collateral projections from nucleus reuniens of thalamus ...vertes/re-dl-published.pdf ·...

ORIGINAL ARTICLE

Collateral projections from nucleus reuniens of thalamusto 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

� 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 hippo-

campus (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. Pro-

nounced 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 peri-

reuniens nucleus (pRE), whereas those projecting to HF

occupied a wide mediolateral cross sectional area of RE

lying between cells projecting to the mPFC. Approxi-

mately, 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

labeled neurons. The nucleus reuniens has been shown to

be a vital link in limbic subcortical–cortical communica-

tion and recent evidence indicates a direct RE involvement

in hippocampal and medial prefrontal cortical-dependent

behaviors. The present findings indicate that RE is criti-

cally 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

W. B. Hoover � R. P. Vertes (&)

Center for Complex Systems and Brain Sciences,

Florida Atlantic University, Boca Raton, FL 33431, USA

e-mail: [email protected]

123

Brain Struct Funct (2012) 217:191–209

DOI 10.1007/s00429-011-0345-6

RH Rhomboid nucleus of thalamus

slm Stratum lacunosum moleculare

SMT Submedial nucleus of thalamus

SUB,v Subiculum, ventral division

VS Vegetative state

3V Third ventricle

Introduction

The nucleus reuniens (RE) lies ventrally on the midline of

the thalamus, above the third ventricle, and extends lon-

gitudinally 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 hip-

pocampus 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 en-

torhinal 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 mo-

leculare (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

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 retro-

grade 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-associ-

ated’ 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 contingen-

cies, 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-asso-

ciated 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 pri-

marily localized to RE regions projecting to the hippo-

campus 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

192 Brain Struct Funct (2012) 217:191–209

123

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 pro-

jecting 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 Labo-

ratories, 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, Bowd-

oin, 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 tran-

scardially 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

slides and coverslipped using DPX media (BDH Labora-

tories, 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 Photo-

Shop 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 hippo-

campal 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 repre-

sentative sections evenly spaced throughout the rostro-

caudal extent of RE. Cells were classified as single labeled

if they were excited (epi-fluorescence) with one set of fil-

ters (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 schemati-

cally 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).

Brain Struct Funct (2012) 217:191–209 193

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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 sche-

matically 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 pre-

limbic 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 hippo-

campus (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 dif-

ferential 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

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 rostrocau-

dally 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

A

B

E

F

C

D

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


123

pRE) which almost entirely consisted of FG labeled neu-

rons. 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 ro-

strocaudal 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. Asso-

ciated 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 later-

ally 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

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


123

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 ro-

strocaudally 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 num-

bers 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).

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

1

Case 15 (FG-mPFC; FR CA1v)section

2

3

4

5

6

n = 164, 46.2% n = 191, 53.8% DB: n = 17, 4.8%Total number of cells labeled = 355


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 = 436n = 184, 49.9% n = 185, 50.1% DB: n = 21, 5.7%Total number of cells labeled = 369


1

Case 21 (FG-mPFC; FR CA1d)section

2

3

4

5

6

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 = 0Total number of cells labeled = 170n = 261, 94.2% n = 16, 5.8% DB: n = 1, 0.36%Total number of cells labeled = 277


n = 147, 86% n = 24, 14% DB: n = 2, 1.2%Total number of cells labeled = 171

n = 72, 81.8% n = 16, 18.2 % DB: n = 1, 0.81%

Total number of cells labeled = 88

Fluorogold labeledFluororuby labeledDouble labeled

A

B

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


123

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 propor-

tionally 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

Fig. 4 Schematic representation of locations and patterns of Fluoro-

gold (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


123

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.

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

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. Scalebar 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 leftrectangle in Fig. 5, and a cluster

of Fluororuby labeled neurons

laterally in RE (b) in the region

depicted by the right rectanglein Fig. 5. Scale bar 100 lm


123

injection of case 22 was caudal that of case 25, sug-

gesting 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).





mPFC and FR injections in dorsal CA1 for case 21. Note the sparse

FR labeling in RE with the dorsal CA1 injection


123

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 (pro-

jecting 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)

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 neu-

rons (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











for cases 22 and 25. The FR

injections for these cases were

made in the ventral subiculum

of the hippocampus


123

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 distribu-

tion 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





mPFC and FR injections in the ventral subiculum for case 22


123

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

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 medio-

lateral cross sectional area of RE lying between cells dis-

tributing to the mPFC; (3) with the exception of the dorsal

Fig. 10 Photomicrographs

depicting Fluorogold (FG) and

Fluororuby (FR) double labeled

(FG ? FR) neurons (openarrows) 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. Scalebar for a–c and d–f 20 lm


123

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 approx-

imately 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











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


123

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 out-

numbered FG? cells by approximately 60/40%, the num-

bers 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

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


123

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).

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 downwardvertical arrows is shown at

higher magnification in

Fig. 14a, while the region

denoted by the diagonal arrowsis shown at higher magnification

in Fig. 14b. Scale bar 200 lm


123

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

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 peri-

reuniens 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) injec-

tions 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 neu-

rons 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 (Herken-

ham, 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).

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 verticalarrows 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


123

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 dis-

tributed throughout RE. Although labeled cells spread

mediolaterally across RE, they were most densely con-

centrated 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 infra-

limbic region. Using PHA-L, Risold et al. (1997) subse-

quently described (at best) moderate RE projections to the

ventral mPFC. In a recent examination of efferent projec-

tions 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%).

Unlike the present demonstration of significant per-

centages 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 exam-

ination of RE/midline thalamic projections to the amyg-

dala, 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 hippocam-

pus; 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 (col-

lateral 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 (Wouter-

lood 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 informa-

tion and its subsequent transfer to limbic forebrain struc-

tures. 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,


123

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. Spe-

cifically, RE lesioned rats spent considerably less time in

the correct quadrant of the pool than controls which nor-

mally would be interpreted as a hippocampal-dependent (or

memory associated) deficit. The authors, however, viewed

this as a non-mnemonic (or prefrontal-associated impair-

ment) 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 acqui-

sition 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 pre-

frontal 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 pro-

found 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 inter-

mittent periods of awareness of self and environment

(Schiff et al. 2007). While early attempts to restore

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 improve-

ments. 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 arousal-

related 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’ pre-

viously 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 rel-

atively significant percentage of RE neurons project to both

structures via axon collaterals. RE is thus critically posi-

tioned to influence limbic forebrain structures, particularly

the HF and the mPFC, and the functions associated with

them.

Acknowledgments This research was supported by National Sci-

ence Foundation grant IOS 0820639 to RPV.

References

Bertram EH, Zhang DX (1999) Thalamic excitation of hippocampal

CA1 neurons: a comparison with the effects of CA3 stimulation.

Neuroscience 92:15–26

Bokor H, Csaki A, Kocsis K, Kiss J (2002) Cellular architecture of the

nucleus reuniens thalami and its putative aspartatergic/glutama-

tergic projection to the hippocampus and medial septum in the

rat. Eur J Neurosci 16:1227–1239

Canteras NS, Goto M (1999) Connections of the precommissural

nucleus. J Comp Neurol 408:23–45

Castaigne P, Lhermitte F, Buge A, Escourolle R, Hauw JJ, Lyon-Caen

O (1981) Paramedian thalamic and midbrain infarct: clinical and

neuropathological study. Ann Neurol 10:127–148

Cavdar S, Onat FY, Cakmak YO, Yananli HR, Gulcebi M, Aker R

(2008) The pathways connecting the hippocampal formation, the

thalamic reuniens nucleus and the thalamic reticular nucleus in

the rat. J Anat 212:249–256

Davoodi FG, Motamedi F, Naghdi N, Akbari E (2009) Effect of

reversible inactivation of the reuniens nucleus on spatial learning

and memory in rats using Morris water maze task. Behav Brain

Res 198:130–135

Davoodi FG, Motamedi F, Akbari E, Ghanbarian E, Jila B (2011)

Effect of reversible inactivation of reuniens nucleus on memory

processing in passive avoidance task. Behav Brain Res 221:1–6

Deliac P, Richer E, Berthomieu J, Paty J, Cohadon F, Bensch C

(1993) Electrophysiological development under thalamic


123

stimulation of post-traumatic persistent vegetative states. Apro-

pos of 25 cases. Neurochirurgie 39:293–303

Dolleman-Van der Weel MJ, Lopes da Silva FH, Witter MP (1997)

Nucleus reuniens thalami modulates activity in hippocampal

field CA1 through excitatory and inhibitory mechanisms.

J Neurosci 17:5640–5650

Dolleman-van der Weel MJ, Morris RG, Witter MP (2009) Neuro-

toxic lesions of the thalamic reuniens or mediodorsal nucleus in

rats affect non-mnemonic aspects of watermaze learning. Brain

Struct Funct 213:329–342

Dollerman-Van der Weel MJ, Witter MP (1996) Projections from

nucleus reuniens thalami to the entorhinal cortex, hippocampal

field CA1, and the subiculum in the rat arise from different

populations of neurons. J Comp Neurol 364:637–650

Hembrook JR, Mair RG (2011) Lesions of reuniens and rhomboid

nuclei impair radial arm maze win-shift performance. Hippo-

campus 21:815–826

Herkenham M (1978) The connections of the nucleus reuniens

thalami: evidence for a direct thalamo-hippocampal pathway in

the rat. J Comp Neurol 177:589–610

Krout KE, Belzer RE, Loewy AD (2002) Brainstem projections to

midline and intralaminar thalamic nuclei of the rat. J Comp

Neurol 448:53–101

Lanciego JL, Wouterlood FG (2006) Multiple neuronal tract tracing:

approaches for multiple tract tracing. In: Zaborszky L, Wouterlood

FG, Lanciego JL (eds) Neuroanatomical tract-tracing 3: molecules

neurons and systems. Springer, New York, pp 336–365

McKenna JT, Vertes RP (2004) Afferent projections to nucleus

reuniens of the thalamus. J Comp Neurol 480:115–142

Ohtake T, Yamada H (1989) Efferent connections of the nucleus

reuniens and the rhomboid nucleus in the rat: an anterograde

PHA-L study. Neurosci Res 6:556–568

Olucha-Bordonau FE, Teruel V, Barcia-Gonzalez J, Ruiz-Torner A,

Valverde-Navarro AA, Martınez-Soriano F (2003) Cytoarchi-

tecture and efferent projections of the nucleus incertus of the rat.

J Comp Neurol 464:62–97

Plum F (1991) Vulnerability of the brain and heart after cardiac arrest.

N Engl J Med 324:1278–1280

Risold PY, Thompson RH, Swanson LW (1997) The structural

organization of connections between hypothalamus and cerebral

cortex. Brain Res Rev 24:197–254

Schiff ND, Plum F (2000) The role of arousal and ‘‘gating’’ systems

in the neurology of impaired consciousness. J Clin Neurophysiol

17:438–452

Schiff ND, Giacino JT, Kalmar K, Victor JD, Baker K, Gerber M,

Fritz B, Eisenberg B, O’Connor J, Kobylarz EJ, Farris S,

Machado A, McCagg C, Plum F, Fins JJ, Rezai AR (2007)

Behavioural improvements with thalamic stimulation after

severe traumatic brain injury. Nature 448:600–603

Schmued LC, Kyriakidis K, Heimer L (1990) In vivo anterograde and

retrograde axonal transport of the fluorescent rhodamine-dex-

tran-amine, Fluororuby, within the CNS. Brain Res 526:127–134

Shah SA, Schiff ND (2010) Central thalamic deep brain stimulation

for cognitive neuromodulation—a review of proposed mecha-

nisms and investigational studies. Eur J Neurosci 32:1135–1144

Su HS, Bentivoglio M (1990) Thalamic midline cell populations

projecting to the nucleus accumbens, amygdala, and hippocam-

pus in the rat. J Comp Neurol 297:582–593

Swanson LW (2004) Brain maps: structure of the rat brain. Elsevier,

New York

Van der Werf YD, Witter MP, Groenewegen HJ (2002) The

intralaminar and midline nuclei of the thalamus. Anatomical

and functional evidence for participation in processes of arousal

and awareness. Brain Res Brain Res Rev 39:107–140

Vertes RP (2002) Analysis of projections from the medial prefrontal

cortex to the thalamus in the rat, with emphasis on nucleus

reuniens. J Comp Neurol 442:163–187

Vertes RP (2004) Differential projections of the infralimbic and

prelimbic cortex in the rat. Synapse 51:32–58

Vertes RP (2006) Interactions among the medial prefrontal cortex,

hippocampus and midline thalamus in emotional and cognitive

processing in the rat. Neuroscience 142:1–20

Vertes RP, Hoover WB, Do Valle AC, Sherman A, Rodriguez JJ

(2006) Efferent projections of reuniens and rhomboid nuclei of

the thalamus in the rat. J Comp Neurol 499:768–796

Vertes RP, Hoover WB, Szigeti-Buck K, Leranth C (2007) Nucleus

reuniens of the midline thalamus: link between the medial

prefrontal cortex and the hippocampus. Brain Res Bull

71:601–609

Viana Di Prisco G, Vertes RP (2006) Excitatory actions of the ventral

midline thalamus (rhomboid/reuniens) on the medial prefrontal

cortex in the rat. Synapse 60:45–55

Witter MP, Groenewegen HJ, Lopes da Silva FH, Lohman AH (1989)

Functional organization of the extrinsic and intrinsic circuitry of

the parahippocampal region. Prog Neurobiol 33:161–253

Wouterlood FG (1991) Innervation of entorhinal principal cells by

neurons of the nucleus reuniens thalami. Anterograde PHA-L

tracing combined with retrograde fluorescent tracing and intra-

cellular injection with Lucifer yellow in the rat. Eur J Neurosci

3:641–647

Wouterlood FG, Saldana E, Witter MP (1990) Projection from the

nucleus reuniens thalami to the hippocampal region: light and

electron microscopic tracing study in the rat with the anterograde

tracer Phaseolus vulgaris-leucoagglutinin. J Comp Neurol

296:179–203

Yamamoto T, Katayama Y (2005) Deep brain stimulation therapy for

the vegetative state. Neuropsychol Rehabil 15:406–413


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