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Afferent and efferent connections of the nucleus sphericus in the snakeThamnophis sirtalis Convergence of olfactory and vomeronasal information in the lateral cortex and the amygdala

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THE JOURNAL OF COMPARATIVE NEUROLOGY 385:627–640 (1997)
Afferent and Efferent Connections
of the Nucleus Sphericus in the Snake
Thamnophis sirtalis: Convergence of
Olfactory and Vomeronasal Information
in the Lateral Cortex and the Amygdala
ENRIQUE LANUZA1,2 AND MIMI HALPERN1*
of Anatomy and Cell Biology, Health Science Center at Brooklyn,
State University of New York, Brooklyn, New York 11203
2Department de Biologia Animal, Unitat de Morfologia Microscòpica, Facultat de Ciències
Biològiques. Universitat de València. Burjassot 46100 Spain
1Department
ABSTRACT
This paper is an account of the afferent and efferent projections of the nucleus sphericus
(NS), which is the major secondary vomeronasal structure in the brain of the snake
Thamnophis sirtalis. There are four major efferent pathways from the NS: 1) a bilateral
projection that courses, surrounding the accessory olfactory tract, and innervates several
amygdaloid nuclei (nucleus of the accessory olfactory tract, dorsolateral amygdala, external
amygdala, and ventral anterior amygdala), the rostral parts of the dorsal and lateral cortices,
and the accessory olfactory bulb; 2) a bilateral projection that courses through the medial
forebrain bundle and innervates the olfactostriatum (rostral and ventral striatum); 3) a
commissural projection that courses through the anterior commissure and innervates mainly
the contralateral NS; and 4) a meager bilateral projection to the lateral hypothalamus. On the
other hand, important afferent projections to the NS arise solely in the accessory olfactory
bulb, the nucleus of the accessory olfactory tract, and the contralateral NS.
This pattern of connections has three important implications: first, the lateral cortex
probably integrates olfactory and vomeronasal information. Second, because the NS projection to the hypothalamus is meager and does not reach the ventromedial hypothalamic
nucleus, vomeronasal information from the NS is not relayed directly to that nucleus, as
previously reported. Finally, a structure located in the rostral and ventral telencephalon, the
olfactostriatum, stands as the major tertiary vomeronasal center in the snake brain. These
three conclusions change to an important extent our previous picture of how vomeronasal
information is processed in the brain of reptiles. J. Comp. Neurol. 385:627–640,
1997. r 1997 Wiley-Liss, Inc.
Indexing terms: chemosensory systems; vomeronasal projections; biotinylated dextran amine;
reptiles
In most tetrapod vertebrates, there are two main sensory systems that are capable of relaying information
about the chemicals present in the environment: the
olfactory system, which detects volatile chemicals, and the
vomeronasal system, which is specialized to detect complex chemicals of high molecular weight (e.g., pheromones). Both chemosensory systems are very well developed in squamate reptiles (lizards and snakes), given the
fact that chemical stimuli are very important in the
ecology of these groups (for recent reviews, see Mason,
1992; Halpern, 1992). In reptiles, as in all other tetrapods
that possess a vomeronasal system, olfactory information
r 1997 WILEY-LISS, INC.
and vomeronasal information are processed separately in
the brain (mammals: Scalia and Winans, 1975; reptiles:
Halpern, 1976; amphibians: Scalia et al., 1991). Olfactory
Grant sponsor: NIH; Grant number DC00104; Grant sponsor: Valencian
IVGI; Grant number: 0021076.
*Correspondence to: Dr. Mimi Halpern. Department of Anatomy and
Cell Biology, Box 5, Health Science Center at Brooklyn, State University
of New York, 450 Clarkson Avenue, Brooklyn, NY 11203. E-mail:
mhalpern@netmail.hscbklyn.edu
Received 11 November 1996; Revised 14 April 1997; Accepted 17 April
1997
628
E. LANUZA AND M. HALPERN
information enters the brain through the main olfactory
bulb (MOB), from where it is relayed to the lateral
(pyriform) cortex (LC), which is the main secondary olfactory structure in the brain. On the other hand, vomeronasal information enters the brain through the accessory
olfactory bulb (AOB), which projects almost exclusively to
the nucleus sphericus (NS; for a review of the olfactory and
vomeronasal projections in reptiles, see Lohman and
Smeets, 1993).
The NS, therefore, is the main secondary vomeronasal
structure in the brain of squamate reptiles. In the garter
snake (Thamnophis sirtalis), it is the most prominent
structure of the telencephalon, occupying almost the entire
caudal half of the telencephalic hemisphere. The development of the NS in different reptilian species appears to be
strongly correlated with the development of the vomeronasal system in each particular species (Schwenk, 1993). In
species without a functional vomeronasal system (some
agamid and chamaleonid lizards, e.g., Chamaleo chamaleo), the NS is not recognizable in the brain (Northcutt,
1978); in species with a poorly developed vomeronasal
organ and a small accessory olfactory bulb, like some
iguanid lizards (e.g., Iguana iguana and Dipsosaurus
dorsalis), the NS is small (Northcutt, 1978); and, in species
with a well-developed vomeronasal organ and a large
accessory olfactory bulb, like most snakes, the NS occupies
almost the entire caudal half of the subcortical telencephalon (Halpern, 1980). Therefore, knowledge of the connections of the NS is essential to understand how vomeronasal information is processed in the reptilian brain.
Somewhat surprisingly, almost no information is available on the connections of the NS other than its wellknown input from the accessory olfactory bulb (for review,
see Lohman and Smeets, 1993), its connection with the
contralateral NS (Halpern, 1980), and what is generally
believed to be the efferent projection to the ventromedial
hypothalamic nucleus (VMH; Voneida and Sligar, 1979;
Halpern, 1980; Martı́nez-Garcı́a et al., 1993b). However,
recent studies in lizards (Bruce and Neary, 1995a; Lanuza
et al., 1997) and our own observations in snakes (Lanuza
and Halpern, unpublished observations) strongly suggest
that the projection to the VMH actually originates in the
posterior part of the dorsal ventricular ridge and the
lateral amygdala rather than in the NS. Therefore, vomeronasal information is not directly relayed to the VMH. If the
NS does not project to the VMH, then almost nothing is
known about its efferent projections, and there are not
enough data to suggest a hypothesis about how vomeronasal information is processed in the brain.
An important question that the present scheme of
connections of the NS leaves unresolved is whether the
olfactory and vomeronasal information converge in the
brain in later stages of information processing. Both kinds
of chemosensory stimuli are important in several crucial
behaviors, such as prey detection and mate recognition, as
well as in species-typical social behaviors (for reviews, see
Burghardt, 1970; Halpern, 1992). Thus, it is reasonable to
suggest that olfactory and vomeronasal information should
interact in the brain in order to give the animal a complete
picture of its chemical environment.
To fill this gap in our knowledge of the tertiary connections of the vomeronasal system, we have reinvestigated
the afferent and efferent projections of the NS in the garter
snake (T. sirtalis) by means of the sensitive tract-tracing
technique of intraaxonal transport of the modern tracer
biotinylated dextran amine (BDA). Although BDA has
been reported to be mainly an anterograde tracer (Veenman et al., 1992), in our experimental conditions, it also
gives rise to consistent retrograde transport.
MATERIALS AND METHODS
Adult specimens of T. sirtalis (both sexes), with body
weights between 18 g and 75 g, were purchased from a
licensed dealer. Animals were maintained in terraria
under a natural day/night cycle at 22–30°C, given water ad
libitum, and fed weekly.
For surgery, animals were cooled prior to anesthetization with an intramuscular injection of 0.003 ml of 0.5%
brevital sodium (methohexital sodium; Eli Lilly and Co.,
Indianapolis, IN) per gram of body weight. Iontophoretic
injections of BDA (MW 10,000; Molecular Probes, Eugene,
OR) dissolved 10% in Tris buffer (0.05 M), pH 7.6 (Veenman et al., 1992), were made in the nucleus sphericus
(n 5 8). For controls, some injections were made in the
medial (n 5 2), dorsal (n 5 3), and lateral cortices (n 5 5).
Abbreviations
ac
ADVR
AOB
aot
apc
BNST
ch
CoApm
DBB
DC
DLA
DMA
dRF
ExA
gl
gr
H
LA
LC
lfb
LHA
LPA
anterior commissure
anterior dorsal ventricular ridge
accessory olfactory bulb
accessory olfactory tract
anterior pallial commissure
bed nucleus of the stria terminalis
corticohypothalamic tract
posteromedial cortical nucleus of the amygdala
nucleus of the diagonal band of Broca
dorsal cortex
dorsolateral amygdaloid nucleus
dorsomedial anterior thalamic nucleus
dorsal retrobulbar formation
external amygdala
glomerular layer of the AOB
granular layer of the AOB
habenula
lateral amygdala
lateral cortex
lateral forebrain bundle
lateral hypothalamic area
lateral preoptic area
LTM
m
MA
MC
mfb
MOB
NAcc
Naot
NS
OS
OT
ot
PDVR
S
sh
Si
St
v
VAA
VMH
VPA
lateral tuberomammillary area
mitral cell layer of the AOB
medial amygdala
medial cortex
medial forebrain bundle
main olfactory bulb
nucleus accumbens
nucleus of the accessory olfactory tract
nucleus sphericus
olfactostriatum
olfactory tubercle
optic tract
posterior dorsal ventricular ridge
septum
septohypothalamic tract
nucleus septalis impar
striatum
ventricle
ventral anterior amygdala
ventromedial hypothalamic nucleus
ventral posterior amygdala
TERTIARY VOMERONASAL CONNECTIONS IN SNAKES
629
Fig. 1. Camera lucida drawings showing the location of the injection sites in the eight specimens with injections in the nucleus
sphericus (NS). The sections are arranged from rostral (A) to caudal
(H). Hatched areas indicate the extent of the injections sites. The fiber
labeling adjacent to the injection site is also depicted to give an idea of
the area of diffusion of the tracer. A: Specimen 9640. B: Specimen
9620. C: Specimen 9637. D: Specimen 9616. E: Specimen 9636. F:
Specimen 9610. G: Specimen 9621. H: Specimen 9607. For abbreviations, see list. Scale bar 5 500 µm.
The injections were made by using glass micropipettes
with inner tip diameters of 20–40 µm. A positive DC
current of 3–5 µA was applied during 5–15 minutes (pulse
7 seconds on/off).
After a survival period ranging from 8 to 12 days,
animals were deeply anesthetized with brevital sodium
and transcardially perfused with 30–40 ml of saline solution (0.9% NaCl), followed by 60–80 ml of fixative (4%
paraformaldehyde) in 0.1 M phosphate buffer (PB), pH 7.4.
Brains were removed from the skull, postfixed in the same
fixative for 4–12 hours at 4°C, cryoprotected in 30%
sucrose in PB at 4°C until they sank, and cut at 35 µm in
the frontal plane on a freezing microtome. The sections
were collected in three matching series.
For BDA histochemical detection, endogenous peroxidase activity was first inhibited by incubating the sections
for 30 minutes in a 0.3% H2O2 solution in saline Tris buffer
(0.05 M, pH 7.6, 0.9% NaCl). Sections were then incubated
in avidin-biotin complex, usually one series in ‘‘elite’’ ABC
(ABC Elite Vectastain kit; Vector Laboratories, Burlingame, CA) and another in ‘‘standard’’ ABC (ABC Standard
Vectastain kit; Vector Laboratories), and were diluted in
Tris-buffered saline with 0.3% Triton X-100 as a permeating agent for 1–2 hours at room temperature. The reason
for using the two types of ABC kits was that the elite ABC
gave an amplified signal that made it difficult to determine
the limits of the injection site and also yielded considerable
background, even with very short developing times. In
contrast, the standard ABC gave a more precise picture of
the extent of the injection site and yielded much less
background, with the disadvantage of being somewhat less
sensitive.
The resulting peroxidase label was revealed directly by
using 3,38-diaminobenzidine (DAB; Sigma, St. Louis, MO)
as the chromogen diluted at 0.02% in Tris buffer, pH 8.0,
with nickel salts (0.4%) and H2O2 (0.01%). Sections were
subsequently counterstained with acidic toluidine blue.
The research reported herein was performed according to
the guidelines of the Animal Care and Utilization Act,
under the supervision of the SUNY Health Science Center
Institutional Animal Committee.
RESULTS
Injections into the NS
We made injections into different parts of the dorsoventral, rostrocaudal, and mediolateral extent of the NS (Fig.
1). All of the injections into the NS gave rise to a similar
pattern of anterograde labeling. The anterograde labeling
found in specimen 9637 (Fig. 1C) is described and illustrated (Fig. 2) as an example.
To some extent, all of the injections into the NS also
affected the overlying medial cortex (MC) or dorsal cortex
(DC), depending on the rostrocaudal location of the injection (Fig. 1). Therefore, some control injections into the
cortex were made to distinguish the labeling due to the
cortical neurons affected by the injection. In some injec-
630
E. LANUZA AND M. HALPERN
tions into the NS, only a number of labeled fibers and a few
labeled neurons appeared in the cortex (Fig. 1B–E), which
facilitated the recognition of the labeling due to NS
neurons. In any case, all of the observed labeling is
described in Results, and the fiber labeling that was
probably due to the affected portion of the cortex is
subtracted later (see Discussion). In the present work, we
have followed the nomenclature of Halpern (1980) for the
telencephalon of snakes, with the exception of the amygdaloid complex, for which the nomenclature of Lanuza and
Halpern (1997) has been used.
Anterograde transport
Intratelencephalic labeling. Following injections centered in the NS, anterogradely labeled fibers leave the NS
through three main pathways. The most important projection courses to surround the accessory olfactory tract (aot),
the second projection courses via the medial forebrain
bundle (mfb), and the third projection courses via the
anterior commissure (ac).
The projection that courses along the external border of
the aot gives rise to abundant terminal-like labeling in the
nucleus of the accessory olfactory tract (Naot) throughout
its rostrocaudal extent, especially dorsal and medial to the
tract (Figs. 2C–E, 4B,E). Most of the labeled fibers avoid
the center of the tract, leaving it relatively free of fibers
(Figs. 2D, 4B,E). These fibers continue rostrally to retrobulbar levels, where they surround the olfactory ventricle
dorsally (Fig. 2B) and give rise to a dense plexus of
terminal-like labeling in the granular layer of the accessory olfactory bulb (AOB; Figs. 2A,B, 4A).
In the rostral half of the telencephalon, from the retrobulbar formation to the anterior commissure, many labeled
fibers leave the aot in a dorsal and lateral direction (Fig.
2C–G). At rostral levels, these fibers reach the dorsal
retrobulbar formation (dRF), where they give rise to a
dense plexus of fiber and terminal-like labeling (Fig. 2C).
At the same rostral levels, other fibers leave the aot in a
dorsolateral direction, giving rise to a light plexus of fibers
in the lateral cortex (LC) (Fig. 2C). More caudally, labeled
fibers innervate the outer half of the external plexiform
layer of the DC (Figs. 2D–F, 4D) and the internal plexiform
and cellular layers of the LC (Fig. 2E, F, 4C). The fiber
labeling is denser at rostral levels (level of the retrobulbar
formation; Fig. 2C) and becomes lighter caudally. At
precommissural levels (Fig. 2F,G), the labeled fibers that
leave the aot in a dorsolateral direction give rise to dense,
terminal-like labeling in several amygdalar nuclei: the
dorsolateral amygdala (DLA), external amygdala (ExA),
and ventral anterior amygdala (VAA; Figs. 2F,G, 4B).
Some labeled fibers also appear in the caudal lateral
amygdala (LA), mainly at the level of the injection site
(Fig. 2H–J).
At caudal telencephalic levels, the hilus and the mural
layer of the injected NS contain a considerable amount of
anterograde labeling (Fig. 2J,K). The set of projections
that courses via the aot, which will be described later, is
bilateral, with ipsilateral predominance. The commissural
fibers cross the midline via the anterior commissure.
The fibers that course through the mfb exit the NS
medially and appear to innervate the bed nucleus of the
stria terminalis (BNST) and the medial amygdala (MA;
Fig. 2H). More medially, these fibers gather together above
the lateralmost part of the ac (Fig. 2H). Then, they travel
rostrally through the mfb (Fig. 2F,G), traversing the
ventral telencephalon to finally give rise to a dense field of
terminal-like labeling in the olfactostriatum (Figs. 2C–E,
4E; terminology of Halpern, 1980; following Durward,
1930). This area is limited dorsally by the rostralmost part
of the nucleus accumbens (as identified by Smeets, 1988),
laterally by the Naot, ventrally by the olfactory tubercle
(OT) and the horizontal limb of the nucleus of the diagonal
band of Broca (DBB), and medially by the vertical limb of
the DBB. At the level of the retrobulbar formation, the
labeled fibers continue rostrally just medial to the olfactory ventricle, until they reach the granular layer of the
AOB. Therefore, the NS appears to reach the AOB through
two different projections, a lateral one via the aot and a
medial one via the mfb.
The fibers that course through the mfb do not form a
compact tract but, rather, are loosely arranged. Some of
them enter the vertical limb of the DBB and the nucleus
accumbens (NAcc; Fig. 2F). They exhibit varicosities along
their way through the ventral telencephalon that may
represent boutons en passant mainly on neurons of the bed
nucleus of the mfb (Fig. 2F). This projection is also
bilateral with ipsilateral predominance.
The third intratelencephalic projection is a commissural
projection that courses via the ac (Fig. 2I). After crossing
the commissure, a few fibers enter the contralateral mfb
and travel rostrally, innervating the olfactostriatum (Fig.
2D–F). The majority of the commissural fibers continue
laterally and give rise to terminal-like labeling mainly in
the external, mural, and submural layer of the contralateral NS (Fig. 2J,K), always in a position symmetric to the
site of injection. In the case illustrated in Figure 2, the
injection site is rostral and dorsal in the NS, and the
resulting fiber labeling in the contralateral NS is most
abundant in the dorsal and rostral areas (Fig. 2I–L). The
fibers destined for the lateral part of the contralateral NS
tend to course over the dorsal NS instead of crossing
through it. Numbers of labeled fibers continue rostrally,
innervating the Naot (Fig. 2F–H) and also the same
amygdalar nuclei that receive a NS projection on the
ipsilateral side: the DLA (Fig. 2H), the VAA, and the ExA
(Fig. 2F,G). Whereas the contralateral innervation of the
ExA and the VAA is very light, the innervation of the
contralateral DLA is remarkable. More rostrally, labeled
fibers are observed in the contralateral rostral LC (Fig. 2E)
and in the granular layer of the contralateral AOB (Fig.
2A,B). Therefore, all of the intratelencephalic efferent
projections of the NS are bilateral, although they have a
strong ipsilateral predominance.
In addition to the already described labeling, anterogradely labeled fibers and terminals appeared in the
medial and dorsal cortices at the level of the injection site
(Fig 2H–J) and in the septum at most of it rostrocaudal
extent, but especially caudally (Fig. 2H,I). Some of the
labeled fibers in the septum cross the midline through the
anterior pallial commissure (apc; Fig. 2I), reaching the
contralateral septum and also the contralateral dorsomedial cortex (Fig. 2J,K). This labeling was very light in the
injections in which only a few cortical neurons were
affected.
Extratelencephalic labeling. All of the anterograde
labeling found in extratelencephalic areas was located in
the hypothalamus. Following injections centered in the
NS, a small bundle of fibers leaves the NS in the ventromedial direction, surrounds the lfb dorsally (Fig. 2H), and
turns ventrally, crossing the lateral preoptic area (LPA;
TERTIARY VOMERONASAL CONNECTIONS IN SNAKES
Fig. 2. A–L: Camera lucida drawings of transverse sections through
the brain of a snake showing the distribution of biotinylated dextran
amine (BDA)-labeled fibers and neurons (solid circles) after an injection centered into the nucleus sphericus (NS). Injection site depicted in
631
H and I (hatched area). Arrowhead in H indicates the bundle of fibers
destined for the hypothalamus. A is rostral, L is caudal. For abbreviations, see list. Scale bar 5 200 µm.
632
E. LANUZA AND M. HALPERN
Fig. 2I). Some scattered fibers are observed to reach the
medial preoptic area (Fig. 2H,I). During its dorsoventral
course through the LPA (Fig. 2I), this bundle of fibers splits
apart and is no longer visible as a unit but, rather, as
solitary fibers travelling in a ventral direction located
lateral to the lfb (Fig. 2J, 4F). They traverse the lateral
hypothalamic area (LHA; at the level of the anterior
hypothalamus; Fig. 2J) and, apparently, terminate in a
cell-poor area located just lateral to the ventromedial
hypothalamic nucleus (VMH; Fig. 2K). This area extends
caudally to the level of the mammillary bodies (Fig. 2L).
Because it is the lateral area of the tuberal part of the
hypothalamus, and it reaches the level of the mammillary
bodies, we have called it the lateral tuberomammillary
area (LTM). Most of the fibers that course through the
lateral hypothalamus exhibit varicosities along their route
that may represent boutons en passant on cells of the
LHA.
In the contralateral hypothalamus, some (although very
few) fibers can be observed following the same pathway as
that described on the ipsilateral side. In addition to the
described labeling, some of the fibers that cross the septum
continue farther ventrally and reach the hypothalamus
(Fig. 2H,I), where they course both medially and laterally
and, apparently, terminate in the periventricular area of
the anterior and tuberal hypothalamus and in the LHA
(Fig. 2J,K).
Retrograde transport
Following injections centered in the NS, the majority of
the retrogradely labeled neurons were found in the mitral
cell layer of the AOB (Fig. 2A,B) and in the Naot, mainly in
its medial part (Figs. 2C–F, 4H). In addition, retrogradely
labeled cells appeared scattered in the nucleus of the
diagonal band (Fig. 2G), in the vicinity of the lateral
forebrain bundle (Fig. 2G), and, more caudally, in the
nucleus of the ac (Fig. 2H). Retrogradely labeled neurons
also appeared in the DC and the MC.
Control injections into the DC
We describe here only the efferent projections of the DC
that coincided with those described in the injection centered in the NS. Following injections into the DC (Fig. 3B),
in the telencephalon, numerous labeled fibers appeared in
the septum, the DBB, the DLA, and the LA (Fig. 3A,B). In
addition, a few anterogradely labeled fibers appeared in
the granular layer of the accessory olfactory bulb, the
rostral LC, and the nucleus accumbens.
In the diencephalon, anterogradely labeled fibers reach
the hypothalamus via two pathways, one medial and one
lateral. The medial pathway, which crosses through the
septum, is actually formed by a rostral component, the
precommissural fornix (Lohman and Van Woerden-Verkley, 1972, 1976), called septohypothalamic tract (sh) by
Hoogland and Vermulen-Vanderzee (1989; Fig. 3A), and a
caudal component, the postcommissural fornix (Lohman
and Van Woerden-Verkley, 1972, 1976), called corticohypothalamic tract (ch) by Hoogland and Vermulen-Vanderzee
(1989; Fig. 3C). The lateral pathway is formed by fibers
that curve around the lateral corner of the lateral ventricle
and course medially through the amygdaloid complex (Fig.
3A).
The precommissural fornix and the lateral tract join in
the lateral preoptic area (Fig. 3B) and innervate the
lateral hypothalamus (lateral preoptic area: Fig. 3A,B;
lateral hypothalamic area: Fig. 3C,D). The postcommissural fornix enters the hypothalamus more caudally (Fig.
3C) and innervates the periventricular and medial hypothalamus at anterior and tuberal levels (Fig. 3C,D).
Injections into the LC
We wanted to confirm the projection of the NS to the LC
by retrograde transport after injections into the LC. Somewhat surprisingly, retrogradely labeled cells after injections in the LC consistently appeared only in the lateral
wall of the mural layer of the NS and not in the medial
layer (Fig. 4G). The injections into the LC were located
rostrally (at the levels of Fig. 2E–G); therefore, we can be
sure that the retrograde labeling in the NS was not due to
medial diffusion of the tracer.
DISCUSSION
The results of the present work reveal (after subtraction
of the projection from the cortex; see below) that the
nucleus sphericus of the snake T. sirtalis has four major
efferent projections: 1) a projection to the accessory olfactory bulb, rostral DC, rostral LC, and several nuclei of the
amygdaloid complex that courses along the external border of the accessory olfactory tract; 2) a projection to the
olfactostriatum that courses via the mfb; 3) a projection to
the contralateral hemisphere that reaches the contralateral NS plus all of the targets of the ipsilateral projections
through the aot (AOB, Naot, DC, LC, and amygdaloid
nuclei) and through the mfb (OS). This projection crosses
the midline through the anterior commissure; and 4) a
projection to the hypothalamus that courses as a bundle of
fibers medial to the lfb. On the other hand, all of the NS
inputs arise in other vomeronasal structures: in the AOB
(the major input), the Naot, and the contralateral NS. It
may also receive a light projection from the DBB.
This pattern of afferent and efferent projections of the
NS, as will be discussed later, suggests a new scheme for
the processing of vomeronasal information in the snake
brain. This is particularly relevant, because snakes are
probably the animals with the best developed vomeronasal
system, and, because of this, they have been used extensively as models for the study of the structure, function,
and behavioral roles of this particular sensory system
(Halpern, 1992; Halpern and Holtzman, 1993). Before
discussing other aspects of the results, a comparison of the
projections of the NS and the DC is necessary, because the
injections into the NS always affected part of the overlying
cortex.
Subtraction of DC projections
It was easy to differentiate the telencehalic projections
of the DC from those that originated in the NS. However, it
was somewhat more difficult to distinguish between the
corticohypothalamic projections and the projections to the
hypothalamus that originated in the NS.
The clearest case of anterograde labeling found after
injections aimed at the NS that actually corresponded to
the cortical areas affected is the case of the anterograde
labeling found in the septum. Both DC and MC, as
described previously (for review, see Ulinski, 1990), project
to the septum, and the anterograde labeling that we found
in the septum following our injections into the NS is
exactly the same as the labeling found in the septum after
cortical injections (see Figs. 2H,I, 3B,C). Therefore, the
Fig. 3. A–D: Camera lucida drawings of transverse sections through the brain showing the
distribution of BDA-labeled fibers after an injection restricted to the dorsal cortex (DC). Injection site is
depicted in B (hatched area). For conventions, see Figure 2. A is rostral, D is caudal. For abbreviations, see
list. Scale bar 5 200 µm.
634
Fig. 4. Photomicrographs illustrating the anterograde (A–F) and
retrograde (H) labeling following BDA injections in the NS and
retrograde labeling after an injection into the lateral cortex (G). A:
Anterograde labeling in the granular layer of the accessory olfactory
bulb (AOB). The position of the anterograde labeling is marked by
arrowheads. B: Detail of the innervation of the nucleus of the
accessory olfactory tract (Naot), the external amygdala (ExA), and the
ventral anterior amygdala (VAA). C: High-magnification photomicrograph showing the plexus of beaded fibers that innervate the lateral
cortex (LC). Arrowheads indicate some of the varicosities of the fibers.
E. LANUZA AND M. HALPERN
D: Detail of the innervation of the external plexiform layer of the
rostral DC. The position of the anterograde labeling is indicated by
arrowheads. E: Heavy anterograde labeling in the olfactostriatum
(OS). F: Scattered fibers in the lateral hypothalamic area (LHA). G:
Retrogradely labeled neurons in the lateral mural layer of the NS after
an injection into the LC. H: Retrogradely labeled neuron (arrowhead)
in the Naot after an injection into the NS. For abbreviations, see list.
Scale bars 5 200 µm in A (also applies to B,D,E,G), 40 µm in C (also
applies to F,H).
TERTIARY VOMERONASAL CONNECTIONS IN SNAKES
fiber labeling found in the septum after the injections into
the NS that affected the overlying cortex should be considered part of the efferent projections of the cortex. Similarly,
the fiber labeling found in the DC and the MC at the level
of the injection site (Fig. 2H,I) and more caudally (Fig.
2J,K), as well as the few fibers found in the contralateral
DC and MC (Fig. 2J,K) very probably arise from the cortex
overlying the NS and not from the NS itself. In addition,
the following structures showed anterograde labeled fibers
both after injections into the NS and after injections into
the DC: the AOB, LC, rostral DC, DLA, LA, DBB, and the
nucleus accumbens in the ventral telencephalon.
The projection from the NS to the AOB is very clear (Fig.
4A). It was described previously in the snake T. sirtalis
(Halpern, 1980) and in the lizards Tupinambis nigropunctatus (Voneida and Sligar, 1979) and Podarcis hispanica
(Martı́nez-Garcı́a et al., 1991). The main difference between the projection to the AOB that originates in the NS
and the one that originates in the DC is that the former is
relatively massive, whereas the latter is very meager. Both
terminate in the granular layer of the AOB, and the
appearance of the centrifugal fibers originating from both
the NS and the DC is indistinguishable.
The anterograde labeling found in the LC and in rostral
DC following injections into the NS clearly originated in
the NS. Even after very large injections into the cortex
(affecting most of the caudal part of both the DC and MC),
the fiber labeling found in the LC and in rostral DC is
sparse. Moreover, after injections into the NS, the course of
the fibers reaching the rostral DC and LC can be followed
from the aot to these cortical structures (Fig. 2C–F).
Furthermore, injections into the LC give rise to retrogradely labeled cells in the NS, thus confirming the
projection from the NS to the LC.
The DLA and LA are both densely innervated by the DC
(Fig. 3). The fiber labeling in the LA found after injections
into the NS is quite sparse and is limited mainly to the
levels of the injection site (Fig. 2H–J). Therefore, we
believe that there is no projection from the NS to the LA,
and it is more likely that the fiber labeling found in the LA
is due to the projection from the DC as well as to some
diffusion of the tracer from the injection site. Moreover, no
contralateral projection to the LA has been observed in
contrast to the rest of the amygdaloid nuclei innervated by
the NS, which are all bilaterally innervated.
On the other hand, the DLA is probably densely innervated by both the DC and the NS. Anterograde labeling in
the DLA after injections into the NS is bilateral and is also
found in animals in which the injections included only the
MC and the NS. The MC has not been found to project to
the DLA (Olucha et al., 1988; Ulinski, 1990; Hoogland and
Vermeulen-Vanderzee, 1993). Moreover, one injection centered in the DLA (which also affected the caudal DC) gave
rise to retrogradely labeled somata in the NS (not charted).
Regarding the projection to the ventral telencephalon,
the few labeled fibers that appear in the NAcc following
injections in the NS are probably due to cortical neurons
affected by the injection (even in the animals with large
injections into the NS, only a very few fibers appear in the
NAcc, whereas, in animals with large injections into the
DC, many labeled fibers appear in the NAcc). It is more
difficult to distinguish the projection of the DC and the NS
to the DBB. Large injections into the DC result in a heavy
projection to the DBB, whereas large injections into the NS
give rise to only a relatively small number of fibers in the
635
DBB. Therefore, if there is a projection from the NS to the
DBB, then it is a light one.
It is difficult to distinguish between the corticohypothalamic projections and the projections to the hypothalamus
that originate in the NS. The corticohypothalamic projections of the DC are much more extensive than those of the
NS and, apparently, also innervate most of the areas
reached by the NS projection. In agreement with the
results of Hoogland and Vermeulen-Vanderzee (1989) in
the lizard Gekko gecko, we have found that the DC projects
to the hypothalamus via two pathways: a medial pathway
that courses through the septum (actually formed by the
septohypothalamic tract and the medial corticohypothalamic tract; see Fig. 3) and a lateral pathway that courses
through the amygdaloid complex, between the Naot and
the anterior dorsal ventricular ridge (ADVR; Fig. 3A). The
MC has been reported to project to the medial hypothalamus via the medial corticohypothalamic tract (Halpern,
1980), although this projection has not been found in
lizards (Olucha et al., 1988; Ulinski, 1990; Hoogland and
Vermeulen-Vanderzee, 1993).
The projection through the medial corticohypothalamic
tract terminates mainly in the periventricular areas of the
anterior and tuberal hypothalamus. In our opinion, the
labeled fibers that we find in the periventricular hypothalamus following injections centered in the NS are probably
due to this corticohypothalamic projection. In fact, some of
these fibers could be followed back to the septum.
The other DC projections to the hypothalamus (through
the septohypothalamic tract and through the lateral pathway) follow a similar course within the hypothalamus to
one of the fibers that leaves the NS: All three projections
enter the hypothalamus rostrally, innervating the lateral
preoptic area, and extend ventrally, innervating the lateral hypothalamic area of the anterior and tuberal hypothalamus, including the LTM. Accordingly, the DC projects
to the LHA and to the area located lateral to the VMH in
the lizard G. gecko (Hoogland and Vermeulen-Vanderzee,
1989). However, anterogradely labeled fibers in the LHA
and the LTM appeared in all of the animals with injections
into the NS, including those in which only the NS and the
MC were affected (see Fig. 1D,E,H). Therefore, part of the
innervation of the lateral hypothalamus (LPA, LHA, and
LTM) probably arises in the NS.
Intratelencephalic projections of the NS
The major intratelencephalic projections of the NS reach
the AOB, the Naot, and the olfactostriatum (OS). These
structures appear to form a heavily interconnected circuit
for the processing of the vomeronasal information. The
connections between the AOB and the NS are massive and
reciprocal (Halpern, 1980; Lanuza and Halpern, 1997), as
are the connections between the Naot and the NS (this
report) and between the Naot and the AOB (Lanuza and
Halpern, 1997). The projection from the NS to the OS is
also massive, although it is not reciprocal. The OS, in turn,
projects to the AOB (Lanuza and Halpern, 1997). Therefore, several possibilities exist for feed-back regulation of
vomeronasal input, and it seems that the vomeronasal
information is highly processed within this circuitry before
it is integrated with other sensory modalities (e.g., in the
LC and the amygdala).
A projection from the NS to the NAcc is reported but is
not illustrated in Martı́nez-Garcı́a et al. (1993b). However,
in agreement with our data, González et al. (1990) did not
636
find retrogradely labeled cells in the NS after Fluoro-Gold
injections into the NAcc of the lizard G. gecko. In the work
of Voneida and Sligar (1979), the projections from the NS
are described based on an injection clearly centered in the
DLA (see their Fig. 4), rostral and dorsal to the NS.
Anterograde labeling after this injection is located in the
caudal NAcc, that is, caudal to the olfactostriatum (Voneida and Sligar, 1979). Therefore, we believe that the NS
in lizards probably also projects to the olfactostriatum
(medial and ventral to the NAcc) and not to the NAcc itself.
The basal forebrain in snakes is a cytoarchitectonically
complex area, and its connections and neurochemistry are
still poorly studied. The olfactostriatum, as a result, has
been considered to be part of the striatum, without specifically naming it, by most authors (Ulinski, 1978; Smeets et
al., 1986a). We have chosen to keep the name ‘‘olfactostriatum,’’ because it was already present in the literature
(Durward, 1930; Halpern, 1980). However, a study of the
distribution of dopamine in the brain of another snake
(Python regius) suggests that the OS may be part of a
complex dopaminergic area that is probably equivalent to
the NAcc/striatum of other squamate reptiles (Smeets,
1988).
Neurophysiological studies in the green iguana (I.
iguana) demonstrated that electrical stimulation of neurons in the olfactostriatum elicited strong tongue-flicking
responses (Distel, 1978). The same results were obtained
following stimulation of the NS. However, these data
should be interpreted with caution, because stimulation of
the NAcc also elicited tongue-flicking responses. It is
known that garter snakes respond to the presence of
significant chemical stimuli in the environment (e.g., prey
extracts) with an increase in tongue-flick rate (Halpern
and Kubie, 1983). The projection from the NS to the
olfactostriatum is probably implicated in this behavior,
which has been extensively used as a way to monitor
vomeronasal stimulation in behavioral tasks (for review,
see Halpern, 1992). On the other hand, tongue-flicking
activity is observed in a wide variety of behaviors; therefore, it is not surprising that it can be elicited by stimulating other nuclei, such as the NAcc, that are not directly
implicated in the vomeronasal system.
The other efferent intratelencephalic projections of the
NS reach the rostral DC, the rostral LC, and several nuclei
of the amygdaloid formation: the ExA, VAA, and DLA. The
projections to the olfactory areas (LC, ExA, and VAA) will
be discussed below in the section about the convergence of
olfactory and vomeronasal information. The NS projection
to the DLA is strong and bilateral (Fig. 2G,H). It has also
been reported in the lizard P. hispanica (Martı́nez-Garcı́a
et al., 1993b). In both lizards and snakes, the DLA does not
receive (directly) either olfactory or vomeronasal input
(Martı́nez-Garcı́a et al., 1991; Lohman and Smeets, 1993;
Lanuza and Halpern, 1997), and, in P. hispanica, it has
been shown to project bilaterally to the striatum, including
the NAcc, and to receive a putative cholinergic afferent
from the basal forebrain (Martı́nez-Garcı́a et al., 1993b).
On the basis of these projections plus the strong acetylcholinesterase reactivity of its neuropil, the DLA has been
compared to the mammalian basolateral nucleus of the
amygdala (Martı́nez-Garcı́a et al., 1993b; for a review of
the amygdaloid projections in mammals, see Price et al.,
1987). In mammals, this nucleus has been implicated in
stimulus-reward associations, especially via its projections
to the NAcc of the ventral striatum (Everitt and Robbins,
1992). The DLA of T. sirtalis also appears to project to the
E. LANUZA AND M. HALPERN
NAcc (Lanuza and Halpern, unpublished observations).
Moreover, vomeronasal stimuli in snakes are known to
have an important reinforcing value, so that they can be
used as unconditioned or reinforcing stimuli to condition a
response to an arbitrary stimulus (Halpern, 1988). Therefore, it seems likely that the projection from the NS to the
DLA is providing vomeronasal information to the amygdalostriatal circuit, which is probably implicated in mediating the reinforcing value of vomeronasal stimuli.
The projection to the rostral part of the DC (Fig. 4D) has
not been shown in other reptiles, but its presence was
suggested in the lizard P. hispanica (Martı́nez-Garcı́a et
al., 1986; Martı́nez-Garcı́a and Olucha, 1988). In the lizard
G. gecko, the projection from the LC to the MC has been
suggested to give rise to boutons en passant in the DC
(Hoogland and Vermeulen-Vanderzee, 1995). If this is the
case, then the rostral DC could be integrating olfactory
and vomeronasal information that is received directly by
the secondary olfactory and vomeronasal centers, respectively. No other sensory input to the DC is known in any
reptile, with the notable exception of the geniculocortical
projection in turtles (for review, see Ulinski, 1990). These
cases seem to be examples of divergent evolution in
different reptilian orders.
The projection from the NS to the DC may be an
important pathway for vomeronasal information to reach
the hypothalamus, because the NS does not project to the
VMH, and the projection to other hypothalamic areas
(LHA and LTM) is sparse (see Results). The DC has strong
projections to both the periventricular and the lateral
hypothalamus (see Results). Furthermore, in the lizard G.
gecko, most of the projection from the DC to the hypothalamus originates in the rostral part of the DC (Hoogland and
Vermeulen-Vanderzee, 1989). Alternative pathways
through which vomeronasal information may reach the
hypothalamus will be discussed below. On the other hand,
the DC receives an important input from the dorsolateral
anterior thalamic nucleus (Bruce and Butler, 1984) that is
probably multimodal that does not carry chemosensory
information (Hoogland, 1981, 1982; Martı́nez-Garcı́a and
Lorente, 1990; Martı́nez-Garcı́a et al., 1993a; Kenigfest et
al., 1997). The DC also receives other inputs from the
hypothalamus and brainstem (Bruce and Butler, 1984).
Therefore, the DC should not be considered to be a tertiary
area in the vomeronasal system but, rather, a limbic cortex
(Hoogland and Vermeulen-Vanderzee, 1989) or a ‘‘general’’
cortex (nonhippocampal, nonolfactory; Bruce and Neary,
1995b) that receives a tertiary vomeronasal input from the
NS and a multimodal input from the dorsal thalamus.
Descending projection to the hypothalamus
The main discrepancy between this study and the
previous reports (Voneida and Sligar, 1979; Halpern, 1980;
Martı́nez-Garcı́a et al., 1993b) is that we have not found a
projection from the NS to the VMH but, instead, a meager
projection to the LHA and to the LTM, a cell-poor area
located lateral to the VMH. Voneida and Sligar (1979) and
Martı́nez-Garcı́a et al. (1993b) reported that the NS projected to the shell (the outer rim) of the VMH, whereas
Halpern (1980) found that the projection reached the
whole VMH. The projection to the VMH actually originates
in the structures located medial and lateral to the NS, the
posterior part of the dorsal ventricular ridge (PDVR;
medially adjacent to the NS) and the lateral amygdala
(laterally adjacent to the NS). This has been demonstrated
recently in the lizards G. gecko (Bruce and Neary, 1995a)
TERTIARY VOMERONASAL CONNECTIONS IN SNAKES
and P. hispanica (Lanuza et al., 1997), and we have
confirmed it in our own laboratory in the snake T. sirtalis
(Lanuza and Halpern, unpublished observations). It can
also be observed in the work of Voneida and Sligar (1979),
in which the injection sites located in the DVR (anterior or
posterior) revealed a projection to the core of the VMH (see
their Figs. 2 and 3), whereas the projection to the shell of
the VMH was found after an injection that, in our opinion,
affected the DLA and the LA (see their Fig. 4) more than
the NS. The PDVR and the LA were very likely affected by
the lesions of Halpern (1980; see her Fig. 7) and by the
horseradish peroxidase injections of Martı́nez-Garcı́a et
al., 1993b; F. Martı́nez-Garcı́a, personal communication;
see also their Fig. 2).
Therefore, the main efferent projection of the NS is not
to the hypothalamus, as was previously believed, but,
rather, to the Naot, the amygdala (DLA, ExA, and VAA),
rostral DC and LC, and the olfactostriatum (see Results
and Fig. 2). The projection to the hypothalamus is actually
quite meager, and this fact changes to an important extent
our previous conception of how vomeronasal information is
processed in the brain of squamate reptiles. If the vomeronasal information was directly relayed to the VMH, then
the NS would probably serve mainly as a simple relay, and
the VMH nucleus would be dominated very strongly by
vomeronasal input. This appeared to be a logical conclusion, because vomeronasal information is very important
in the ecology of most squamate reptiles. However, the
telencephalic projection to the VMH appears to originate
to an important extent from two structures that receive
direct input from neither the main nor the accessory
olfactory bulbs: the PDVR and the LA; therefore, the
descending projection to the VMH from these nuclei does
not appear to carry any direct chemosensory information.
There are several pathways through which vomeronasal
information reaches the hypothalamus. One possibility, as
discussed above, is through the projection from the NS to
the DC, which, in turn, projects to the periventricular and
lateral hypothalamus. A more direct pathway would be via
the projections of the Naot and medial amygdala (MA) to
the hypothalamus. These projections are not well studied,
but retrogradely labeled neurons appear in the Naot and
MA after injections into the VMH in the lizards G. gecko
(Bruce and Neary, 1995a) and P. hispanica (Lanuza et al.,
1997). The Naot receives a strong projection from the AOB
(Lanuza and Halpern, 1997) and from the NS (this study);
therefore, its neurons are strongly driven by vomeronasal
stimuli. The MA receives a light projection from the AOB
(Lanuza and Halpern, 1997) and the NS (this study), but it
projects massively to the VMH (Bruce and Neary, 1995a;
Lanuza et al., 1997), therefore influencing strongly the
VMH activity. The BNST also receives a projection from
the NS, and it may project to the hypothalamus, although
the projections of this nucleus are still unexplored in
reptiles.
Another possible pathway for vomeronasal information
to reach the hypothalamus is through the projection from
the NS to the LC. The ventral part of the LC projects to the
PDVR in T. sirtalis (Lanuza and Halpern, unpublished
observations) and in the lizard G. gecko (Hoogland and
Vermeulen-Vanderzee, 1995). Although the NS projects
mainly to the dorsal part of the LC, this structure may be
relaying integrated olfactory and vomeronasal information to the PDVR. The PDVR of lizards has been shown to
receive a convergent projection from the different sensory
areas of the ADVR (Font et al., 1995; Andreu et al., 1996);
637
therefore, it would be receiving visual, somatosensory, and
auditory information from the ADVR and olfactory and
vomeronasal information from the LC.
Therefore, two putative multimodal projections reach
the hypothalamus, both of which may carry integrated
vomeronasal information: the projection from the DC and
the projection from the PDVR. We want to point out that
the two projections terminate in different hypothalamic
areas in a complementary fashion. The DC innervates
mainly the periventricular and lateral hypothalamus,
whereas the PDVR innervates mainly the VMH.
The projection from the NS to the LHA and to the LTM
appears to be quite sparse (see Fig. 2). However, we should
keep in mind that the NS in T. sirtalis is a very large
structure, and our tracer injections, although they were
relatively large, always affected only a fraction of the cells
that form the mural layer of the entire NS (Fig. 1).
Therefore, it is possible that the whole projection is more
important than it appears.
Convergence of olfactory and vomeronasal
information in the rostral LC
and olfactory amygdala
It is well known that the olfactory projection in reptiles
terminates in the external half of the external plexiform
layer of the LC (for review, see Lohman and Smeets, 1993).
The bilateral vomeronasal projection that originates in the
NS terminates mainly in the internal plexiform layer and
in the cell layer of the LC. Therefore, there is not a direct
overlap between the two chemosensory projections. However, it is likely that at least some neurons of the LC
integrate both types of chemosensory information. The
morphology of the neurons of the LC in snakes has been
studied by Ulinski and Rainey (1980), and there are two
main neuronal types in the rostral part of the LC, ‘‘bowl’’
neurons and double pyramidal neurons. The bowl neurons,
so called because their dendritic trees curve toward the
pial surface in a configuration that resembles a bowl, have
their somata located in the cell layer of the LC. It has been
demonstrated that they receive olfactory input on their
distal dendrites in the external plexiform layer (Ulinski
and Rainey, 1980). Many (probably most) of these neurons
project their axons to the MC. The other type of cell, the
double pyramidal neuron, has a somata located either in
the inner part of the cell layer or in the internal plexiform
layer of the LC. The dendrites of the double pyramidal
neurons originate from both poles of the somata, forming
pyramidal dendritic fields located in both plexiform layers
of the LC. The dendritic fields of these neurons span the
whole depth of the LC (see Fig. 12 in Ulinski and Rainey,
1980). We retrogradely labeled some of these double
pyramidal cells after an injection that affected the DC and
the dorsal part of the LC (Fig. 5A; only the proximal
dendrites are drawn, because there is a dense plexus of
fiber labeling that made it impossible to distinguish the
distal dendrites). The double dendritic morphology allows
these neurons to receive direct olfactory input on their
external dendritic tree and vomeronasal input from the NS
on the internal dendritic tree, as represented schematically in Figure 5B. In accordance with this hypothesis, the
double pyramidal neurons are more frequent in the dorsal
and rostral part of the LC (Ulinski and Rainey, 1980),
where the vomeronasal projection from the NS mainly
terminates. In any case, electron microscopic studies are
needed to confirm the possibility of olfactory and vomeronasal convergence onto individual double pyramidal cells of
638
E. LANUZA AND M. HALPERN
Fig. 5. A: Camera lucida drawing of a double pyramidal neuron of
the LC that was retrogradely labeled after an injection affecting both
the DC and the LC. The distal dendrites could not be drawn, because a
dense plexus of labeled fibers occupied the plexiform layers. Numbers
1, 2, and 3 designate the external, cell, and internal layer of the LC,
respectively. The approximate location of this neuron is shown in B. B:
Schematic drawing of the rostral LC as seen in a frontal section
showing the terminal field of the projection from the main olfactory
bulb (hatched area) and the terminal field of the projection from the
NS (represented by arrowheads). A double pyramidal neuron is
probably receiving both kinds of chemosensory inputs. For abbreviations, see list. Scale bars 5 40 µm in A, 200 µm in B.
the LC. The targets of the axons of these neurons are
unknown.
The ventral part of the LC, as stated above, projects
heavily to the PDVR in the lizard G. gecko (Hoogland and
Vermeulen-Vanderzee, 1995). It will be important to investigate whether the double pyramidal neurons project to
this structure, which, in turn, projects to the VMH.
A bilateral projection from the NS to the LC also has
been found in the lizards P. hispanica (Martı́nez-Garcı́a et
al., 1986, 1993b) and G. gecko (Bruce and Butler, 1984).
Martı́nez-Garcı́a et al. have suggested the possibility of
integration of olfactory and vomeronasal information in
the LC. Therefore, the integration of olfactory and vomeronasal information in the LC appears to be a general
feature of squamate reptiles. This fact changes considerably our previous picture of how olfactory and vomeronasal information are processed in the reptilian brain. The
LC should not be considered to be a pure olfactory struc-
ture (the olfactory cortex) but, rather, a chemosensory
cortex that integrates both kinds of chemosensory information.
On the other hand, the main olfactory bulb gives rise in
T. sirtalis to a light (bilateral) projection to the two nuclei
of the rostral amygdala, the ExA and the VAA (Lanuza and
Halpern, 1997). Given the strong innervation of these two
nuclei by the NS, the olfactory and vomeronasal projections do overlap directly in these areas. The situation is
opposite to that found in the LC: In the ExA and the VAA,
the predominant input is vomeronasal (from the NS), and
there is only a light olfactory input. In the LC, the olfactory
input is much stronger than the vomeronasal input.
Nothing is known about the connections of the ExA, but
the VAA appears to be reciprocally connected with the LC
(Martı́nez-Garcı́a et al., 1986) in the lizard P. hispanica,
and it has been recently shown in P. hispanica to project
(relatively lightly) to the lateral hypothalamus (Lanuza et
TERTIARY VOMERONASAL CONNECTIONS IN SNAKES
al., 1997). Therefore, this nucleus would be a direct relay of
olfactory and vomeronasal information to the hypothalamus, avoiding the multimodal areas (PDVR and DC).
Heterogeneity in the NS
Curiously enough, retrogradely labeled cells in the NS
after injections into the LC consistently appeared only in
the lateral part of the NS, thus suggesting that the
projection from the NS to the LC originates only in
neurons of the lateral (not of the medial) mural layer of the
NS. Similar results have been reported for the lizard G.
gecko (Bruce and Butler, 1984). This is not the only case of
differences between the lateral and medial part of the NS.
In the present study, one injection centered in the DLA
nucleus (that also affected the DC) gave rise to retrogradely labeled neurons only in the medial and dorsal
parts of the NS (not illustrated). Although we only made a
single injection into the DLA, it is worth noting that
retrograde labeling did not appear in the part of the NS
closest to the injection site (the dorsolateral quadrant). In
the lizard P. hispanica, injections of BDA into the preoptic
hypothalamus give rise to retrogradely labeled cells located mainly in the ventromedial part of the NS, whereas
injections into the anterior hypothalamus (that affect the
stria terminalis) give rise to retrogradely labeled cells
mainly in the lateral mural layer of the NS (Lanuza et al.,
1997). These results also suggest that different aspects of
the NS have differential projections. More data are needed
to confirm this hypothesis.
There are also some chemoarchitectonic differences between the medial part and the lateral part of the NS of T.
sirtalis. Only the ventral and medial parts contain steroid
hormone-accumulating cells (Halpern et al., 1982), and
only the medial half of the hilus is positive for acetyl
cholinesterase reactivity (Lanuza and Halpern, unpublished observations). In the snake P. regius (Smeets, 1988)
and the lizard G. gecko (Smeets et al., 1986b) the dorsal
part of the NS has a dense dopaminergic innervation,
whereas the ventral part receives only a sparse dopaminergic innervation. The commissural projections that link the
two, as we have demonstrated, are organized in a topographical fashion (i.e., they show a rough point-to-point
topography). In conclusion, the NS should be considered to
be a heterogenous structure, although we do not know yet
the functional significance of these intranuclear differences.
The question of the origin of the dopaminergic innervation of the dorsal NS is intriguing, because we have not
found retrogradely labeled cells in any dopaminergic
nucleus after tracer injections into the NS. In fact, all of
the retrograde labeling was restricted to the telencephalon, where there are no dopaminergic cells except for the
main and accessory olfactory bulbs (Smeets, 1988). However, the dopaminergic cells of the bulbs are not mitral
cells but are mainly periglomerular neurons; therefore, it
is unlikely that they are the origin of the dopaminargic
innervation of the NS. This suggests that the retrograde
transport of BDA may not be sensitive enough to detect
relatively sparse projections, such as the one that is the
origin of the dopaminergic innervation of the NS.
Although no topography has been found in the projection
from the AOB to the NS (Lanuza and Halpern, 1997), it
should be kept in mind that, in mammals, the vomeronasal
sensory epithelium has two spatially separated populations of receptor cells (Shinohara et al., 1992; Halpern et
al., 1995) that project differentially to the AOB (Jia and
639
Halpern, 1996). It is not known whether the same is true
for reptiles. However, because the vomeronasal system
appears to be conserved through evolution, the compartmentalization of the NS suggests that the AOB and the
vomeronasal epithelium may also be heterogeneous structures in reptiles. If such a heterogeneity exists in squmate
reptiles, then this would suggest that different attributes
of the vomeronasal stimuli may be processed in different
parts of the NS.
Comparative remarks
The NS of reptiles has been considered to be homologous
to the posteromedial cortical amygdaloid nucleus (CoApm)
of mammals (Ulinski and Peterson, 1981; Martı́nez-Garcı́a
et al., 1991, 1993b) on the basis that both nuclei receive a
vomeronasal input from the AOB, project back to the AOB,
and have commissural projections with its contralateral
equivalent (for the connections of the mammalian CoApm,
see de Olmos et al., 1985; Price et al., 1987; Canteras et al.,
1992). The CoApm was also believed to project to the shell
of the VMH (Krettek and Price, 1978; de Olmos et al.,
1985), as was the NS, and this feature also sustained the
homology between the two structures. This work and
recent studies in the lizards G. gecko (Bruce and Neary,
1995a) and P. hispanica (Lanuza et al., 1997) have shown
that the NS does not project to the VMH, and, based on
this result, Bruce and Neary (1995a,b) suggested that the
NS is a specialization of the squamate brain and does not
really have a homologue in mammals. However, Canteras
et al. (1992) have shown that the CoApm actually does not
project to the hypothalamus. Therefore, the putative homology between the NS and the CoApm appears to be well
sustained. Moreover, Canteras et al. (1992) have also
found that the CoApm provides an appreciable input to the
piriform and entorhinal areas, which may be equivalent to
the projection from the NS to the LC.
ACKNOWLEDGMENTS
This research was supported in part by an NIH grant
(DC00104) and by a grant from the Valencian IVEI (Institut Valencià d’Estudis i Investigació; 002/076). EL was
supported by a grant from the ‘‘la Caixa’’ Foundation.
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amygdalar, efferent, afferent, vomeronasal, informatika, connection, sphericus, snakethamnophis, lateral, sirtalis, corte, nucleus, olfactory, convergence
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