Brain Struct Funct (2012) 217:191–209
DOI 10.1007/s00429-011-0345-6
ORIGINAL ARTICLE
Collateral projections from nucleus reuniens of thalamus
to hippocampus and medial prefrontal cortex in the rat:
a single and double retrograde fluorescent labeling study
Walter B. Hoover • Robert P. Vertes
Received: 12 May 2011 / Accepted: 18 August 2011 / Published online: 15 September 2011
Ó Springer-Verlag 2011
Abstract The nucleus reuniens (RE) of the midline
thalamus has been shown to strongly innervate structures of
the limbic forebrain, prominently including the hippocampus (HF) and the medial prefrontal cortex (mPFC) and
to exert pronounced excitatory effects on HF and mPFC. It
was unknown, however, whether RE projections to, and
hence actions on, the HF and mPFC originate from a
common or largely separate groups of RE neurons. Using
fluorescent retrograde tracing techniques, we examined the
patterns of distribution of RE cells projecting to HF, to the
mPFC or to both sites via axon collaterals. Specifically,
injections of the retrograde tracers Fluorogold (FG) or
Fluororuby (FR) were made in the mPFC and in various
subfields of HF and patterns of single (FG or FR) or double
labeled (FG ? FR) cells in RE were determined. Pronounced numbers of (single) labeled neurons were present
throughout RE with FG or FR injections, and although
intermingled in RE, cells projecting to the mPFC were
preferentially distributed along the midline or in the perireuniens nucleus (pRE), whereas those projecting to HF
occupied a wide mediolateral cross sectional area of RE
lying between cells projecting to the mPFC. Approximately, tenfold more labeled cells were present in RE with
ventral compared to dorsal CA1 injections. Like single
labeled neurons, double labeled cells were found
throughout RE, but were most densely concentrated in
areas of greatest overlap of FG? and FR? neurons or
mainly in the lateral one-third of RE, medial to pRE.
Depending on specific combinations of injections, double
labeled cells ranged from approximately 3–9% of the
W. B. Hoover R. P. Vertes (&)
Center for Complex Systems and Brain Sciences,
Florida Atlantic University, Boca Raton, FL 33431, USA
e-mail: vertes@ccs.fau.edu
labeled neurons. The nucleus reuniens has been shown to
be a vital link in limbic subcortical–cortical communication and recent evidence indicates a direct RE involvement
in hippocampal and medial prefrontal cortical-dependent
behaviors. The present findings indicate that RE is critically positioned to influence the HF and mPFC, and their
associated behaviors, via separate or collateral projections
to these sites.
Keywords Infralimbic cortex Prelimbic cortex Entorhinal cortex Subiculum of hippocampus Spatial
learning Arousal Attention Consciousness
Abbreviations
CA1,d,v Field CA1 of Ammon’s horn, dorsal, ventral
division
CA3
Field CA3 of Ammon’s horn
DB
Double labeled cell
DBS
Deep brain stimulation
EC, l, m Entorhinal cortex, lateral, medial division
FG
Fluorogold
FR
Fluororuby
HF
Hippocampal formation
IL
Infralimbic cortex
MCS
Minimally conscious state
mPFC
Medial prefrontal cortex
mt
Mammillothalamic tract
PFC
Prefrontal cortex
PL
Prelimbic cortex
pRE
Perireuniens nucleus of thalamus
PT
Paratenial nucleus of thalamus
PV
Paraventricular nucleus of thalamus
PVHy
Paraventricular nucleus of hypothalamus
RAM
Radial arm maze
RE
Nucleus reuniens of thalamus
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192
RH
slm
SMT
SUB,v
VS
3V
Brain Struct Funct (2012) 217:191–209
Rhomboid nucleus of thalamus
Stratum lacunosum moleculare
Submedial nucleus of thalamus
Subiculum, ventral division
Vegetative state
Third ventricle
Introduction
The nucleus reuniens (RE) lies ventrally on the midline of
the thalamus, above the third ventricle, and extends longitudinally virtually throughout the thalamus (Swanson
2004; Vertes et al. 2006). RE is reciprocally connected
with the hippocampus (HF) and the medial prefrontal
cortex (mPFC) (Herkenham 1978; Wouterlood et al. 1990;
Dollerman-Van der Weel and Witter 1996; Risold et al.
1997; Bokor et al. 2002; McKenna and Vertes 2004; Vertes
2002, 2004, 2006; Cavdar et al. 2008), and as such appears
to be critically involved in the two way communication
between these structures.
RE is a major route through which the mPFC influences
the hippocampus. Specifically, HF distributes to the mPFC,
but there are no direct return projections from the mPFC to
the hippocampus. Accordingly, mPFC effects on the hippocampus appear to be mainly relayed through RE, thus
completing an important loop between these structures:
HF [ mPFC [ RE [ HF (Vertes et al. 2006, 2007). At the
ultrastructural level, mPFC fibers have been shown to
synaptically connect with RE neurons projecting to the
hippocampus (Vertes et al. 2007). In addition to RE,
another route from the mPFC to HF is through the entorhinal cortex (Witter et al. 1989; Vertes 2004).
The few reports that have examined the physiological
effects of RE on the HF and mPFC have shown that RE
exerts strong excitatory actions on both structures. With
regard to HF, Dolleman-Van der Weel et al. (1997) showed
that RE stimulation produced large amplitude negative
going evoked responses (sink) at stratum lacunosum moleculare (slm) of CA1 as well as paired pulse facilitation at
CA1. Bertram and Zhang (1999) confirmed these findings
and further demonstrated that the excitatory actions of RE
at CA1 were equivalent to, or greater than, those of CA3 on
CA1, leading them to conclude that the RE projection to
the hippocampus ‘‘allows for the direct and powerful
excitation of the CA1 region’’ which ‘‘by passes the
trisynaptic/commissural pathway that has been thought to
be the exclusive excitatory drive to CA1’’. With respect to
the mPFC, we showed that RE stimulation produced short
latency (monosynaptic), large amplitude evoked potentials
at mPFC, with the largest effects at inner layers (5/6) of the
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ventral mPFC (Viana Di Prisco and Vertes 2006). The
foregoing indicates that RE distributes to, and significantly
effects, the hippocampus and medial prefrontal cortex.
A currently unresolved issue, however, is whether RE
projections to, and hence actions on, the HF and mPFC
originate from a common group of cells or alternatively
from separate populations of RE neurons. Previous work
suggests that RE projections to its primary targets are
essentially segregated within RE. Using fluorescent retrograde tracers, Dollerman-Van der Weel and Witter (1996)
reported that RE projections to CA1 and to the subiculum
of HF, to the entorhinal cortex (EC) and to the perirhinal
cortex mainly arose from separate groups of RE neurons.
Specifically, RE projections: (1) to CA1 originated from
the dorsolateral RE; (2) to the subiculum, from the lateral
RE (3) to the medial EC (ECm), from the medial RE; (4) to
the lateral EC (ECl) from the ventral half of RE, and (5) to
the perirhinal cortex from the perireuniens (pRE) nucleus
(or lateral wings of RE). In a similar manner, we showed
that RE fibers to the orbital cortex arose from pRE, to the
ECm mainly from the rostral RE and to ECl from the
caudal RE (Vertes et al. 2006). This indicates a segregation
of RE output to its main targets, and suggests the same may
be true for RE projections to the HF and mPFC.
The prospect of segregated RE outputs gains support
from recent examinations of the effects of RE lesions on
behavior. While few reports have examined the behavioral
effects of RE lesions, the findings to date conflict with
regard to whether RE lesions produce ‘prefrontal-associated’ or ‘hippocampal-dependent’ deficits. In an initial
study, Dolleman-Van der Weel et al. (2009), using water
maze tasks, reported that RE lesions produced deficits in
shifting strategies to changing environmental contingencies, but had little effect on spatial memory. Specifically, in
a probe test following training (escape platform removed),
rats with RE lesions initially swam to the correct quadrant,
indicating memory was intact, but quickly abandoned this
behavior, favoring one of ‘search over all the pool’ for the
missing platform. This was viewed as an inflexible strategy
to an environmental change, or a prefrontal cortical-associated deficit. In contrast to this, Davoodi et al. (2009)
reported that the reversible suppression of RE disrupted
reference and working memory tasks on the water maze,
while Hembrook and Mair (2011) showed that RE lesioned
rats displayed marked deficits on delayed non-match to
sample radial arm maze (RAM) tasks.
While several factors could account for the differing
results including choice of tasks, it is also possible that RE
lesions differed with respect to whether they were primarily localized to RE regions projecting to the hippocampus or to the mPFC—if, in fact, RE cells projecting to
these two structures are segregated within RE. The present
reports addresses this issue, that is, whether, or to what
Brain Struct Funct (2012) 217:191–209
degree, RE cells projecting to the HF and to the mPFC
originate from the same or largely separate populations of
RE neurons.
In brief, we showed that RE cells projecting to the HF
and the mPFC were intermingled within RE, but with
clusters distributing selectively to each site. RE cells projecting to HF were mainly located lateral to the midline
within the medial two-thirds of RE, while those distributing
to the mPFC were predominantly located in the lateral
one-third of RE extending to the lateral wings of RE,
particularly at caudal levels of RE. In addition, relatively
significant percentages of RE cells (3–9%) projected via
collaterals to the HF and mPFC. Double labeled cells were
mainly situated on the midline and in mid-mediolateral
regions of RE.
Materials and methods
Twenty-seven male Sprague–Dawley rats (Harlan Laboratories, Indianapolis, IN) weighing 350–425 g were
injected with two retrograde fluorescent tracers, Fluorogold
(Fluorochrome, Denver, CO) and Fluororuby (Invitrogen,
Carlsbad, CA). These experiments were approved by the
Florida Atlantic University Institutional Animal Care and
Use Committee and conform to all federal regulations and
National Institutes of Health guidelines for the care and use
of laboratory animals.
Fluorogold (FG) and Fluororuby (FR) were dissolved in
a 0.1 M sodium acetate buffer (pH 3.5 to 4.5) to yield an
8% concentration. Rats were anesthetized for surgery using
an 80 mg/kg dose of Ketamine and 10 mg/kg dose of
Xylazine. FG or FR was iontophoretically deposited into
the hippocampus or into the medial prefrontal cortex using
glass micropipettes with an outside tip diameter of
75–100 lm. Retrograde tracer injections were made: (1)
into the prelimbic (PL) and infralimbic (IL) cortices of the
mPFC, or (2) into the dorsal or ventral CA1 or the ventral
subiculum of the hippocampus. Positive direct current
(8–10 lA) was applied through a Grass stimulator (model
88) coupled with a high-voltage stimulator (FHC, Bowdoin, ME) at 2 s ‘‘on’’/2 s ‘‘off’’ intervals for 2–10 min.
Following a survival time of 7 days, rats were deeply
anesthetized with sodium pentobarbital and perfused transcardially with 100 ml of heparinized saline wash followed
by 450 ml of fixative (4% paraformaldehyde in 0.1 M
sodium phosphate buffer (PB), pH 7.4). The brains were
then removed and postfixed in 4% paraformaldehyde—
0.1 M PB solution at 48C for 24 h. Fifty micron transverse
sections were collected in 0.1 M PB (pH 7.4) using a
vibrating microtome and stored at 48C. Representative
sections were mounted onto chrome–alum gelatin coated
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slides and coverslipped using DPX media (BDH Laboratories, Poole, England). An adjacent series of sections was
stained with cresyl violet for anatomical reference.
Sections were examined with epi-fluorescent techniques
using appropriate filters for FG (excitation 350–395 nm;
emission 530–600 nm) and FR (excitation 540–560 nm;
emission 580 nm).
Photomicrographs
Photomicrographs of injection sites and labeled cells were
taken with a QImaging (Q ICAM) camera mounted on a
Nikon Eclipse E600 microscope using Nikon Elements 3.0
imaging software. Using Elements software, monochrome
micrographs were color corrected to reflect the appropriate
tracer (green for FG and red for FR). The color adjusted
micrographs were also used for cell counts and for the
schematic depiction of labeled cells. Micrographs were
adjusted for brightness and contrast using Adobe PhotoShop 7.0 (Mountain View, CA). Some micrographs were
also overlaid to depict double labeled cells utilizing the
layering capabilities of Adobe PhotoShop 7.0.
Cell counts
All 27 cases were analyzed for numbers and patterns of
single and double labeled cells in RE following hippocampal and mPFC injections. Fourteen of 27 cases had
particularly well placed injections of retrograde tracers in
both the HF and mPFC. Seven of these 14 cases were
selected for cell counting based on optimal injections in
representative regions of the hippocampus: dorsal and
ventral CA1, the ventral subiculum and spanning ventral
CA1 and the ventral subiculum. Counts of single (FG or
FR) and double labeled cells were taken from six representative sections evenly spaced throughout the rostrocaudal extent of RE. Cells were classified as single labeled
if they were excited (epi-fluorescence) with one set of filters (FG or FR) but not the other, and double labeled if they
were excited (epi-fluorescence) using the FG and FR filters
in the same focal plane at 200 and 4009 magnification.
Schematics
Four of the 7 cases used for cell counting were schematically illustrated. The medial prefrontal injections of these 4
cases were placed in the ventral mPFC, approximately on
the border of the the prelimbic and infralimbic cortices.
The HF injections for these 4 cases were: (1) ventral CA1
(case 15); (2) dorsal CA1 (case 21); (3) ventral subiculum
(case 22); and (4) spanning the ventral CA1 and the ventral
subiculum (case 27).
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Brain Struct Funct (2012) 217:191–209
Results
Injections of the retrograde tracers FG or FR were made
into the ventral mPFC and into various regions of the
hippocampus, and numbers and locations of retrogradely
labeled neurons in RE containing one (FG of FR) or both
tracers were determined. Seven cases with injections
optimally placed in the mPFC and hippocampal subfields
are described in detail and four of these cases are schematically illustrated.
Figure 1 schematically depicts sites of injections for the
seven cases. As shown (Fig. 1) the mPFC injections were
situated in the ventral mPFC, localized to the IL or prelimbic cortices. The mPFC injections could essentially be
divided into three groups: a ventral group centered in IL
with extensions dorsally to PL (cases 9, 15 and 27); an
intermediate group centered in PL with spread ventrally to
IL (cases 21, 25, and 26) and a dorsal injection (case 22)
restricted to PL. Hippocampal injections were placed in
three subfields of the HF: (1) CA1, dorsally (case 21) and
ventrally (case 15); (2) the ventral subiculum (cases 22,
and 25); and (3) the ventral CA1/ventral subiculum—or
spanning the two fields (cases 9, 26, and 27). For six of
seven of these cases, FG was injected in the mPFC and FR
in the hippocampus.
Figure 2 shows sites of injections in the mPFC and HF
for cases 15 and 27. As depicted, the mPFC injections were
confined to IL/PL (Fig. 2a, c), whereas the HF injection for
case 15 was centered in the slm of CA1 of the ventral HF
(Fig. 2b) and that for case 27 was localized to slm at the border
of CA1/subiculum of the ventral HF (Fig. 2d). The slm is the
terminal destination of RE fibers distributing to the hippocampus (Wouterlood et al. 1990; Vertes et al. 2006).
mPFC and dorsal and ventral CA1 injections (cases 21
and 15)
Figure 3a shows the number and relative percentages of
single and double retrogradely labeled cells at six rostral to
caudal levels of RE following injections in the mPFC and
in CA1 of the ventral hippocampus (case 15). As depicted,
this mPFC-ventral CA1 pair of injections gave rise to
marked numbers of labeled cells in RE (range 193–438
cells) with the greatest numbers at mid-levels of RE (levels
2–4)—which is the largest expanse of RE. With the
exception of level 6, there were more labeled cells in RE
with HF than with mPFC injections with the greatest differential at levels 2 (61.6%, HF; 38.4%, mPFC) and 4
(60.1%, HF; 39.9%, mPFC). Interestingly, the ratio of
labeled cells was reversed at the caudal RE (level 6) such
that a greater percentage of cells were labeled with mPFC
(62.7%) than with HF (37.3%) injections. The foregoing
indicates proportionally stronger projections from rostral
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C
A
D
B
E
F
Fig. 1 Schematic representation of paired injections of Fluorogold
(FG) in the infralimbic (IL) and prelimbic (PL) cortices of the medial
prefrontal cortex (mPFC) (a, b), and Fluororuby (FG) injections in the
dorsal or ventral CA1, ventral subiculum (SUBv) or spanning ventral
CA1/SUBv (c–f) in cases 15, 21, 22, 25, 26 and 27, and paired injections
of FR in the mPFC (a, b) and FG in CA1/SUBv (d) for case 9
RE (levels 1–4) to the hippocampus and from the caudal
RE (levels 5 and 6) to the mPFC. Relatively pronounced
numbers of double labeled (DB) neurons were observed at
all levels of RE ranging from 3.7 to 8.4%, with the largest
percentage of DBs at a rostral (level 2, 6.2%) and caudal
level (level 5, 5.7%) of RE.
Figure 4 schematically depicts the locations of single
(FG, green dots; FR, red dots) and double labeled
(FG ? FR, black triangles) cells at six rostrocaudal levels
of RE for case 15 (mPFC-ventral CA1). As shown, FG-and
FR-labeled neurons were largely intermingled rostrocaudally throughout RE, with a tendency for FR-labeled cells
(projecting to HF) to be located medially in RE and FG
labeled cells (projecting to mPFC) to reside laterally in
RE at the rostral RE (Fig. 4b, c). This medial to lateral
segregation became more pronounced caudally in RE
(Fig. 4d–f), particularly within the lateral wings of RE (or
Brain Struct Funct (2012) 217:191–209
195
Fig. 2 a, b Sites of paired injections of Fluorogold in the infralimbic/
prelimbic cortex of the mPFC (a) and Fluororuby in CA1 of the
ventral hippocampus (b) for case 15. c, d Sites of paired injections of
Fluorogold in the infralimbic/prelimbic cortex of the mPFC (c) and
Fluororuby at the border of CA1 and the subiculum of the ventral
hippocampus (d) for case 27. Note that the ventral hippocampal
injections (b, d) are centered in the stratum lacunosum moleculare
(slm) of HF. Scale bar for a, c 1000 lm; for b,d 1120 lm
pRE) which almost entirely consisted of FG labeled neurons. This pattern of caudal RE labeling (Fig. 4d, e) is shown
in the photomicrographs of Figs. 5 and 6. As depicted, clusters
of FG? cells are present on the midline and in the lateral
wings of RE (Figs. 5, 6a), whereas FR? neurons are mainly
located lateral to the midline (Fig. 5, right rectangle) between
the clusters of FG? cells (Figs. 5, 6b). As further illustrated
(Fig. 5), the region just lateral to the midline mainly contained
FR? cells, while that farther laterally (or medial to pRE)
contained a greater mixture of FR? and FG? cells.
While the percentage of double labeled cells neurons (to
total numbers) was relatively constant rostrocaudally
throughout RE with case 15 (see Fig. 3a), ranging from 3.7
to 6.2%, more were present medially than laterally in RE,
particularly at caudal levels of RE (Fig. 4d–f), probably
owing to a greater intermingling of FG-and FR-labeled
neurons in the medial than lateral RE.
Figure 3b shows the numbers and relative percentages
of single and double retrogradely labeled cells at six rostrocaudal levels of RE following an injection in the mPFC
and in CA1 of the dorsal hippocampus (case 21). As
depicted, there was a greater percentage of FG? than FR?
cells at all levels of RE (Fig. 3b). This weighting in favor
of FG? cells (to the mPFC) results from considerably
fewer labeled cells in RE with dorsal CA1 (case 21, Fig. 7)
than with ventral CA1 (case 15, Fig. 4) injections. Associated with this, exceedingly few DB cells were observed
at any level of RE with case 21 (Figs. 3b, 7); or only eight
double labeled cells were present throughout RE. Similar,
however, to case 15 in which the mPFC injection was only
slightly ventral to that of case 21 (Fig. 1), marked numbers
of FG? cells were present in RE (range 72–261 over the
6 levels) (Fig. 3b) and more densely concentrated laterally than medially in RE, particularly at caudal levels of
RE, or within pRE (Fig. 7c–f). In addition, there was a
tendency, at least rostrally, for FG? cells to form a
midline and a lateral group with sparser labeling between
them (Fig. 7a–d).
mPFC and ventral subiculum injections
(cases 22 and 25)
Figure 8a depicts the number and relative percentages of
single and double labeled cells at six rostral to caudal
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196
Fig. 3 Numbers and relative
percentages of Fluorogold (FG)
labeled cells (green) with
medial prefrontal cortical
injections, Fluororuby (FR)
labeled cells (red) with
hippocampal injections and
double labeled cells (FG ? FG)
(black) at six rostral to caudal
levels (1–6) of nucleus reuniens
for cases 15 and 21. The FR
injection for case 15 was made
in the ventral CA1 of the
hippocampus (HF), for case 21
in dorsal CA1 of HF. Note the
significantly greater number of
FR-labeled cells in RE with the
ventral than with the dorsal CA1
injection
Brain Struct Funct (2012) 217:191–209
A
Case 15 (FG-mPFC; FR CA1v)
section
Fluorogold labeled
Fluororuby labeled
Double labeled
n = 164, 46.2% n = 191, 53.8% DB: n = 17, 4.8%
1 Total number of cells labeled = 355
n = 168, 38.4% n = 270, 61.6% DB: n = 27, 6.2%
2 Total number of cells labeled = 438
3
n = 180, 47.2% n = 201, 52.8% DB: n = 18, 4.7%
Total number of cells labeled = 381
n = 174, 39.9% n = 262, 60.1% DB: n = 16, 3.7%
Total number of cells labeled = 436
n = 184, 49.9% n = 185, 50.1% DB: n = 21, 5.7%
5
Total number of cells labeled = 369
4
6
B
n = 121, 62.7% n = 72, 37.3% DB: n = 8, 4.1%
Total number of cells labeled = 193
Case 21 (FG-mPFC; FR CA1d)
section
1
n = 171, 90.5% n = 18, 9.5 % DB: n = 2, 1.0%
Total number of cells labeled= 189
n = 157, 92.4% n = 13, 7.6 % DB: n = 0
Total number of cells labeled = 170
n = 261, 94.2% n = 16, 5.8% DB: n = 1, 0.36%
3
Total number of cells labeled = 277
2
n = 153, 87.4% n = 22, 12.6% DB: n = 2, 1.1%
Total number of cells labeled = 175
n = 147, 86% n = 24, 14% DB: n = 2, 1.2%
5
Total number of cells labeled = 171
n = 72, 81.8% n = 16, 18.2 % DB: n = 1, 0.81%
4
6
Total number of cells labeled = 88
levels of RE following an injection in the mPFC and in the
ventral subiculum (SUBv) of HF (case 22). Pronounced
numbers of retrogradely labeled neurons were found rostrocaudally throughout RE with total numbers of cells
(FG ? FR) at the six levels ranging from 258 at level 6 to
709 cells at level 2. There were roughly equivalent numbers of FG (projecting to mPFC) and FR (projecting to HF)
cells in RE, with larger percentages of FR? neurons at a
rostral (level 1) and at caudal levels of RE (4–6), and larger
percentages of FG? cells at intermediate levels of RE (2,
3). Percentages of DB cells (to total numbers) were fairly
constant across rostrocaudal levels of RE, ranging from
2.1% at level 2 to 3.9% at level 6, but overall percentages
of DB cells were lower for this case (mPFC/SUB injection)
than for the mPFC-ventral CA1 injection (case 15)—as
well as for the other mPFC-subicular injection (case 25)
(Fig. 8b).
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Figure 9 schematically depicts the locations of single and
double labeled cells at six rostral to caudal levels of RE for
case 22 (Fig. 8a). As shown for cases 15 and 21, considerably
greater numbers of FG? neurons were present laterally than
medially in RE, particularly at caudal regions of RE where
they were mainly found on the lateral border of RE,
extending laterally to pRE. In addition, and as generally seen
with other cases, FR? cells were densely packed within an
intermediate (mediolateral) zone of RE (Fig. 9a–f), with
some extension to the midline at the caudal RE (Fig. 9d, e).
At rostral levels (Fig. 9a, b), these patterns of labeling
resulted in a mid-lateral core of FR-labeled cells surrounded
by FG? neurons, laterally and medially. Finally, FR? cells
were considerably more densely concentrated dorsally (or
dorsolaterally) than ventrally in RE. As shown in Fig. 8a,
double labeled neurons were quite evenly distributed
throughout RE, with largest percentages at the caudal RE (or
Brain Struct Funct (2012) 217:191–209
197
Fig. 4 Schematic representation of locations and patterns of Fluorogold (FG) labeled cells (green dots), Fluororuby (FR) labeled cells
(red dots) and double labeled cells (black triangles) at six rostral to
caudal levels of nucleus reuniens (a–f) following FG injections in the
mPFC and FR injections in ventral CA1 for case 15
levels 4–6, Fig. 9d–f), and ranged from 2.1–3.9% of labeled
neurons (Fig. 8a). DB cells were fairly tightly clustered
dorsoventrally, extending from the midline to pRE, within
mid regions of RE (Fig. 9c–f).
Figure 8b depicts the number and relative percentages of
single and double labeled cells at six rostral to caudal levels
of RE following an injection in the mPFC and in the ventral
subiculum of HF (case 25). As shown, there were proportionally more FR than FG labeled cells at all levels of RE
with the greatest differential at the caudal RE: a 70/30%
ratio (FR/FG) at level 5. The percentage of DB cells was
relatively high ranging from 4.0 to 7.4% of labeled neurons,
with the largest percentages at the rostral RE: 7.4% at level
1 and 7.3% at level 2. There were quite marked differences
in the patterns of labeling for the two mPFC/ventral SUB
cases (cases 22 and 25). For instance, more labeled cells
(FG ? FR) were observed in RE with case 22 than with
case 25 (Fig. 8a, b) which could involve relative sizes and/
or locations of the injections in the two cases. With regard to
the mPFC injections, the FG injection of case 22 was just
dorsal to, but only slightly larger than, that of case 25
(Fig. 1a, b), suggesting that locations rather than the sizes of
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Brain Struct Funct (2012) 217:191–209
Fig. 5 Low magnification
photomicrograph of a transverse
section through the thalamus
depicting the distribution of
Fluorogold and Fluororuby
labeled cells at a mid
rostrocaudal level of nucleus
reuniens (RE) following a FG
injection in the mPFC and a FR
injection in CA1 of the ventral
hippocampus for case 15. Note:
1. clusters of FG labeled
neurons along the midline and
in the lateral wings of RE
(perireuniens nucleus, pRE);
2. clusters of FG labeled cells
lateral to the midline; and
3. a intermingling of FG-and
FR-labeled cells just medial to
pRE. The regions denoted by
the left and right rectangles are
depicted at higher magnification
in Fig. 6a, b, respectively. Scale
bar 200 lm
Fig. 6 High magnification
photomicrographs depicting a
cluster of Fluorogold labeled
cells on the midline (a) in RE in
the region depicted by the left
rectangle in Fig. 5, and a cluster
of Fluororuby labeled neurons
laterally in RE (b) in the region
depicted by the right rectangle
in Fig. 5. Scale bar 100 lm
injections mainly contributed to the differences in labeling
in the two cases. Or, in effect, RE distributes more heavily
to the dorsal than to the ventral PL.
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The same distinction appears to apply to the subicular
injections in the two cases; that is, the SUB injections for
cases 22 and 25 were of equivalent size (Fig. 1e, f), but the
Brain Struct Funct (2012) 217:191–209
199
Fig. 7 Schematic representation of locations and patterns of Fluorogold (FG) labeled cells (green dots), Fluororuby (FR) labeled cells
(red dots) and double labeled cells (black triangles) at six rostral to
caudal levels of nucleus reuniens (a–f) following FG injections in the
mPFC and FR injections in dorsal CA1 for case 21. Note the sparse
FR labeling in RE with the dorsal CA1 injection
injection of case 22 was caudal that of case 25, suggesting a stronger RE output to the caudal-ventral SUB
(case 22) than to the rostral SUB (case 25). Despite
greater numbers of labeled neurons in RE with case 22
than with case 25, considerably greater percentages of
cells were double labeled with case 25 than with case 22.
This would appear to indicate a more pronounced
branching of RE cells (collateral projections) to the
ventral PL/rostral SUB (case 25) than to the dorsal PL/
caudal SUB (case 22). Figure 10 depicts DB cells in RE
for case 25 (Fig. 10a–c).
mPFC and ventral CA1/ventral subiculum (cases 9, 26,
and 27)
The hippocampal injections of cases 9, 26 and 27 in part
encompassed CA1 and subicular regions of the ventral
hippocampus (Fig. 1d); that is, situated ventral to the
ventral CA1 case (case 15, Fig. 1d) and dorsal to the
ventral subicular cases (cases 22, and 25, Fig. 1e, f).
The ventral CA1/SUB injections (cases 9, 26, 27) were
slightly larger than the ventral subicular injections (cases
22, and 25).
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200
Brain Struct Funct (2012) 217:191–209
Fig. 8 Numbers and relative
percentages of Fluorogold (FG)
labeled cells (green) with
medial prefrontal cortical
injections, Fluororuby (FR)
labeled cells (red) with
hippocampal injections and
double labeled cells (FG ? FG)
(black) at six rostral to caudal
levels (1–6) of nucleus reuniens
for cases 22 and 25. The FR
injections for these cases were
made in the ventral subiculum
of the hippocampus
Figure 11a shows the number and relative percentages
of single and double labeled cells at six rostrocaudal levels
of RE with an injection in the mPFC and in the ventral
CA1/SUB of HF (case 9). As shown, the tracers were
reversed for case 9; that is, FR was injected into the mPFC
and FG into the hippocampus. Interestingly, proportionally
more FR? (projecting to HF) than FG? neurons (projecting to mPFC) were found at the rostral RE (levels 1, 2),
which contrasts with a reversal of ratios at the caudal RE,
most pronounced at the caudal pole of RE (levels 5, and 6).
Specifically, 70.3% of the cells at level 5 and 76.2% at
level 6 were FR? neurons. Although case 9 contained a
lower percentage of DB cells than found with the other
ventral CA1/SUB cases, percentages were moderate
(2.4–4.9%), and DB cells were fairly evenly distributed
throughout RE, with the largest concentration at the caudal
RE (Fig. 11a, levels 4–6).
Figure 11b shows the number and relative percentages
of single and double labeled cells at six rostral to caudal
levels of RE following injections in the mPFC and in the
ventral CA1/SUB of HF (case 26). Interestingly for this
case, unlike the other cases, a greater percentage of FG?
(projecting to mPFC) than FR? cells (projecting to HF)
123
were observed at all levels of RE. The ratios of FG?/FR?
neurons were relatively constant across levels ranging from
52.6/47.4% (level 3) to 57.5/42.5% (level 2). Possibly
related to the greater proportion of FG? to FR? cells
throughout RE, a large percentage of cells were double
labeled (8.1–11.1%), and were quite evenly distributed
throughout RE with highest percentages at the very rostral
(level 1, 10.2%) and caudal RE (level 6, 11.1%). This
represented the highest percentage of DB cells of all cases.
Figure 11c depicts the number and relative percentages
of single and double labeled cells at six rostral to caudal
levels of RE following an injection in the mPFC and the
ventral CA1/ventral subiculum of HF (case 27). As shown,
pronounced numbers of single and double labeled cells
were present at all levels of RE with this pair of injections.
While rostral levels of RE (levels 1, and 2) contained
proportionally more FR? than FG labeled cells (62/38%),
relatively equal numbers of FG? and FR? cells were
present in remaining (or caudal) regions of RE (levels 3–6).
There was a considerably greater number of labeled neurons (FG?FR) with case 27 (total, 3,138) than with the
other mPFC/ventral CA1/SUB cases: case 9 (total 1,975);
case 26 (total 2,287) (Fig. 11). This would appear to
Brain Struct Funct (2012) 217:191–209
201
Fig. 9 Schematic representation of locations and patterns of Fluorogold (FG) labeled cells (green dots), Fluororuby (FR) labeled cells
(red dots) and double labeled cells (black triangles) at six rostral to
caudal levels of nucleus reuniens (a–f) following FG injections in the
mPFC and FR injections in the ventral subiculum for case 22
involve larger FG (Fig. 1a, b) as well as FR (Fig. 1d)
injections with case 27 than for the other two cases.
A large percentage of cells were double labeled with
case 27 (Figs. 11c, 12). They were quite evenly distributed
throughout RE, ranging from 4.3 to 7.7% —with largest
percentages at the rostral RE (levels 1, 2). The percentage
of DB cells, however, was lower for case 27 than for the
other ventral CA1/SUB cases (cases 9, 26).
Figure 12 schematically depicts the pattern of distribution of single and double labeled cells at six levels of RE
for case 27. Similar to other cases, FG? neurons
(projecting to mPFC) were most densely concentrated
along the midline and within the lateral wings of RE
(Figs. 13, 14b). FR? cells (projecting to HF) were most
densely packed in an intermediate zone positioned between
the medially and laterally located FG? cells (Figs. 13,
14a), and were mainly localized to the ventral half of RE,
rostrally (Fig. 12a, b) and the dorsal two-thirds of RE,
caudally (Fig. 12c–e). As described (Fig. 11c), DB cells
were fairly evenly distributed rostrocaudally throughout
RE, with the heaviest concentration in the rostral pole of
RE (Fig. 12a, b). Two relatively distinct populations of DB
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Brain Struct Funct (2012) 217:191–209
Fig. 10 Photomicrographs
depicting Fluorogold (FG) and
Fluororuby (FR) double labeled
(FG ? FR) neurons (open
arrows) for case 25 (a–c) and
case 27 (d–f). a and d show FG
labeled cells (green), b and
e show FR-labeled neurons
(red) and c and f show double
labeled cells (yellow) for each
case. Closed arrow in b denotes
a ‘‘FR cell’’ that was below
threshold for counting, while the
closed arrow in d denotes a
‘‘FG cell’’ that was below
threshold for counting. Scale
bar for a–c and d–f 20 lm
cells were detected: a collection on the midline, most
prominent at mid to rostral levels of RE (Fig. 12a–d), and a
mid-dorsoventral group that extended mediolaterally across
the central RE (Fig. 12c–f). Double labeled cells of the
central RE (medial to pRE) (Fig. 13) are depicted in the
photomicrographs of Fig. 10d–f.
Discussion
Using double retrograde fluorescent techniques, we
describe patterns of projections from the RE of the midline
123
thalamus: (1) to subfields of the hippocampus; (2) to the
ventral medial prefrontal cortex; and (3) to both regions via
axon collaterals.
The main findings were: (1) pronounced numbers of
retrogradely labeled neurons (single labeled) were present
throughout RE with injections in the ventral mPFC or in
subfields of HF; (2) although intermingled in RE, cells
projecting to the mPFC were preferentially distributed
along the midline or in the perireuniens nucleus (pRE),
whereas those projecting to HF occupied a wide mediolateral cross sectional area of RE lying between cells distributing to the mPFC; (3) with the exception of the dorsal
Brain Struct Funct (2012) 217:191–209
203
Fig. 11 Numbers and relative
percentages of Fluorogold (FG)
labeled cells (green) with
medial prefrontal cortical
injections, Fluororuby (FR)
labeled cells (red) with
hippocampal injections and
double labeled cells (FG ? FG)
(black) at six rostral to caudal
levels (1–6) of nucleus reuniens
for cases 9, 26, and 27. The
hippocampal (HF) injections for
each of these cases spanned
CA1 and the ventral subiculum
of HF. Unlike the other
illustrated cases, FG was
injected in CA1/SUBv and FR
in the mPFC for case 9
CA1 injection, there were considerably more labeled cells
in the rostral than caudal half of RE with mPFC or HF
injections; (4) ventral CA1 injections gave rise to approximately 10 times greater numbers of labeled neurons in
RE than did dorsal CA1 injections; (5) comparable to
single labeled neurons, double labeled cells were found
throughout RE, but were most densely concentrated in the
areas of greatest overlap of FG? and FR? cells—on the
midline and in the lateral one-third of RE, medial to pRE;
and (6) depending on specific combinations of injections,
double labeled cells ranged from approximately 3–9% of
the labeled neurons.
Methodological considerations
As described, two retrograde fluorescent tracers were used,
FG and FR. In preliminary work, we determined that the
optimal pairing of the two tracers was to inject FG in the
mPFC and FR in the hippocampus. The reason was that, in
our hands, FR was the (slightly) better retrograde tracer and
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204
Brain Struct Funct (2012) 217:191–209
Fig. 12 Schematic representation of locations and patterns of
Fluorogold (FG) labeled cells (green dots), Fluororuby (FR) labeled
cells (red dots) and double labeled cells (black triangles) at six rostral
to caudal levels of nucleus reuniens (a–f) following a FG injection in
the mPFC and a FR injection spanning CA1 and the ventral
subiculum for case 27
as such FR was deposited into the site requiring the most
precise positioning of injections (or less margin for error),
which was the outer molecular layer of CA1/subiculum.
RE projections to the hippocampus terminate within the
slm of CA1 and the subiculum (Wouterlood et al. 1990;
Vertes et al. 2006). This pairing (FG in mPFC and FR in
HF) was, however, not used for all cases. It was reversed in
about 10% of the cases including case 9.
If, as indicated, FR is a more effective retrograde tracer
than FG, it might be expected that FR injections would
produce proportionally more labeled cells in RE than
would FG injections—and thus possibly over represent
numbers of FR? compared to FG? neurons in RE. This
was not, however, borne out by the findings. With the
possible exception of case 25 in which FR? cells outnumbered FG? cells by approximately 60/40%, the numbers of FR and FG labeled neurons were largely equivalent
across RE for all cases. And for cases 9 and 26, there were
proportionally more FG? than FR? cells at all levels of
RE.
Two other factors could have possibly influenced the
relative percentages of FR/FG neurons in RE; that is, size
of injections and the differential strength of RE projections
to mPFC or to HF. Regarding injection size, it is well
recognized that the magnitude of labeling varies quite
directly with size of (retrograde) injections. With some
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Brain Struct Funct (2012) 217:191–209
205
Fig. 13 Low magnification
photomicrograph of a transverse
section through the thalamus
showing patterns of Fluorogold
and Fluororuby labeled cells at a
mid rostrocaudal level of
nucleus reuniens (RE) following
a FG injection in the mPFC and
a FR injection in CA1 of the
ventral hippocampus of case 27.
Note a cluster of FG labeled
neurons in the lateral wings of
RE and prominent populations
of FR-labeled cells extending
medially from the lateral wings
to the midline of RE. The region
denoted by the downward
vertical arrows is shown at
higher magnification in
Fig. 14a, while the region
denoted by the diagonal arrows
is shown at higher magnification
in Fig. 14b. Scale bar 200 lm
variation, FG and FR injections were of equivalent size, but
as group FR injections were slightly larger than FG
injections. Regarding differential strength of RE-mPFC
and RE-ventral HF projections, previous studies have
demonstrated massive RE projections to the hippocampus
(Herkenham 1978; Risold et al. 1997; Wouterlood et al.
1990; Bokor et al. 2002; Vertes et al. 2006, 2007) and
pronounced but less dense RE projections to the mPFC
(Herkenham 1978; Risold et al. 1997; Vertes et al. 2006).
In effect, then, each of the foregoing factors (relative
effectiveness of tracers, size of injections and differential
strength of RE projections to targets) would seem to favor
FR over FG labeling, but as mentioned there was a fairly
equal distribution of the two types of labeled cells, with
only a minor shift toward FR? cells.
With all retrograde tracers, there is the possibility of
uptake of tracers not only by fibers terminating at the site of
injection but also by those passing through the injection—
the fibers of passage problem. This is considerably less an
issue here in that: (1) RE projections to the present sites of
retrograde injections (mPFC and CA1/subiculum) have
been previously demonstrated with anterograde tracers
(Herkenham 1978; Wouterlood et al. 1990; Vertes et al.
2006); and (2) essentially the sole terminal destination of
fibers passing through the ventral mPFC would be more
rostral levels of the mPFC (Vertes et al. 2006) and those
passing through the CA1/subiculum of ventral HF would
be more caudal levels of the subiculum. Accordingly, if
there was a minor uptake of either tracer by damaged fibers
coursing through the sites of injection (to the rostral mPFC
or to the caudal subiculum), this would not noticeably alter
the present findings of single or collateral RE projections to
the mPFC and to the CA1/subiculum. In addition, the
present retrograde tracers, FG and FR, appear to be among
the least susceptible to uptake by the fibers of passage
(Schmued et al. 1990; Lanciego and Wouterlood 2006).
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206
Fig. 14 High magnification photomicrographs depicting a cluster of
Fluororuby labeled neurons laterally in nucleus reuniens (RE), medial
to the lateral wings of RE (a), in the region depicted by the vertical
arrows of Fig. 13 and a cluster of Fluorogold labeled cells in the
lateral wings of RE (b) in the region depicted by the diagonal arrows
of Fig. 13. Scale bar for a, b 100 lm
RE projections to subfields of the hippocampus: dorsal
and ventral CA1, ventral subiculum and ventral CA1/
subiculum (single labeled neurons). Comparison
with previous studies
Hippocampal injections gave rise to pronounced numbers
of (single) labeled cells within RE. This supports previous
results showing that RE strongly targets the hippocampus
(Herkenham 1978; Risold et al. 1997; Wouterlood et al.
1990; Bokor et al. 2002; Vertes et al. 2006, 2007).
Although spread throughout RE, labeled cells were most
densely concentrated in the intermediate mediolateral RE,
just lateral to the midline, rostrally, and on the medial
border of the perireuniens nucleus (or lateral wings) of RE,
caudally. Although relatively significant numbers of
labeled cells were also present on the midline, few were
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Brain Struct Funct (2012) 217:191–209
observed in pRE with HF injections. Previous reports have
similarly shown that RE cells projecting to HF are mainly
located laterally/dorsolaterally in RE (Su and Bentivoglio
1990; Dollerman-Van der Weel and Witter 1996; Bokor
et al. 2002).
In an examination of collateral RE projections to the
hippocampus and entorhinal cortex, Dollerman-Van der
Weel and Witter (1996) also noted a virtual absence of
labeled cells in pRE with HF injections. Interestingly, this
differed from their demonstration of ‘‘an exceptionally
large number of retrogradely labeled cells in the perireuniens nucleus’’ with injections in the perirhinal cortex.
By contrast, however, with the paucity of labeled neurons
in pRE with HF injections (Dollerman-Van der Weel and
Witter 1996, present results), anterograde (PHA-L) injections in pRE were shown to produce relatively substantial
terminal labeling in the outer moleculare layer of
CA1/subiculum of the ventral HF (Vertes et al. 2006).
The foregoing might suggest, then, that the pRE output to
HF originates from a restricted population of pRE cells
with fibers that branch extensively within slm of the ventral
HF.
Although labeled cells extended throughout RE with HF
injections, more were observed in the rostral than caudal
half of RE—with approximate rostral/caudal ratios of
60/40%. Dollerman-Van der Weel and Witter (1996)
similarly showed that RE cells projecting to HF (and to
EC) mainly originate from the rostral half of RE. These
findings would appear mainly due to the fact that RE is
larger rostrally and narrows caudally (Swanson 2004).
There were approximately ten times more labeled neurons in RE with ventral CA1 (case 15) than with dorsal
CA1 (case 21) injections. Since dorsal and ventral CA1
injections were of equivalent size and positioned in the
same subfields of CA1, this would indicate considerably
stronger RE projections to the ventral than to dorsal CA1.
This is supported by previous findings, using anterograde
tracers, showing a much greater density of labeled fibers in
the ventral than dorsal CA1 with RE injections (Herkenham, 1978; Ohtake and Yamada 1989; Wouterlood et al.
1990; Risold et al. 1997; Vertes et al. 2006).
Injections in various regions of the ventral hippocampus
(CA1, subiculum, CA1/SUB) produced generally similar
numbers of labeled cells in RE. Nonetheless, injections in
the ventral subiculum (cases 22, and 25) produced more
labeled neurons in RE than did ventral CA1 or CA1/SUB
injections. The single exception to this was case 27 which
was a large injection (of ventral CA1/SUB) and mainly
localized to the subiculum. This indicates stronger RE
projections to the subiculum than to CA1 of the ventral
hippocampus, and is generally consistent with previous
reports using anterograde tracers (Wouterlood et al. 1990;
Risold et al. 1997; Vertes et al. 2006).
Brain Struct Funct (2012) 217:191–209
RE projections to the ventral mPFC (single labeled
neurons). Comparison with previous studies
Injections in the ventral mPFC (IL and PL) gave rise to
significant numbers of retrogradely labeled neurons distributed throughout RE. Although labeled cells spread
mediolaterally across RE, they were most densely concentrated in the lateral wings of RE (pRE), and secondarily
along the midline and on the medial border of pRE.
Comparatively, fewer labeled cells were present just lateral
to the midline—or in the region of dense concentration of
labeled cells with HF injections. Similar to HF injections,
there were proportionally more labeled cells in the rostral
than in caudal half of RE with mPFC injections but relative
differences were less for mPFC than for HF injections,
likely owing to the fact that pRE is most fully expressed
caudally in RE.
Compared to reports examining RE-HF projections, few
studies have described RE projections to the mPFC. In an
early report using tritiated amino acids, Herkenham (1978)
showed that RE fibers spread rather diffusely to the medial
wall of ventral mPFC terminating in what was termed the
infraradiate area (corresponding to PL) and in the infralimbic region. Using PHA-L, Risold et al. (1997) subsequently described (at best) moderate RE projections to the
ventral mPFC. In a recent examination of efferent projections of RE (and the dorsally adjacent rhomboid nucleus)
using PHA-L, we found that RE strongly targets the mPFC
with fibers densely concentrated in layers 1 and 5/6 of IL
and PL (Vertes et al. 2006).
Collateral RE projections to the mPFC
and to the hippocampus. Comparison with previous
studies
With the exception of case 21 in which the percentage of
double labeled cells was less than 1% due to the sparse
retrograde labeling in RE with the dorsal CA1 injection,
the percentages of DB cells to total numbers of labeled
neurons ranged from approximately 3 to 9.25%. Excluding
case 21, the percentage of DB cells in 5 of 6 of the cases
was 3–6% of labeled neurons. The percentage of DB cells
for case 26 was 9.25%, or considerably higher than for the
other cases. Case 26 involved a mPFC injection spanning
PL/IL (Fig. 1a) and a HF injection in ventral CA1/SUB
(Fig. 1d). It is presently unclear why this particular pairing
of injections gave rise to such a large percentage of DBs. It
was not, for instance, the fact that case 26 contained more
labeled cells (and hence more DBs) than did the other
cases. There was no relationship between percentages of
DBs and total numbers of labeled cells. In fact, case 22
contained the most labeled neurons (3,248), but the lowest
percentage of DBs (3.01%).
207
Unlike the present demonstration of significant percentages of DB cells (3–9%) with HF and mPFC injections,
previous reports failed to show similarly high percentages
of DBs with various combinations of injections in HF and
in other forebrain structures. For example, an early examination of RE/midline thalamic projections to the amygdala, nucleus accumbens (ACC) and ventral HF (Su and
Bentivoglio 1990) showed that separate, only minimally
overlapping, populations of RE cells distribute to each site.
Virtually no double labeled neurons were found. In like
manner, Dollerman-Van der Weel and Witter (1996)
reported that RE projections to the entorhinal cortex, to
CA1 and to the subiculum arose from distinct regions of
RE. Finally, Bokor et al. (2002) described separate origins
of RE cells distributing to the septum and the hippocampus; that is, ventromedially in RE to the septum and
dorsolaterally in RE to HF. Based on their findings and
those of previous reports, Bokor et al. (2002) concluded
that: ‘‘it is likely that distinct cell populations form clusters
at various subregions in the RE, the clusters giving rise to
projections to well defined target areas in the limbic
system.’’
Consistent with the foregoing, we presently describe
distinct clusters of RE neurons distributing to either HF or
to the mPFC, but in contrast to earlier findings have
identified significant numbers of RE cells with branching
(or collateral) projections to HF and to the mPFC. As has
been noted, RE projects strongly to the HF and to mPFC,
and as such RE may exert a greater dual influence (collateral projections) on major targets than on secondary
ones.
Functional considerations
The nucleus reuniens of the midline thalamus receives a
diverse and widely distributed set of afferent projections,
mainly from limbic/limbic related structures (Risold et al.
1997; Canteras and Goto 1999; Krout et al. 2002; Vertes
2002; Olucha-Bordonau et al. 2003; McKenna and Vertes
2004) and distributes fairly selectively to the hippocampus/
parahippocampus and to the orbitomedial PFC (Wouterlood et al. 1990; Wouterlood 1991; Vertes et al. 2006).
Accordingly, RE appears to be an important interface in
limbic subcortical–cortical communication, that is, a site of
convergence (and integration) of limbic afferent information and its subsequent transfer to limbic forebrain structures. RE is thought to be critically involved in processes of
arousal and attentional or in gating the flow of information
to the limbic forebrain (Van der Werf et al. 2002; Vertes
2006, 2007).
Although not extensively examined, a few recent reports
have described the effects of RE lesions on behavior
(Dolleman-Van der Weel et al. 2009; Davoodi et al. 2009,
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208
2011; Hembrook and Mair 2011). In an initial study, using
a water maze task, Dolleman-Van der Weel et al. (2009)
reported that rats with RE lesions showed no deficits on the
acquisition phase of the task but impairments on the probe
test (escape platform removed) following training. Specifically, RE lesioned rats spent considerably less time in
the correct quadrant of the pool than controls which normally would be interpreted as a hippocampal-dependent (or
memory associated) deficit. The authors, however, viewed
this as a non-mnemonic (or prefrontal-associated impairment) in that the lesioned rats initially swam to the correct
quadrant but quickly abandoned this behavior, favoring one
of ‘search over all the pool’ for the missing platform. This
rapid switch in strategy was described as inflexible (or an
impulsive) adaptation to an environmental change—or a
PFC deficit.
By contrast with the foregoing, subsequent reports have
described hippocampal-dependent deficits with RE lesions
(Davoodi et al. 2009, 2011; Hembrook and Mair 2011).
Davoodi et al. (2009, 2011) initially showed that the
reversible suppression of RE with tetracaine significantly
impaired performance on reference and working memory
tasks on the water maze (WM), and subsequently that
inactivating RE prior to, or immediately after, the acquisition of a passive avoidance task disrupted performance on
this task when tested 24 h later.
Supporting this, Hembrook and Mair (2011) recently
demonstrated that rats with lesions of RE (and the dorsally
adjacent rhomboid nucleus) exhibited significant deficits in
spatial learning on a delayed non-match to sample (DNMS)
radial arm maze (RAM) task, but none on reaction time
(RT) tasks. Hembrook and Mair (2011) proposed that RE
lesions would have a much greater disruptive effect on
tasks involving both the hippocampus and the PFC (delay
RAM tasks), than on those separately affecting the PFC
(RT tasks) or the hippocampus (reference memory in the
WM). They stated that RE ‘‘may play a more specific role
affecting interactions between hippocampus and PFC,
activating them in concert and thus serving more as a
gating function’’. And accordingly, RE may be ‘‘critical for
tasks that require the coordinated activation of the prefrontal cortex and the hippocampal system.’’ The presently
identified RE cells with collateral projections to HF and
mPFC may be pivotally involved in functions requiring the
combined actions of the HF and mPFC.
On the human level, it is well established that bilateral
lesions of the midline (or central) thalamus produces profound alterations of consciousness ranging from vegetative
states (VS) to coma (Castaigne et al. 1981; Plum 1991;
Schiff and Plum 2000). Part of this continuum is the
minimally conscious state (MCS), characterized by intermittent periods of awareness of self and environment
(Schiff et al. 2007). While early attempts to restore
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Brain Struct Funct (2012) 217:191–209
purposeful, self directed, behavior with stimulation of the
central thalamus in MCS or VS patients were unsuccessful
(Deliac et al. 1993; Yamamoto and Katayama 2005), Schiff
and colleagues (Schiff et al. 2007) recently demonstrated
that deep brain stimulation (DBS) of the central thalamus
in a MCS patient produced striking behavioral improvements. Specifically, with DBS the patient regained the
ability to follow verbal commands, purposively manipulate
objects, intelligibly communicate, and orally consume food
(Schiff et al. 2007). Regarding possible mechanisms for
these effects, Shah and Schiff (2010) suggested that the
central thalamus is instrumental in the transfer of arousalrelated information to the forebrain which is critical for
maintaining requisite levels of cortical activation for
effective cognitive functioning. In effect, DBS of intact
regions of the midline thalamus serves to ‘reactivate’ previously dormant regions of cortex to restore levels of
consciousness necessary for purposeful behavior.
In summary, the present results show that RE strongly
targets the hippocampus and the mPFC, with separate
populations favoring one or the other site, and that a relatively significant percentage of RE neurons project to both
structures via axon collaterals. RE is thus critically positioned to influence limbic forebrain structures, particularly
the HF and the mPFC, and the functions associated with
them.
Acknowledgments This research was supported by National Science Foundation grant IOS 0820639 to RPV.
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