Rapp et al., 1994). The notable
exception was the Purkinje neuron in the humpback whale, which
exhibited straighter, more vertically oriented tertiary dendritic
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Jacobs et al. Neuronal morphology in cerebellar cortex
FIGURE 10 | Neurolucida tracings of neurons in the cerebellar cortex of
the humpback whale (top) and giraffe (bottom) indicating relative soma
depth from the pial surface (in µm). Basket neurons (A–E; U–X); stellate
neurons (F–H; R–T); Lugaro neurons (I–L; Y–AA); Golgi neurons (P,Q;
BB–EE); granule neurons (M–O; FF–HH). Axons, when present, are indicated
in red. Scale bar: 100μm.
arbors than any of the other species examined. This dendritic
pattern differed from the other aquatic mammal in the cur-
rent study (e.g., the manatee) and from the other cetartiodactyl
(e.g., the giraffe). Humpback whale Purkinje neurons actually
resembled those observed in mormyrid electric fish (Meek and
Nieuwenhuys, 1991; Meek, 1992; Han et al., 2006), with a pal-
isade pattern of relatively unbranched, molecular layer dendritic
arbors that extend in parallel to the pial surface (Nieuwenhuys
and Nicholson, 1967; Meek and Nieuwenhuys, 1991). Visual
observation of our Golgi stains suggests, however, that the
humpback whale may have substantially fewer palisade den-
drites than the ∼50 noted in mormyrid Purkinje neurons (Meek
and Nieuwenhuys, 1991). Direct comparison within cetaceans is
problematic because only Adanina (1965) provides any images
of Golgi impregnated cerebellar cortical neurons in cetaceans
(specifically, Tursiops truncatus and Delphinus delphis). Adanina
suggests that there is heterogeneity in the somata of Purkinje neu-
rons, with some being pear-shaped and others being fusiform.
Unfortunately, although Purkinje neuron dendritic arbors in the
dolphin may be consistent with our observations in the hump-
back whale, Adanina’s impregnation is insufficient for a definitive
conclusion. Further research is necessary to confirm whether
Purkinje neurons in mysticetes are morphologically different
from those in other cetaceans, or from mammals in general.
NEURONAL MORPHOLOGY: QUANTITATIVE OBSERVATIONS
With the exception of Purkinje neuron reconstructions and a few
measurements of individual dendritic segments, there appear to
be very limited quantitative dendritic data on cerebellar cortical
neurons (Palay and Chan-Palay, 1974; Braak and Braak, 1983).
There is, however, a small number of digital reconstructions of
molecular layer interneurons (classical stellate and basket neu-
rons) in the rat based on rapid Golgi impregnations (Sultan and
Bower, 1998). Not surprisingly, soma size (69μm2) in these rat
neurons was smaller than in stellate neurons from all species in
the present sample. The number of primary branches (3.1) in the
rat was also near the minimum of the current sample (tiger: 2.8
primary branches in stellate neurons; whale: 3.0 primary branches
in basket neurons). Similarly, the dendritic length for rat molec-
ular interneurons (1189μm) was shorter than the lowest values
for stellate (leopard TDL = 1367μm) and basket neurons (tiger
TDL = 1285μm) in the current sample (see Figure 12), although
it should be noted that Sultan and Bower (1998) sectioned their
tissue at 100μm rather than 120μm, which could result in more
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Jacobs et al. Neuronal morphology in cerebellar cortex
FIGURE 11 | Neurolucida tracings of neurons in the cerebellar cortex of
the human (top) and chimpanzee (bottom) indicating relative soma depth
from the pial surface (in µm). Basket neurons (A–C; S–V); stellate neurons
(D–F; P–R); Lugaro neurons (G–I; W–AA); Golgi neurons (J–L;GG–JJ); granule
neurons (M–O; BB–FF). Axons, when present, are indicated in red. Note that
the axons for basket neurons (B) and (U) followed the curvature of the folia in
their original sections for a long distance, and thus incorrectly appear, here in
the schematic, to extend to or beyond the pial surface. Scale bar: 100μm.
attenuated dendritic length values. To the extent that we can gen-
eralize from the present sample, it appears that, among molecular
layer interneurons, the more superficial stellate neurons tended
to be smaller than the deeper basket neurons for most dendritic
measures, a finding consistent with observations in the cerebral
neocortex, where deeper neurons tend to be larger than more
superficial neurons (Jacobs et al., 1997, 2001). In the granule cell
layer, Lugaro and Golgi neurons were typically similar in overall
size (e.g., dendritic Vol and TDL); however, they exhibited vastly
different morphologies, as reflected in typically greater MSL val-
ues for Lugaro neurons (indicating long, unbranched dendrites)
and higher DSC values for Golgi neurons (suggesting a more
complex dendritic branching pattern).
Variability in neuronal measures across species was much
smaller than that observed for brain mass (64-fold) and cere-
bellar volume (103-fold). In general, dendritic measurements
and soma size tended to be positively correlated with cerebel-
lar volume for most neuron types, although this tendency was
skewed by the large size of elephant neurons. There was a 3.08-
fold difference in soma size among the species in the current
sample, with the elephant having the largest values across all neu-
ron types. Moreover, most neuron types were characterized by
a 3.5- to 5-fold range of variation in dendritic measures across
species. The Lugaro neuron, however, averaged a 7.25-fold vari-
ation across species, mainly because of the extraordinary size of
Lugaro neurons in the elephant (Maseko et al., 2012a)—note
that excluding the elephant data resulted in a 3.19-fold varia-
tion in Lugaro neurons. Although the length of Lugaro dendrites
in the present sample appears to be within the range of what
has been reported in rats (i.e., from 100 to 700μm from the
soma; Lainé and Axelrad, 1996), it is the measure of Lugaro
dendritic Vol. that especially differentiates the elephant from
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Jacobs et al. Neuronal morphology in cerebellar cortex
FIGURE 12 | (Continued)
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Jacobs et al. Neuronal morphology in cerebellar cortex
FIGURE 12 | (Continued)
Frontiers in Neuroanatomy www.frontiersin.org April 2014 | Volume 8 | Article 24 | 20
Jacobs et al. Neuronal morphology in cerebellar cortex
FIGURE 12 | Bar graphs indicated the relative values of four dependent
measures (Volume, Total Dendritic Length, Mean Segment Length,
and Dendritic Segment Count) for stellate (A–D), basket (E–H), Lugaro
(I–L), Golgi (M–P), and granule (Q–T) neurons across the eight species
(Continued)
FIGURE 12 | Continued
in the current study. The eight species are arranged from left to right on
the abscissa in a fixed order, from the smallest (clouded leopard) to the
largest brain (elephant). Phylogenetic relationships among species are color
coded as follows: afrotherians (dark blue = elephant; light blue = manatee);
carnivores (black = tiger; gray = leopard); cetartiodactyls (dark brown =
humpback; light brown = giraffe); and primates (dark green = human; light
green = chimpanzee). Note the following: (1) tortuosity measures are not
illustrated here; (2) granule neurons in afrotherians, and Golgi neurons in
the manatee are not illustrated here because they did not stain; and (3) the
ordinate scale for granule neurons is much smaller than the scale for other
neuron types. Error bars = s.e.m.
other species. For dendritic Vol, there was an average 14.43-fold
increase across species in the current sample, as opposed to an
average 2.81-fold increase for all other dendritic measures. This
suggests that dendritic Vol might scale more steeply than other
dendritic measures for cross-species comparisons in the cerebel-
lum, an observation that seems to have been confirmed in the
current MARSplines analysis, which indicated that dendritic Vol
was the most consistently used variable for interspecies differen-
tiation. These findings also appear consistent with the suggestion
that there is a positive relationship between brain mass and
dendritic extent in the neocortex (Elston et al., 2006; Herculano-
Houzel et al., 2006; Sarko et al., 2009; Jacobs et al., 2011; Manger
et al., 2013), a corollary being that, similar to the cerebral cortex
(Haug, 1967, 1987), neuronal density in the cerebellum appears
to be inversely related to brain mass (Lange, 1975; Maseko et al.,
2012a).
INFERENTIAL STATISTICAL COMPARISONS ACROSS SPECIES
One goal of the present investigation was to compare neuronal
morphology in the cerebellar cortex across several large brained
mammals not previously examined. We tested for species dif-
ferences using MARSplines analyses, which indicated that there
were significant differences in dendritic measures (and soma
size) among all species. Moreover, this analysis revealed not only
which measures were most important for differentiating individ-
ual species, but also the unique combinations and weightings
of these measures (Table 4). In future studies, a data set with a
much larger number of neurons, and with all neuron types repre-
sented for every species, would enable a more detailed evaluation
of the relative importance of neuron types (e.g., Lugaro vs. Golgi)
to species differentiation. At this point, an interesting question
is whether the evolutionary, ecological, and behavioral adapta-
tions that influence brain mass and cerebellar volume, might also
shape aspects of the somatodendritic morphology in neurons
themselves.
FUNCTIONAL SPECULATIONS
The large-brained mammals in the current sample represent a
diverse range of ecological, somatic, and behavioral adaptations.
Here, we can only speculate very generally how these adapta-
tions may relate to factors such as cerebellar volume and neuronal
morphology in these species. Insofar as the cerebellum has tra-
ditionally been implicated in motor control (Fulton and Dow,
1937; Marr, 1969; Glickstein and Yeo, 1990), the motor system
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Jacobs et al. Neuronal morphology in cerebellar cortex
FIGURE 13 | Sholl analyses of five neuron types (stellate: A–D; basket:
E–H; Lugaro: I–L; Golgi: M–P; granule: Q–S), arranged by taxonomic
groupings (afrotherians: elephant and manatee; carnivores: tiger and
clouded leopard; cetartiodactyls: humpback whale and giraffe; and
primates: human and chimpanzee), indicating relative dendritic
complexity of branching patterns. Dendritic intersections were quantified
at 20-μm intervals using concentric rings. Note that granule neurons in
afrotherians, and Golgi neurons in the manatee are not illustrated here
because they did not stain, and that the ordinate scale for granule neurons is
much smaller than the scale for other neuron types.
of a particular species is often the initial focal point (Onodera
and Hicks, 1999). For example, the elephant possesses the largest
absolute and relative cerebellar volume of any mammal inves-
tigated to date (Shoshani et al., 2006; Maseko et al., 2012b), a
finding typically explained with reference to the fine motor con-
trol demands of its trunk (Endo et al., 2001; Maseko et al., 2012b).
A more integrative, and perhaps parsimonious, perspective sug-
gests that the cerebellum is not involved exclusively with motor
control, but rather that it is engaged in monitoring and adjusting
the acquisition of sensory information for the rest of the nervous
system (Bower, 1992, 1997; Gao et al., 1996). As such, one factor
contributing to the large elephant cerebellum may be the doc-
umented importance of the trunk in multi-sensory exploration
of the environment (Rasmussen and Munger, 1996; Bagley et al.,
2006; Foerder et al., 2011). If this theory is extended to the domain
of communication, the auditory-tactile infrasound information
perceived through the elephant’s feet may also contribute to its
enlarged cerebellum (Garstang, 2004; Bouley et al., 2007; Soltis,
2009).
Additional investigations into the sensory role of cerebellum
may provide insight into the other species examined in the cur-
rent study. For example, imaging research has revealed that the
lateral cerebellar hemispheres are involved in sensory acquisition
and discrimination in humans (Parsons et al., 1997), a finding
that may also apply to chimpanzees. Certainly, this is consistent
with the expansion of the lateral cerebellum in hominoids rela-
tive to other primates (Rilling and Insel, 1998; MacLeod et al.,
2003; Rilling, 2006). Electrophysiological research has shown that
cats have larger tactile representations of the forelimbs in the lat-
eral cerebellar hemispheres than do rodents (e.g., mice, rats, and
guinea pigs) because cats use their forelimbs more for sensory
exploration of their environment than do rodents, which depend
more on tactile information from the face region (Welker, 1964;
Bower, 1997, 2011). Speculatively, cerebellar sensory representa-
tions for the manatee may resemble those of rodents insofar as
the manatee has a sensitive, perioral tactile system for explor-
ing its aquatic environment (Marshall et al., 1998, 2003; Reep
et al., 2001). In contrast, the felines of the current study may be
characterized by strong forelimb representation in the lateral cere-
bellum. Finally, such a sensory focus on cerebellar processing may
help clarify why there is a relative increase in cerebellar volume in
microchiropterans and odontocete cetaceans (Baron et al., 1996;
Marino et al., 2000) vis-à-vis primates (Maseko et al., 2012b).
From the perspective ofmotor control, this observation is difficult
Frontiers in Neuroanatomy www.frontiersin.org April 2014 | Volume 8 | Article 24 | 22
Jacobs et al. Neuronal morphology in cerebellar cortex
Table 4 | Correct-incorrect confusion matrices for differentiation of species.
Predicted Predictor importancea
99.6% correctb Elephant Other species Row totalc Vol TDL MSL DSC Tor Soma size
African elephant Elephant 19 1 20 16 17 5 0 8 5
Other species 0 235 235
Column total 19 236 255O
bs
er
ve
d
Florida manatee 98.5% correct Manatee Other species Row total Vol TDL MSL DSC Tor Soma size
Manatee 11 4 15 5 10 7 0 7 15
Other species 0 254 254
Column total 11 258 269O
bs
er
ve
d
Siberian tiger 91.8% correct Tiger Other species Row total Vol TDL MSL DSC Tor Soma size
Tiger 10 23 33 3 5 16 7 7 3
Other species 3 281 284
Column total 13 304 317O
bs
er
ve
d
Clouded leopard 92.1% correct Leopard Other species Row total Vol TDL MSL DSC Tor Soma size
Leopard 8 1 9 9 2 7 0 8 6
Other species 24 284 308
Column total 32 285 317O
bs
er
ve
d
Humpback whale 89.3% correct Whale Other species Row total Vol TDL MSL DSC Tor Soma size
Whale 13 34 47 2 4 6 3 10 1
Other species 0 270 270
Column total 13 304 317O
bs
er
ve
d
Giraffe 90.2% correct Giraffe Other species Row total Vol TDL MSL DSC Tor Soma size
Giraffe 27 29 56 7 3 4 6 6 7
Other species 2 259 261
Column total 29 288 317O
bs
er
ve
d
Human 93.7% correct Human Other species Row total Vol TDL MSL DSC Tor Soma size
Human 8 20 28 8 9 1 8 6 1
Other species 0 289 289
Column total 8 309 317O
bs
er
ve
d
Common chimpanzee 85.5% correct Chimpanzee Other species Row total Vol TDL MSL DSC Tor Soma size
Chimpanzee 63 23 86 6 2 1 3 2 9
Other species 23 208 231
Column total 86 231 317O
bs
er
ve
d
aPredictor importance indicates how many times each measure appears in the regression analysis for that particular species. See text for more details.
bBold numbers represent the correct predictions. The percentage correct is calculated by dividing the sum of the two bold numbers by the total number of neurons
examined. So, for the elephant: (19 + 235)/255 = 0.996.
cNote that the total number of neurons for elephants and manatees is not 317, as for all other species, because some neuron types did not stain in the two
afrotherians.
to explain insofar as primates arguably have greater fine motor
dexterity (Darian-Smith et al., 2007; Kaas, 2008). However, both
microchiropterans and odontocetes rely extensively on the coor-
dinated use of sensory surfaces when exploring their environment
with echolocation (Norris et al., 1961; Ghose et al., 2006; Surlyke
et al., 2009; Akamatsu et al., 2010), and this may contribute to
an expansion in cerebellar tissue. By extension, the humpback
whale cerebellum may also be affected by the whale’s extensive
vocal repertoire (Mercado et al., 2010; Garland et al., 2011) which,
similar to echolocation, may serve as a type of sonar that pro-
vides sensory information about its aquatic world (Frazer and
Mercado, 2000).
Frontiers in Neuroanatomy www.frontiersin.org April 2014 | Volume 8 | Article 24 | 23
Jacobs et al. Neuronal morphology in cerebellar cortex
Finally, although the basic circuitry of the cerebellar cortex is
fairly well documented in a limited number of species, discern-
ing structure-function relationships can be challenging (Sultan
and Glickstein, 2007; Schilling et al., 2009). This is especially true
when direct electrophysiological experimentation on a species is
not possible. There are, however, two morphological findings of
particular functional interest in the present study: (1) the distinc-
tive morphology and large size of Lugaro neurons in the elephant
cerebellum, and (2) the presence of a palisade dendritic pattern
for Purkinje neurons in the humpback whale. With regards to
first observation, what remains unclear is whether the elephant
Lugaro neurons are functionally connected in the samemanner as
demonstrated in other species, that is, whether they receive sero-
tonergic input (Dieudonné and Dumoulin, 2000; Geurts et al.,
2003), input from Purkinje neuron collaterals (Lainé and Axelrad,
1996; Geurts et al., 2003), and/or whether they project to molec-
ular layer interneurons (Flace et al., 2004; Ambrosi et al., 2007)
and to Golgi neurons (Melik-Musyan and Fanardzhyan, 1998;
Dumoulin et al., 2001; Crook et al., 2006). The expansive den-
dritic arbors of elephant Lugaro neuorons would suggest a broad
sampling of local input but, because we have no information on
their axonal projections, it is unclear to what extent they exert
inhibitory feedback on Purkinje neurons, modulate mossy fiber
input, and/or contribute to long-term depression (Geurts et al.,
2003; Melik-Musyan and Fanardzhyan, 2004).With regards to the
humpback whale Purkinje neuron dendrites, any functional spec-
ulation would be premature until future research confirms the
current, tentative findings. If such Purkinje cell dendritic mor-
phology actually obtains in mysticetes, or in cetaceans in general,
then the next question would be whether the acoustic world of
cetaceans and the electrosensory system in mormyrids have any
neurofunctional commonalities. Only more detailed comparative
research will address such issues.
ACKNOWLEDGMENTS
Partial support for this work was provided by Colorado College’s
divisional research funds (Bob Jacobs), the James S. McDonnell
Foundation (grant 22002078, to Chet C. Sherwood, Patrick
R. Hof; grant 220020293 to Chet C. Sherwood), National
Science Foundation (BCS-0827531 to Chet C. Sherwood), and
the South African National Research Foundation (Paul R.
Manger; FA2005033100004). We would also like to thank the
Danish Cardiovascular Research Program, especially Emil Toft-
Brøndum, for allowing us to obtain the specimens of giraffe
brains.
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Conflict of Interest Statement: The authors declare that the research was con-
ducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Received: 31 October 2013; accepted: 01 April 2014; published online: 23 April 2014.
Citation: Jacobs B, Johnson NL, Wahl D, Schall M, Maseko BC, Lewandowski A,
Raghanti MA, Wicinski B, Butti C, Hopkins WD, Bertelsen MF, Walsh T, Roberts
JR, Reep RL, Hof PR, Sherwood CC and Manger PR (2014) Comparative neuronal
morphology of the cerebellar cortex in afrotherians, carnivores, cetartiodactyls, and
primates. Front. Neuroanat. 8:24. doi: 10.3389/fnana.2014.00024
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Wicinski, Butti, Hopkins, Bertelsen,Walsh, Roberts, Reep, Hof, Sherwood andManger.
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