Copeia, 2000(1), pp.

265269

Noninvasive High Field MRI Brain Imaging of the Garter Snake

(Thamnophis sirtalis)
CHARLES L. ANDERSON, GEORGE W. KABALKA, DONNA G. LAYNE,
JONATHAN P. DYKE, AND GORDON M. BURGHARDT
This report documents the application of high field magnetic resonance imaging
to viewing the central nervous system of garter snakes (1243 g) with emphasis on
the vomeronasal and olfactory systems. Slices in all three planes were taken in lightly
sedated snakes showing many major neural structures with resolution down to less
than 60 mm. The images were obtained as 8 or 16 serial slices with slice spacings
of 0.25 mm and 0.25 mm thick and were processed to a 256 3 256 matrix with a
field of view (FOV) of 14 or 25 mm. The vomeronasal organs, main and accessory
olfactory bulbs, telencephalon, cortex, tectum, medulla, cerebellum, spinal cord,
and other major features of the brain are clearly defined in the images. The value
of noninvasive neural imaging in comparative and experimental studies of squamate
reptiles will become increasingly recognized as the technology is further developed
but can be effectively employed at present.

RADITIONAL methods for the anatomical

study of snakes are invasive in nature. This
is especially true of the brain. Due to their importance in the behavior of snakes, the vomeronasal and olfactory systems of snakes and their
projections in the brain have received considerable attention (Halperin, 1992). Building on
the pioneering work of Wilde (1938), a considerable literature has accumulated on the functioning of the chemical senses in squamate reptiles (Burghardt, 1970; Halpern and Holtzman,
1993; Mason, 1992). Our understanding of the
snake olfactory brain has relied upon traditional lesioning, sectioning, and histological methods, primarily in the Common Garter Snake,
Thamnophis sirtalis (Halpern, 1976; Lanuza and
Halpern, 1997, 1998). However, assessing the
projections and sites of damage has meant killing animals for at least the final confirmation
of projections and extent of experimental insult. Research on the neural systems underlying
behavior would be greatly facilitated if the structure and activity of the animals nervous system
could be rapidly assessed noninvasively before
and after experimental treatment. It would also
be valuable to have a method to evaluate rapidly
the relative development of the CNS portion of
chemosensory systems to extend comparative
studies of squamate chemosensory evolution
(Cooper, 1997; Schwenk, 1993). As an initial
step in this direction, we wish to report the first
detailed noninvasive, Magnetic Resonance Imaging (MRI) study of the main and accessory
olfactory bulbs, vomeronasal organ, and cerebral anatomy in living intact garter snakes
(Thamnophis sirtalis).

MATERIALS

AND

METHODS

The images were acquired using five eastern

garter snakes (Thamnophis sirtalis). Three of the
subjects were from the same litter born in the
laboratory in 1992: 1686F, 1686H, and 1686P.
Snakes ranged in size from 3542 cm snout
vent length, 4452 cm total length, and weighed
from 1220 g. Head width ranged from 9.010.3
mm (measured across the parietal scales just behind the eyes). Head length ranged from 12.5
13.6 mm (measured from the tip of the snout
at the rostral scales to the posterior end of the
parietal scales). Two other wild-caught snakes
came into the lab in 1993: subjects 6076 and
6077. Their measurements were 38.5 and 46 cm
snoutvent length, 49 and 57 cm in total length,
and 23.0 and 42.5 g in weight. Head widths were
9.5 and 12.5 mm and head lengths were 13.9
and 15.7 mm. Over a two-month period in 1994,
each animal was scanned from one to four
times.
To immobilize the animal inside the magnetic bore and keep the area of the head that was
to be scanned from moving, each snake was
anaesthetized using an open-drop technique
(Gandal, 1968; Millichamp, 1988). The snake
was placed in a glass jar (; 500 mL) containing
a small perforated plastic canister containing a
cotton ball saturated with six to eight drops of
Metofanet (methoxyflurane) anaesthesia solution. The snake was determined to be sufficiently anaesthetized for this noninvasive procedure
when it lost the ability to right itself when
turned on its back and only weakly responded
to a tail pinch. Breathing and heart rate were
greatly reduced but still clearly evident.

q 2000 by the American Society of Ichthyologists and Herpetologists

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COPEIA, 2000, NO. 1

Fig. 1. A graphical representation of the SEMS

pulse sequence used in these studies.

The snakes head and 24 cm of its upper

body were placed in an open-ended glass tube
(20 mm internal diameter) and inserted into a
20 mm proton selective saddle coil, head down.
The remainder of the snakes body was supported vertically by a cardboard tube. The caudal end of the animal was secured to the cardboard tube with hypoallergenic tape. The whole
assembly was carefully inserted into the bore of
the magnet to ensure that there was no interference from the interior of the magnet.
The studies were carried out using a Bruker
AMX-400 Wide Bore (89 mm) NMR spectrometer equipped with a Micro Imaging Basis Unit.
The shapes of the amplitude modulated pulses
and the gradient strengths (max 35 G/cm21)
were stored in the three waveform memory
boards. A standard spin-echo multislice (SEMS)
experiment was used for these studies (Callaghan, 1991). The pulse and gradients used to
obtain these images are depicted in Figure 1. In
a SEMS experiment, the following sequence of
events is necessary to obtain the multislice spatial encoded spin-echo. The slice selection is
achieved by restricting the NMR response to a
defined area of a three-dimensional sample.
This is accomplished by the selective application
of the slice gradient (Gz) along the Z axis (Fig.
1). This allows for the selective excitation of the
nuclei within the defined slice by an amplitude
modulated 908 RF pulse. After the 908 RF pulse,
Gz is rephased and the phase encoding (Gy) and
the read (frequency encoding, Gx) gradients are
applied. When phase encoding is complete, all
of the gradients are turned off. The signal is
allowed to dephase for a total encoding and dephasing time of td. Prior to the application of
the frequency modulated 1808 pulse, Gz is applied; after stabilization of the gradient the 1808
refocusing pulse is applied to the dephased signal. Once the pulse has been applied, the slice
gradient is turned off and the read gradient is

applied (Fig. 1). The resultant spin-echo of the

refocused signal is acquired after an interval of
ta, where td 5 td. The time to echo (TE) for the
SEMS experiments varied from 812 ms. The
repetition time (TR) for these studies was found
to be 68 sec. The processed data was transferred to a Sun Sparc Station and further manipulated using NMRi (New Methods Research,
Inc.) image software.
Images with snakes 1686P, 1686H, and 1686G
were acquired using the following parameters.
The images were obtained as eight and 16 serial
slices, with slice spacings of 0.25-mm and 0.25mm thick using a TE of 13.5 ms and a TR of 6
sec. A 512 3 256 matrix was used in the acquisition phase of these studies. The data were processed to a 256 3 256 matrix, which gave 59 mm
resolution in the transverse plane, with a FOV
of 15 mm.
The images from snakes 6076 and 6077 were
acquired using the same parameters with only
slight modifications. The images were obtained
as 16 serial slices with slice spacings of 0.25-mm
and 0.25-mm thick using a TE of 13.5 ms and a
TR of 7 sec. A 512 3 256 matrix was used in the
acquisition phase of these studies. The acquired
data were processed to a 256 3 256 matrix,
which gave 98 mm resolution in the transverse
plane, with a FOV of 25 mm.
RESULTS
Representative images of the brain and chemosensory systems illustrate some of the major
structures observed. We briefly compare our
findings with photomicrographs and reconstruction of the brain structures and chemosensory pathways derived from anterograde degeneration studies reported by Halpern (1976).
More details of the afferent and efferent connections in the main and accessory olfactory systems in garter snakes have been recently reported by (Lanuza and Halpern, 1997, 1998).
Table 1 lists the abbreviations used for various
structures in the figures.
Figure 2 is a multislice dorsal (coronal) image
of snake 6077. The slices progress from most
ventral (2A) to most dorsal (2D). The widest
point of the brain is 5 mm (Fig. 2C). Figure 2A
depicts the base of the brain and the hypothalamus and medulla can be identified. In Figure
2B, the optic chiasm and tract, ventral telencephalon, and spinal cord are additionally
shown. The main olfactory and accessory olfactory bulbs are visible in Figure 2C. The tectum
and cortex can be differentiated from the other
brain structures in Figure 2BD.
Figure 3 is from a multislice sagittal view of

ANDERSON ET AL.SNAKE BRAIN IMAGING

TABLE 1.

ABBREVIATIONS FOR BRAIN STRUCTURES

USED IN FIGURES 24.

Abbreviation

AOB
bas tel
cd tel
cereb
dvr
hyp
lt tel
med
med cortex
MOB
nc
opc
opt
sc
tect
tel
VNO
vt tel

Structure

accessory olfactory bulb

basal telencephalon
caudal telencephalon
cerebellum
dorsal ventricular ridge
hypothalamus
lateral telencephalon
medula
medial cortex
main olfactory bulb
nasal cavity
optic chiasm
optic tract
spinal cord
tectum
telencephalon
vormeronasal organ
ventral telencephalon

267

garter snake 6076 showing two views progressing from the left to right side of the brain. The
length of the brain is about 8 mm from the medulla to the main olfactory bulb, which is visible
in Figure 3A. The cervical portion of the spinal
cord is seen entering the brain in Figure 3B.
The main and accessory olfactory bulbs, telencephalon, tectum, cerebellum, medulla, and hypothalamus are visible. We also took axial slices
of snakes proceeding from the rear of the brain
to the snout, but these are not pictured here.
These slices depicted many structures including
the ventricles, anterior commissure, nucleus
sphericus, optic tract, medial and dorsal cortex,
ventromedial hypothalamus, caudal and rostal
accessory olfactory bulb and tract, main olfactory bulb, and vomeronasal organ.
Figure 4 provides a high resolution, sagittal
view of a smaller snake, 1686H. The main olfactory bulb and the vomeronasal organ are evident along with the nasal cavity, tectum, cortex,
dorsoventricular ridge, basal telencephalon, hypothalamus, and medula.

Fig. 2. A multislice dorsal image of garter snake 6077. Structure labels in Table 1.

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COPEIA, 2000, NO. 1

Fig. 3. Two sagittal views of garter snake 6076. Structure labels in Table 1.

DISCUSSION
These initial studies demonstrate the potential
use of high-field MRI methodology for the noninvasive study of snake anatomy. This technique
provided identifiable images of the vomeronasal
organ, accessory and main olfactory bulbs and
tracts, eyes, optic nerves, cortex, tectum, spinal
cord, and other structures. Although the resolution of detail is far from that obtained with large
brains in larger animals such as mammals, technological advances will certainly make application of these methods in small living animals
much easier and more advanced. Being able to
see minute internal structures (not only of the
brain) is remarkable compared to method available less than a decade ago.

Fig. 4. A higher resolution sagittal view of the olfactory bulb and the vomeronasal organ in garter
snake 1686H. Structure labels in Table 1.

A current assessment of the connections in the

olfactory brain of the common garter snake is
provided in Lanuza and Halpern (1997, 1998).
Chemical cues received by the nasal epithelium
nerve endings of the main olfactory bulb are relayed to the main secondary olfactory structure,
the lateral cortex, as well as the rostral amygdala,
olfactory tubercle, and dorsomedial retrobullar
formation. Vomodors (Cooper and Burghardt,
1990) received by the vomeronasal organ and
the accessory olfactory bulb are relayed via the
accessory olfactory tract to the main secondary
afferent pathway, the nucleus sphericus, a major
amygdaloid structure, as well as the nucleus of
the accessory olfactory tract and the medial
amygdala. The nucleus sphericus has efferent
pathways to amygdaloid nuclei, the olfactostriatum (part of the rostral and ventral telencephalon), the contralateral nucleus sphericus, and the
dorsal and lateral cortex. It is in these cortical
structures that integration of olfactory and vomerolfactory information may occur. The olfactostriatum may be the major tertiary vomeronasal
center. There are also some projections from the
nucleus sphericus to the lateral hypothalamus
but none directly to the ventromedial hypothalamus. Current nomenclature of identical structures differs among authors and homologies
across species are controversial (Butter and
Hodes, 1996). However, the work of Lanuza and
Halpern should bring stability to natricine snake
brain anatomy and allow more detailed functional studies.
Although our results with current imaging
technology do not show the detail found in microscopic histological studies, we do see the
method employed here as valuable as a supplement to histological studies and for facilitating
further research avenues. Many of the major

ANDERSON ET AL.SNAKE BRAIN IMAGING

chemosensory structures in the snake brain can
be seen even without staining or injections, as
needed in some survival imaging methods. Differences among species in the relative development of the different sensory areas of the brain
could be assessed in animals too rare or valuable to sacrifice. Do snakes that rely more on
vision show differences in the size of the various
parts of the brain as compared to less visual species? Do fossorial, aquatic, terrestrial, and arboreal snakes differ? In lizards the range of chemosensory reliance is much greater than in
snakes and brain imaging could provide inexpensive and rapid collection of comparative
data. Studies in progress in our laboratory show
that relative brain volumes can also be calculated from brain images in the three planes. One
could easily develop a large number of hypotheses to evaluate the role of phylogeny and current adaptation.
Noninvasive imaging could also be useful in
studies of lesioned animals where the extent of
damage can be assessed before time-consuming
and expensive behavioral trials are carried out.
On the other hand, monitoring the activity in
intact animals as a way of understanding perceptual, cognitive, motivational and emotional
processes may also become possible. Methods
such as PET scans and functional MRI are available for large animals and those with large
brains; resolution with small brains is not yet
great enough to apply these techniques. However, as technology advances, we should be
ready to exploit them for scientific study of
brain and behavior in often small species critical for resolving basic research questions.
ACKNOWLEDGMENTS
Supported in part by funding from the National Science Foundation (GMB), the U.S. Department of Energy (GWK), and the Robert H.
Cole Foundation (GWK). This research was performed under a protocol approved by the IACUC at the University of Tennessee, Knoxville.
We thank D. Schaeffer (DVM) for assisting with
anesthesia, E. Lanuza for helping identify brain
structures, M. Halpern for reading an earlier version of this paper, and three anonymous reviewers.
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(CLA,GWK,JD) DEPARTMENT OF CHEMISTRY,

UNIVERSITY OF TENNESSEE, KNOXVILLE, TENNESSEE 37996-0900; AND (DLG,GMB) DEPARTMENT OF PSYCHOLOGY, UNIVERSITY OF TENNESSEE, KNOXVILLE, TENNESSEE 37996-0900. PRESENT ADDRESSES: (CL): NMR FACILITIES, DEPARTMENT OF C HEMISTRY , U NIVERSITY OF
HOUSTON, AND HOUSTON, TEXAS 77204;
(DGL) DEPARTMENT OF BIOLOGICAL SCIENCES,
KENT STATE UNIVERSITY, KENT, OHIO 44242;
AND ( JPD) MEMORIAL SLOAN-KETTERING CANCER CENTER, 1225 YORK AVENUE, 1125A/MRI,
NEW YORK, NEW YORK 10028. E-mail: (GWK)
gkabalka@utk.edu; and (GMB) gburghar@
utk.edu. Send reprint requests to GMB. Submitted: 9 July 1998. Accepted: 10 May 1999.
Section editor: R. G. Bowker.