Brain Struct Funct (2012) 217:191–209 DOI 10.1007/s00429-011-0345-6

ORIGINAL ARTICLE

Collateral projections from nucleus reuniens of to and medial 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 labeled neurons. The nucleus reuniens has been shown to thalamus has been shown to strongly innervate structures of be a vital link in limbic subcortical–cortical communica- the limbic forebrain, prominently including the hippo- tion and recent evidence indicates a direct RE involvement campus (HF) and the medial prefrontal cortex (mPFC) and in hippocampal and medial prefrontal cortical-dependent to exert pronounced excitatory effects on HF and mPFC. It behaviors. The present findings indicate that RE is criti- was unknown, however, whether RE projections to, and cally positioned to influence the HF and mPFC, and their hence actions on, the HF and mPFC originate from a associated behaviors, via separate or collateral projections common or largely separate groups of RE neurons. Using to these sites. fluorescent retrograde tracing techniques, we examined the patterns of distribution of RE cells projecting to HF, to the Keywords Infralimbic cortex Á Prelimbic cortex Á mPFC or to both sites via axon collaterals. Specifically, Á of hippocampus Á Spatial injections of the retrograde tracers Fluorogold (FG) or learning Á Arousal Á Attention Á Consciousness Fluororuby (FR) were made in the mPFC and in various subfields of HF and patterns of single (FG or FR) or double Abbreviations labeled (FG ? FR) cells in RE were determined. Pro- CA1,d,v Field CA1 of Ammon’s horn, dorsal, ventral nounced numbers of (single) labeled neurons were present division throughout RE with FG or FR injections, and although CA3 Field CA3 of Ammon’s horn intermingled in RE, cells projecting to the mPFC were DB Double labeled cell preferentially distributed along the midline or in the peri- DBS Deep brain stimulation reuniens nucleus (pRE), whereas those projecting to HF EC, l, m Entorhinal cortex, lateral, medial division occupied a wide mediolateral cross sectional area of RE FG Fluorogold lying between cells projecting to the mPFC. Approxi- FR Fluororuby mately, tenfold more labeled cells were present in RE with HF Hippocampal formation ventral compared to dorsal CA1 injections. Like single IL Infralimbic cortex labeled neurons, double labeled cells were found MCS Minimally conscious state throughout RE, but were most densely concentrated in mPFC Medial prefrontal cortex areas of greatest overlap of FG? and FR? neurons or mt mainly in the lateral one-third of RE, medial to pRE. PFC Prefrontal cortex Depending on specific combinations of injections, double PL Prelimbic cortex labeled cells ranged from approximately 3–9% of the pRE Perireuniens nucleus of thalamus PT Paratenial nucleus of thalamus PV Paraventricular nucleus of thalamus & W. B. Hoover Á R. P. Vertes ( ) PVHy Paraventricular nucleus of Center for Complex Systems and Brain Sciences, Florida Atlantic University, Boca Raton, FL 33431, USA RAM Radial arm maze e-mail: [email protected] RE Nucleus reuniens of thalamus 123 192 Brain Struct Funct (2012) 217:191–209

RH Rhomboid nucleus of thalamus ventral mPFC (Viana Di Prisco and Vertes 2006). The slm Stratum lacunosum moleculare foregoing indicates that RE distributes to, and significantly SMT Submedial nucleus of thalamus effects, the hippocampus and medial prefrontal cortex. SUB,v Subiculum, ventral division A currently unresolved issue, however, is whether RE VS Vegetative state projections to, and hence actions on, the HF and mPFC 3V Third ventricle 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) Introduction reported that RE projections to CA1 and to the subiculum of HF, to the entorhinal cortex (EC) and to the perirhinal The nucleus reuniens (RE) lies ventrally on the midline of cortex mainly arose from separate groups of RE neurons. the thalamus, above the third ventricle, and extends lon- Specifically, RE projections: (1) to CA1 originated from gitudinally virtually throughout the thalamus (Swanson the dorsolateral RE; (2) to the subiculum, from the lateral 2004; Vertes et al. 2006). RE is reciprocally connected RE (3) to the medial EC (ECm), from the medial RE; (4) to with the hippocampus (HF) and the medial prefrontal the lateral EC (ECl) from the ventral half of RE, and (5) to cortex (mPFC) (Herkenham 1978; Wouterlood et al. 1990; the perirhinal cortex from the perireuniens (pRE) nucleus Dollerman-Van der Weel and Witter 1996; Risold et al. (or lateral wings of RE). In a similar manner, we showed 1997; Bokor et al. 2002; McKenna and Vertes 2004; Vertes that RE fibers to the orbital cortex arose from pRE, to the 2002, 2004, 2006; Cavdar et al. 2008), and as such appears ECm mainly from the rostral RE and to ECl from the to be critically involved in the two way communication caudal RE (Vertes et al. 2006). This indicates a segregation between these structures. of RE output to its main targets, and suggests the same may RE is a major route through which the mPFC influences be true for RE projections to the HF and mPFC. the hippocampus. Specifically, HF distributes to the mPFC, The prospect of segregated RE outputs gains support but there are no direct return projections from the mPFC to from recent examinations of the effects of RE lesions on the hippocampus. Accordingly, mPFC effects on the hip- behavior. While few reports have examined the behavioral pocampus appear to be mainly relayed through RE, thus effects of RE lesions, the findings to date conflict with completing an important loop between these structures: regard to whether RE lesions produce ‘prefrontal-associ- HF [ mPFC [ RE [ HF (Vertes et al. 2006, 2007). At the ated’ or ‘hippocampal-dependent’ deficits. In an initial ultrastructural level, mPFC fibers have been shown to study, Dolleman-Van der Weel et al. (2009), using water synaptically connect with RE neurons projecting to the maze tasks, reported that RE lesions produced deficits in hippocampus (Vertes et al. 2007). In addition to RE, shifting strategies to changing environmental contingen- another route from the mPFC to HF is through the en- cies, but had little effect on spatial memory. Specifically, in torhinal cortex (Witter et al. 1989; Vertes 2004). a probe test following training (escape platform removed), The few reports that have examined the physiological rats with RE lesions initially swam to the correct quadrant, effects of RE on the HF and mPFC have shown that RE indicating memory was intact, but quickly abandoned this exerts strong excitatory actions on both structures. With behavior, favoring one of ‘search over all the pool’ for the regard to HF, Dolleman-Van der Weel et al. (1997) showed missing platform. This was viewed as an inflexible strategy that RE stimulation produced large amplitude negative to an environmental change, or a prefrontal cortical-asso- going evoked responses (sink) at stratum lacunosum mo- ciated deficit. In contrast to this, Davoodi et al. (2009) leculare (slm) of CA1 as well as paired pulse facilitation at reported that the reversible suppression of RE disrupted CA1. Bertram and Zhang (1999) confirmed these findings reference and working memory tasks on the water maze, and further demonstrated that the excitatory actions of RE while Hembrook and Mair (2011) showed that RE lesioned at CA1 were equivalent to, or greater than, those of CA3 on rats displayed marked deficits on delayed non-match to CA1, leading them to conclude that the RE projection to sample radial arm maze (RAM) tasks. the hippocampus ‘‘allows for the direct and powerful While several factors could account for the differing excitation of the CA1 region’’ which ‘‘by passes the results including choice of tasks, it is also possible that RE trisynaptic/commissural pathway that has been thought to lesions differed with respect to whether they were pri- be the exclusive excitatory drive to CA1’’. With respect to marily localized to RE regions projecting to the hippo- the mPFC, we showed that RE stimulation produced short campus or to the mPFC—if, in fact, RE cells projecting to latency (monosynaptic), large amplitude evoked potentials these two structures are segregated within RE. The present at mPFC, with the largest effects at inner layers (5/6) of the reports addresses this issue, that is, whether, or to what 123 Brain Struct Funct (2012) 217:191–209 193 degree, RE cells projecting to the HF and to the mPFC slides and coverslipped using DPX media (BDH Labora- originate from the same or largely separate populations of tories, Poole, England). An adjacent series of sections was RE neurons. stained with cresyl violet for anatomical reference. In brief, we showed that RE cells projecting to the HF Sections were examined with epi-fluorescent techniques and the mPFC were intermingled within RE, but with using appropriate filters for FG (excitation 350–395 nm; clusters distributing selectively to each site. RE cells pro- emission 530–600 nm) and FR (excitation 540–560 nm; jecting to HF were mainly located lateral to the midline emission 580 nm). within the medial two-thirds of RE, while those distributing to the mPFC were predominantly located in the lateral Photomicrographs one-third of RE extending to the lateral wings of RE, particularly at caudal levels of RE. In addition, relatively Photomicrographs of injection sites and labeled cells were significant percentages of RE cells (3–9%) projected via taken with a QImaging (Q ICAM) camera mounted on a collaterals to the HF and mPFC. Double labeled cells were Nikon Eclipse E600 microscope using Nikon Elements 3.0 mainly situated on the midline and in mid-mediolateral imaging software. Using Elements software, monochrome regions of RE. 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 Materials and methods schematic depiction of labeled cells. Micrographs were adjusted for brightness and contrast using Adobe Photo- Twenty-seven male Sprague–Dawley rats (Harlan Labo- Shop 7.0 (Mountain View, CA). Some micrographs were ratories, Indianapolis, IN) weighing 350–425 g were also overlaid to depict double labeled cells utilizing the injected with two retrograde fluorescent tracers, Fluorogold layering capabilities of Adobe PhotoShop 7.0. (Fluorochrome, Denver, CO) and Fluororuby (Invitrogen, Carlsbad, CA). These experiments were approved by the Cell counts Florida Atlantic University Institutional Animal Care and Use Committee and conform to all federal regulations and All 27 cases were analyzed for numbers and patterns of National Institutes of Health guidelines for the care and use single and double labeled cells in RE following hippo- of laboratory animals. campal and mPFC injections. Fourteen of 27 cases had Fluorogold (FG) and Fluororuby (FR) were dissolved in particularly well placed injections of retrograde tracers in a 0.1 M sodium acetate buffer (pH 3.5 to 4.5) to yield an both the HF and mPFC. Seven of these 14 cases were 8% concentration. Rats were anesthetized for surgery using selected for cell counting based on optimal injections in an 80 mg/kg dose of Ketamine and 10 mg/kg dose of representative regions of the hippocampus: dorsal and Xylazine. FG or FR was iontophoretically deposited into ventral CA1, the ventral subiculum and spanning ventral the hippocampus or into the medial prefrontal cortex using CA1 and the ventral subiculum. Counts of single (FG or glass micropipettes with an outside tip diameter of FR) and double labeled cells were taken from six repre- 75–100 lm. Retrograde tracer injections were made: (1) sentative sections evenly spaced throughout the rostro- into the prelimbic (PL) and infralimbic (IL) cortices of the caudal extent of RE. Cells were classified as single labeled mPFC, or (2) into the dorsal or ventral CA1 or the ventral if they were excited (epi-fluorescence) with one set of fil- subiculum of the hippocampus. Positive direct current ters (FG or FR) but not the other, and double labeled if they (8–10 lA) was applied through a Grass stimulator (model were excited (epi-fluorescence) using the FG and FR filters 88) coupled with a high-voltage stimulator (FHC, Bowd- in the same focal plane at 200 and 4009 magnification. oin, ME) at 2 s ‘‘on’’/2 s ‘‘off’’ intervals for 2–10 min. Following a survival time of 7 days, rats were deeply Schematics anesthetized with sodium pentobarbital and perfused tran- scardially with 100 ml of heparinized saline wash followed Four of the 7 cases used for cell counting were schemati- by 450 ml of fixative (4% paraformaldehyde in 0.1 M cally illustrated. The medial prefrontal injections of these 4 sodium phosphate buffer (PB), pH 7.4). The brains were cases were placed in the ventral mPFC, approximately on then removed and postfixed in 4% paraformaldehyde— the border of the the prelimbic and infralimbic cortices. 0.1 M PB solution at 48C for 24 h. Fifty micron transverse The HF injections for these 4 cases were: (1) ventral CA1 sections were collected in 0.1 M PB (pH 7.4) using a (case 15); (2) dorsal CA1 (case 21); (3) ventral subiculum vibrating microtome and stored at 48C. Representative (case 22); and (4) spanning the ventral CA1 and the ventral sections were mounted onto chrome–alum gelatin coated subiculum (case 27).

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Results C

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 A labeled neurons in RE containing one (FG of FR) or both tracers were determined. Seven cases with injections D 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- B 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 E 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 F 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 Fig. 1 Schematic representation of paired injections of Fluorogold terminal destination of RE fibers distributing to the hippo- (FG) in the infralimbic (IL) and prelimbic (PL) cortices of the medial prefrontal cortex (mPFC) (a, b), and Fluororuby (FG) injections in the campus (Wouterlood et al. 1990;Vertesetal.2006). 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 mPFC and dorsal and ventral CA1 injections (cases 21 of FR in the mPFC (a, b) and FG in CA1/SUBv (d) for case 9 and 15) RE (levels 1–4) to the hippocampus and from the caudal Figure 3a shows the number and relative percentages of RE (levels 5 and 6) to the mPFC. Relatively pronounced single and double retrogradely labeled cells at six rostral to numbers of double labeled (DB) neurons were observed at caudal levels of RE following injections in the mPFC and all levels of RE ranging from 3.7 to 8.4%, with the largest in CA1 of the ventral hippocampus (case 15). As depicted, percentage of DBs at a rostral (level 2, 6.2%) and caudal this mPFC-ventral CA1 pair of injections gave rise to level (level 5, 5.7%) of RE. marked numbers of labeled cells in RE (range 193–438 Figure 4 schematically depicts the locations of single cells) with the greatest numbers at mid-levels of RE (levels (FG, green dots; FR, red dots) and double labeled 2–4)—which is the largest expanse of RE. With the (FG ? FR, black triangles) cells at six rostrocaudal levels exception of level 6, there were more labeled cells in RE of RE for case 15 (mPFC-ventral CA1). As shown, FG-and with HF than with mPFC injections with the greatest dif- FR-labeled neurons were largely intermingled rostrocau- ferential at levels 2 (61.6%, HF; 38.4%, mPFC) and 4 dally throughout RE, with a tendency for FR-labeled cells (60.1%, HF; 39.9%, mPFC). Interestingly, the ratio of (projecting to HF) to be located medially in RE and FG labeled cells was reversed at the caudal RE (level 6) such labeled cells (projecting to mPFC) to reside laterally in that a greater percentage of cells were labeled with mPFC RE at the rostral RE (Fig. 4b, c). This medial to lateral (62.7%) than with HF (37.3%) injections. The foregoing segregation became more pronounced caudally in RE indicates proportionally stronger projections from rostral (Fig. 4d–f), particularly within the lateral wings of RE (or 123 Brain Struct Funct (2012) 217:191–209 195

Fig. 2 a, b Sites of paired injections of Fluorogold in the infralimbic/ Fluororuby at the border of CA1 and the subiculum of the ventral prelimbic cortex of the mPFC (a) and Fluororuby in CA1 of the hippocampus (d) for case 27. Note that the ventral hippocampal ventral hippocampus (b) for case 15. c, d Sites of paired injections of injections (b, d) are centered in the stratum lacunosum moleculare Fluorogold in the infralimbic/prelimbic cortex of the mPFC (c) and (slm) of HF. Scale bar for a, c 1000 lm; for b,d 1120 lm pRE) which almost entirely consisted of FG labeled neu- cells at all levels of RE (Fig. 3b). This weighting in favor rons. This pattern of caudal RE labeling (Fig. 4d, e) is shown of FG? cells (to the mPFC) results from considerably in the photomicrographs of Figs. 5 and 6. As depicted, clusters fewer labeled cells in RE with dorsal CA1 (case 21, Fig. 7) of FG? cells are present on the midline and in the lateral than with ventral CA1 (case 15, Fig. 4) injections. Asso- wings of RE (Figs. 5, 6a), whereas FR? neurons are mainly ciated with this, exceedingly few DB cells were observed located lateral to the midline (Fig. 5, right rectangle) between at any level of RE with case 21 (Figs. 3b, 7); or only eight the clusters of FG? cells (Figs. 5, 6b). As further illustrated double labeled cells were present throughout RE. Similar, (Fig. 5), the region just lateral to the midline mainly contained however, to case 15 in which the mPFC injection was only FR? cells, while that farther laterally (or medial to pRE) slightly ventral to that of case 21 (Fig. 1), marked numbers contained a greater mixture of FR? and FG? cells. of FG? cells were present in RE (range 72–261 over the While the percentage of double labeled cells neurons (to 6levels)(Fig.3b) and more densely concentrated later- total numbers) was relatively constant rostrocaudally ally than medially in RE, particularly at caudal levels of throughout RE with case 15 (see Fig. 3a), ranging from 3.7 RE, or within pRE (Fig. 7c–f). In addition, there was a to 6.2%, more were present medially than laterally in RE, tendency, at least rostrally, for FG? cells to form a particularly at caudal levels of RE (Fig. 4d–f), probably midline and a lateral group with sparser labeling between owing to a greater intermingling of FG-and FR-labeled them (Fig. 7a–d). neurons in the medial than lateral RE. Figure 3b shows the numbers and relative percentages mPFC and ventral subiculum injections of single and double retrogradely labeled cells at six ro- (cases 22 and 25) strocaudal levels of RE following an injection in the mPFC and in CA1 of the dorsal hippocampus (case 21). As Figure 8a depicts the number and relative percentages of depicted, there was a greater percentage of FG? than FR? single and double labeled cells at six rostral to caudal

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Fig. 3 Numbers and relative Fluorogold labeled percentages of Fluorogold (FG) A Case 15 (FG-mPFC; FR CA1v) Fluororuby labeled labeled cells (green) with section Double labeled medial prefrontal cortical injections, Fluororuby (FR) n = 164, 46.2% n = 191, 53.8% DB: n = 17, 4.8% 1 labeled cells (red) with Total number of cells labeled = 355 hippocampal injections and n = 168, 38.4% n = 270, 61.6% DB: n = 27, 6.2% double labeled cells (FG ? FG) 2 (black) at six rostral to caudal Total number of cells labeled = 438 levels (1–6) of nucleus reuniens n = 180, 47.2% n = 201, 52.8% DB: n = 18, 4.7% 3 for cases 15 and 21. The FR Total number of cells labeled = 381 injection for case 15 was made n = 174, 39.9% n = 262, 60.1% DB: n = 16, 3.7% in the ventral CA1 of the 4 hippocampus (HF), for case 21 Total number of cells labeled = 436 n = 184, 49.9% n = 185, 50.1% DB: n = 21, 5.7% in dorsal CA1 of HF. Note the 5 significantly greater number of Total number of cells labeled = 369 FR-labeled cells in RE with the n = 121, 62.7% n = 72, 37.3% DB: n = 8, 4.1% ventral than with the dorsal CA1 6 Total number of cells labeled = 193 injection B Case 21 (FG-mPFC; FR CA1d) section n = 171, 90.5% n = 18, 9.5 % DB: n = 2, 1.0% 1 Total number of cells labeled= 189 n = 157, 92.4% n = 13, 7.6 % DB: n = 0 2 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 n = 153, 87.4% n = 22, 12.6% DB: n = 2, 1.1% 4 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% 6 Total number of cells labeled = 88

levels of RE following an injection in the mPFC and in the Figure 9 schematically depicts the locations of single and ventral subiculum (SUBv) of HF (case 22). Pronounced double labeled cells at six rostral to caudal levels of RE for numbers of retrogradely labeled neurons were found ro- case 22 (Fig. 8a). As shown for cases 15 and 21, considerably strocaudally throughout RE with total numbers of cells greater numbers of FG? neurons were present laterally than (FG ? FR) at the six levels ranging from 258 at level 6 to medially in RE, particularly at caudal regions of RE where 709 cells at level 2. There were roughly equivalent num- they were mainly found on the lateral border of RE, bers of FG (projecting to mPFC) and FR (projecting to HF) extending laterally to pRE. In addition, and as generally seen cells in RE, with larger percentages of FR? neurons at a with other cases, FR? cells were densely packed within an rostral (level 1) and at caudal levels of RE (4–6), and larger intermediate (mediolateral) zone of RE (Fig. 9a–f), with percentages of FG? cells at intermediate levels of RE (2, some extension to the midline at the caudal RE (Fig. 9d, e). 3). Percentages of DB cells (to total numbers) were fairly At rostral levels (Fig. 9a, b), these patterns of labeling constant across rostrocaudal levels of RE, ranging from resulted in a mid-lateral core of FR-labeled cells surrounded 2.1% at level 2 to 3.9% at level 6, but overall percentages by FG? neurons, laterally and medially. Finally, FR? cells of DB cells were lower for this case (mPFC/SUB injection) were considerably more densely concentrated dorsally (or than for the mPFC-ventral CA1 injection (case 15)—as dorsolaterally) than ventrally in RE. As shown in Fig. 8a, well as for the other mPFC-subicular injection (case 25) double labeled neurons were quite evenly distributed (Fig. 8b). throughout RE, with largest percentages at the caudal RE (or 123 Brain Struct Funct (2012) 217:191–209 197

Fig. 4 Schematic representation of locations and patterns of Fluoro- caudal levels of nucleus reuniens (a–f) following FG injections in the gold (FG) labeled cells (green dots), Fluororuby (FR) labeled cells mPFC and FR injections in ventral CA1 for case 15 (red dots) and double labeled cells (black triangles) at six rostral to levels 4–6, Fig. 9d–f), and ranged from 2.1–3.9% of labeled relatively high ranging from 4.0 to 7.4% of labeled neurons, neurons (Fig. 8a). DB cells were fairly tightly clustered with the largest percentages at the rostral RE: 7.4% at level dorsoventrally, extending from the midline to pRE, within 1 and 7.3% at level 2. There were quite marked differences mid regions of RE (Fig. 9c–f). in the patterns of labeling for the two mPFC/ventral SUB Figure 8b depicts the number and relative percentages of cases (cases 22 and 25). For instance, more labeled cells single and double labeled cells at six rostral to caudal levels (FG ? FR) were observed in RE with case 22 than with of RE following an injection in the mPFC and in the ventral case 25 (Fig. 8a, b) which could involve relative sizes and/ subiculum of HF (case 25). As shown, there were propor- or locations of the injections in the two cases. With regard to tionally more FR than FG labeled cells at all levels of RE the mPFC injections, the FG injection of case 22 was just with the greatest differential at the caudal RE: a 70/30% dorsal to, but only slightly larger than, that of case 25 ratio (FR/FG) at level 5. The percentage of DB cells was (Fig. 1a, b), suggesting that locations rather than the sizes of

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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)inREin 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 The same distinction appears to apply to the subicular in the two cases. Or, in effect, RE distributes more heavily injections in the two cases; that is, the SUB injections for to the dorsal than to the ventral PL. cases 22 and 25 were of equivalent size (Fig. 1e, f), but the

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Fig. 7 Schematic representation of locations and patterns of Fluoro- caudal levels of nucleus reuniens (a–f) following FG injections in the gold (FG) labeled cells (green dots), Fluororuby (FR) labeled cells mPFC and FR injections in dorsal CA1 for case 21. Note the sparse (red dots) and double labeled cells (black triangles) at six rostral to FR labeling in RE with the dorsal CA1 injection injection of case 22 was caudal that of case 25, sug- mPFC and ventral CA1/ventral subiculum (cases 9, 26, gesting a stronger RE output to the caudal-ventral SUB and 27) (case 22) than to the rostral SUB (case 25). Despite greater numbers of labeled neurons in RE with case 22 The hippocampal injections of cases 9, 26 and 27 in part than with case 25, considerably greater percentages of encompassed CA1 and subicular regions of the ventral cells were double labeled with case 25 than with case 22. hippocampus (Fig. 1d); that is, situated ventral to the This would appear to indicate a more pronounced ventral CA1 case (case 15, Fig. 1d) and dorsal to the branching of RE cells (collateral projections) to the ventral subicular cases (cases 22, and 25, Fig. 1e, f). ventral PL/rostral SUB (case 25) than to the dorsal PL/ The ventral CA1/SUB injections (cases 9, 26, 27) were caudal SUB (case 22). Figure 10 depicts DB cells in RE slightly larger than the ventral subicular injections (cases for case 25 (Fig. 10a–c). 22, and 25).

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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 were observed at all levels of RE. The ratios of FG?/FR? of single and double labeled cells at six rostrocaudal levels neurons were relatively constant across levels ranging from of RE with an injection in the mPFC and in the ventral 52.6/47.4% (level 3) to 57.5/42.5% (level 2). Possibly CA1/SUB of HF (case 9). As shown, the tracers were related to the greater proportion of FG? to FR? cells reversed for case 9; that is, FR was injected into the mPFC throughout RE, a large percentage of cells were double and FG into the hippocampus. Interestingly, proportionally labeled (8.1–11.1%), and were quite evenly distributed more FR? (projecting to HF) than FG? neurons (pro- throughout RE with highest percentages at the very rostral jecting to mPFC) were found at the rostral RE (levels 1, 2), (level 1, 10.2%) and caudal RE (level 6, 11.1%). This which contrasts with a reversal of ratios at the caudal RE, represented the highest percentage of DB cells of all cases. most pronounced at the caudal pole of RE (levels 5, and 6). Figure 11c depicts the number and relative percentages Specifically, 70.3% of the cells at level 5 and 76.2% at of single and double labeled cells at six rostral to caudal level 6 were FR? neurons. Although case 9 contained a levels of RE following an injection in the mPFC and the lower percentage of DB cells than found with the other ventral CA1/ventral subiculum of HF (case 27). As shown, ventral CA1/SUB cases, percentages were moderate pronounced numbers of single and double labeled cells (2.4–4.9%), and DB cells were fairly evenly distributed were present at all levels of RE with this pair of injections. throughout RE, with the largest concentration at the caudal While rostral levels of RE (levels 1, and 2) contained RE (Fig. 11a, levels 4–6). proportionally more FR? than FG labeled cells (62/38%), Figure 11b shows the number and relative percentages relatively equal numbers of FG? and FR? cells were of single and double labeled cells at six rostral to caudal present in remaining (or caudal) regions of RE (levels 3–6). levels of RE following injections in the mPFC and in the There was a considerably greater number of labeled neu- ventral CA1/SUB of HF (case 26). Interestingly for this rons (FG?FR) with case 27 (total, 3,138) than with the case, unlike the other cases, a greater percentage of FG? other mPFC/ventral CA1/SUB cases: case 9 (total 1,975); (projecting to mPFC) than FR? cells (projecting to HF) case 26 (total 2,287) (Fig. 11). This would appear to

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Fig. 9 Schematic representation of locations and patterns of Fluoro- caudal levels of nucleus reuniens (a–f) following FG injections in the gold (FG) labeled cells (green dots), Fluororuby (FR) labeled cells mPFC and FR injections in the ventral subiculum for case 22 (red dots) and double labeled cells (black triangles) at six rostral to involve larger FG (Fig. 1a, b) as well as FR (Fig. 1d) (projecting to mPFC) were most densely concentrated injections with case 27 than for the other two cases. along the midline and within the lateral wings of RE A large percentage of cells were double labeled with (Figs. 13, 14b). FR? cells (projecting to HF) were most case 27 (Figs. 11c, 12). They were quite evenly distributed densely packed in an intermediate zone positioned between throughout RE, ranging from 4.3 to 7.7% —with largest the medially and laterally located FG? cells (Figs. 13, percentages at the rostral RE (levels 1, 2). The percentage 14a), and were mainly localized to the ventral half of RE, of DB cells, however, was lower for case 27 than for the rostrally (Fig. 12a, b) and the dorsal two-thirds of RE, other ventral CA1/SUB cases (cases 9, 26). caudally (Fig. 12c–e). As described (Fig. 11c), DB cells Figure 12 schematically depicts the pattern of distribu- were fairly evenly distributed rostrocaudally throughout tion of single and double labeled cells at six levels of RE RE, with the heaviest concentration in the rostral pole of for case 27. Similar to other cases, FG? neurons RE (Fig. 12a, b). Two relatively distinct populations of DB

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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 thalamus: (1) to subfields of the hippocampus; (2) to the prominent at mid to rostral levels of RE (Fig. 12a–d), and a ventral medial prefrontal cortex; and (3) to both regions via mid-dorsoventral group that extended mediolaterally across axon collaterals. the central RE (Fig. 12c–f). Double labeled cells of the The main findings were: (1) pronounced numbers of central RE (medial to pRE) (Fig. 13) are depicted in the retrogradely labeled neurons (single labeled) were present photomicrographs of Fig. 10d–f. 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 Discussion along the midline or in the perireuniens nucleus (pRE), whereas those projecting to HF occupied a wide medio- Using double retrograde fluorescent techniques, we lateral cross sectional area of RE lying between cells dis- describe patterns of projections from the RE of the midline tributing to the mPFC; (3) with the exception of the dorsal

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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 double labeled cells ranged from approximately 3–9% of in the rostral than caudal half of RE with mPFC or HF the labeled neurons. injections; (4) ventral CA1 injections gave rise to approx- imately 10 times greater numbers of labeled neurons in Methodological considerations RE than did dorsal CA1 injections; (5) comparable to single labeled neurons, double labeled cells were found As described, two retrograde fluorescent tracers were used, throughout RE, but were most densely concentrated in the FG and FR. In preliminary work, we determined that the areas of greatest overlap of FG? and FR? cells—on the optimal pairing of the two tracers was to inject FG in the midline and in the lateral one-third of RE, medial to pRE; mPFC and FR in the hippocampus. The reason was that, in and (6) depending on specific combinations of injections, our hands, FR was the (slightly) better retrograde tracer and

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Fig. 12 Schematic representation of locations and patterns of to caudal levels of nucleus reuniens (a–f) following a FG injection in Fluorogold (FG) labeled cells (green dots), Fluororuby (FR) labeled the mPFC and a FR injection spanning CA1 and the ventral cells (red dots) and double labeled cells (black triangles) at six rostral subiculum for case 27 as such FR was deposited into the site requiring the most was not, however, borne out by the findings. With the precise positioning of injections (or less margin for error), possible exception of case 25 in which FR? cells out- which was the outer molecular layer of CA1/subiculum. numbered FG? cells by approximately 60/40%, the num- RE projections to the hippocampus terminate within the bers of FR and FG labeled neurons were largely equivalent slm of CA1 and the subiculum (Wouterlood et al. 1990; across RE for all cases. And for cases 9 and 26, there were Vertes et al. 2006). This pairing (FG in mPFC and FR in proportionally more FG? than FR? cells at all levels of HF) was, however, not used for all cases. It was reversed in RE. about 10% of the cases including case 9. Two other factors could have possibly influenced the If, as indicated, FR is a more effective retrograde tracer relative percentages of FR/FG neurons in RE; that is, size than FG, it might be expected that FR injections would of injections and the differential strength of RE projections produce proportionally more labeled cells in RE than to mPFC or to HF. Regarding injection size, it is well would FG injections—and thus possibly over represent recognized that the magnitude of labeling varies quite numbers of FR? compared to FG? neurons in RE. This directly with size of (retrograde) injections. With some 123 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 the fibers of passage problem. This is considerably less an as group FR injections were slightly larger than FG issue here in that: (1) RE projections to the present sites of injections. Regarding differential strength of RE-mPFC retrograde injections (mPFC and CA1/subiculum) have and RE-ventral HF projections, previous studies have been previously demonstrated with anterograde tracers demonstrated massive RE projections to the hippocampus (Herkenham 1978; Wouterlood et al. 1990; Vertes et al. (Herkenham 1978; Risold et al. 1997; Wouterlood et al. 2006); and (2) essentially the sole terminal destination of 1990; Bokor et al. 2002; Vertes et al. 2006, 2007) and fibers passing through the ventral mPFC would be more pronounced but less dense RE projections to the mPFC rostral levels of the mPFC (Vertes et al. 2006) and those (Herkenham 1978; Risold et al. 1997; Vertes et al. 2006). passing through the CA1/subiculum of ventral HF would In effect, then, each of the foregoing factors (relative be more caudal levels of the subiculum. Accordingly, if effectiveness of tracers, size of injections and differential there was a minor uptake of either tracer by damaged fibers strength of RE projections to targets) would seem to favor coursing through the sites of injection (to the rostral mPFC FR over FG labeling, but as mentioned there was a fairly or to the caudal subiculum), this would not noticeably alter equal distribution of the two types of labeled cells, with the present findings of single or collateral RE projections to only a minor shift toward FR? cells. the mPFC and to the CA1/subiculum. In addition, the With all retrograde tracers, there is the possibility of present retrograde tracers, FG and FR, appear to be among uptake of tracers not only by fibers terminating at the site of the least susceptible to uptake by the fibers of passage injection but also by those passing through the injection— (Schmued et al. 1990; Lanciego and Wouterlood 2006).

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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- Fig. 14 High magnification photomicrographs depicting a cluster of rons in RE with ventral CA1 (case 15) than with dorsal Fluororuby labeled neurons laterally in nucleus reuniens (RE), medial CA1 (case 21) injections. Since dorsal and ventral CA1 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 injections were of equivalent size and positioned in the lateral wings of RE (b) in the region depicted by the diagonal arrows same subfields of CA1, this would indicate considerably of Fig. 13. Scale bar for a, b 100 lm stronger RE projections to the ventral than to dorsal CA1. This is supported by previous findings, using anterograde RE projections to subfields of the hippocampus: dorsal tracers, showing a much greater density of labeled fibers in and ventral CA1, ventral subiculum and ventral CA1/ the ventral than dorsal CA1 with RE injections (Herken- subiculum (single labeled neurons). Comparison ham, 1978; Ohtake and Yamada 1989; Wouterlood et al. with previous studies 1990; Risold et al. 1997; Vertes et al. 2006). Injections in various regions of the ventral hippocampus Hippocampal injections gave rise to pronounced numbers (CA1, subiculum, CA1/SUB) produced generally similar of (single) labeled cells within RE. This supports previous numbers of labeled cells in RE. Nonetheless, injections in results showing that RE strongly targets the hippocampus the ventral subiculum (cases 22, and 25) produced more (Herkenham 1978; Risold et al. 1997; Wouterlood et al. labeled neurons in RE than did ventral CA1 or CA1/SUB 1990; Bokor et al. 2002; Vertes et al. 2006, 2007). injections. The single exception to this was case 27 which Although spread throughout RE, labeled cells were most was a large injection (of ventral CA1/SUB) and mainly densely concentrated in the intermediate mediolateral RE, localized to the subiculum. This indicates stronger RE just lateral to the midline, rostrally, and on the medial projections to the subiculum than to CA1 of the ventral border of the perireuniens nucleus (or lateral wings) of RE, hippocampus, and is generally consistent with previous caudally. Although relatively significant numbers of reports using anterograde tracers (Wouterlood et al. 1990; labeled cells were also present on the midline, few were Risold et al. 1997; Vertes et al. 2006). 123 Brain Struct Funct (2012) 217:191–209 207

RE projections to the ventral mPFC (single labeled Unlike the present demonstration of significant per- neurons). Comparison with previous studies centages of DB cells (3–9%) with HF and mPFC injections, previous reports failed to show similarly high percentages Injections in the ventral mPFC (IL and PL) gave rise to of DBs with various combinations of injections in HF and significant numbers of retrogradely labeled neurons dis- in other forebrain structures. For example, an early exam- tributed throughout RE. Although labeled cells spread ination of RE/midline thalamic projections to the amyg- mediolaterally across RE, they were most densely con- dala, nucleus accumbens (ACC) and ventral HF (Su and centrated in the lateral wings of RE (pRE), and secondarily Bentivoglio 1990) showed that separate, only minimally along the midline and on the medial border of pRE. overlapping, populations of RE cells distribute to each site. Comparatively, fewer labeled cells were present just lateral Virtually no double labeled neurons were found. In like to the midline—or in the region of dense concentration of manner, Dollerman-Van der Weel and Witter (1996) labeled cells with HF injections. Similar to HF injections, reported that RE projections to the entorhinal cortex, to there were proportionally more labeled cells in the rostral CA1 and to the subiculum arose from distinct regions of than in caudal half of RE with mPFC injections but relative RE. Finally, Bokor et al. (2002) described separate origins differences were less for mPFC than for HF injections, of RE cells distributing to the septum and the hippocam- likely owing to the fact that pRE is most fully expressed pus; that is, ventromedially in RE to the septum and caudally in RE. dorsolaterally in RE to HF. Based on their findings and Compared to reports examining RE-HF projections, few those of previous reports, Bokor et al. (2002) concluded studies have described RE projections to the mPFC. In an that: ‘‘it is likely that distinct cell populations form clusters early report using tritiated amino acids, Herkenham (1978) at various subregions in the RE, the clusters giving rise to showed that RE fibers spread rather diffusely to the medial projections to well defined target areas in the limbic wall of ventral mPFC terminating in what was termed the system.’’ infraradiate area (corresponding to PL) and in the infra- Consistent with the foregoing, we presently describe limbic region. Using PHA-L, Risold et al. (1997) subse- distinct clusters of RE neurons distributing to either HF or quently described (at best) moderate RE projections to the to the mPFC, but in contrast to earlier findings have ventral mPFC. In a recent examination of efferent projec- identified significant numbers of RE cells with branching tions of RE (and the dorsally adjacent rhomboid nucleus) (or collateral) projections to HF and to the mPFC. As has using PHA-L, we found that RE strongly targets the mPFC been noted, RE projects strongly to the HF and to mPFC, with fibers densely concentrated in layers 1 and 5/6 of IL and as such RE may exert a greater dual influence (col- and PL (Vertes et al. 2006). lateral projections) on major targets than on secondary ones. Collateral RE projections to the mPFC and to the hippocampus. Comparison with previous Functional considerations studies The nucleus reuniens of the midline thalamus receives a With the exception of case 21 in which the percentage of diverse and widely distributed set of afferent projections, double labeled cells was less than 1% due to the sparse mainly from limbic/limbic related structures (Risold et al. retrograde labeling in RE with the dorsal CA1 injection, 1997; Canteras and Goto 1999; Krout et al. 2002; Vertes the percentages of DB cells to total numbers of labeled 2002; Olucha-Bordonau et al. 2003; McKenna and Vertes neurons ranged from approximately 3 to 9.25%. Excluding 2004) and distributes fairly selectively to the hippocampus/ case 21, the percentage of DB cells in 5 of 6 of the cases parahippocampus and to the orbitomedial PFC (Wouter- was 3–6% of labeled neurons. The percentage of DB cells lood et al. 1990; Wouterlood 1991; Vertes et al. 2006). for case 26 was 9.25%, or considerably higher than for the Accordingly, RE appears to be an important interface in other cases. Case 26 involved a mPFC injection spanning limbic subcortical–cortical communication, that is, a site of PL/IL (Fig. 1a) and a HF injection in ventral CA1/SUB convergence (and integration) of limbic afferent informa- (Fig. 1d). It is presently unclear why this particular pairing tion and its subsequent transfer to limbic forebrain struc- of injections gave rise to such a large percentage of DBs. It tures. RE is thought to be critically involved in processes of was not, for instance, the fact that case 26 contained more arousal and attentional or in gating the flow of information labeled cells (and hence more DBs) than did the other to the limbic forebrain (Van der Werf et al. 2002; Vertes cases. There was no relationship between percentages of 2006, 2007). DBs and total numbers of labeled cells. In fact, case 22 Although not extensively examined, a few recent reports contained the most labeled neurons (3,248), but the lowest have described the effects of RE lesions on behavior percentage of DBs (3.01%). (Dolleman-Van der Weel et al. 2009; Davoodi et al. 2009, 123 208 Brain Struct Funct (2012) 217:191–209

2011; Hembrook and Mair 2011). In an initial study, using purposeful, self directed, behavior with stimulation of the a water maze task, Dolleman-Van der Weel et al. (2009) central thalamus in MCS or VS patients were unsuccessful reported that rats with RE lesions showed no deficits on the (Deliac et al. 1993; Yamamoto and Katayama 2005), Schiff acquisition phase of the task but impairments on the probe and colleagues (Schiff et al. 2007) recently demonstrated test (escape platform removed) following training. Spe- that deep brain stimulation (DBS) of the central thalamus cifically, RE lesioned rats spent considerably less time in in a MCS patient produced striking behavioral improve- the correct quadrant of the pool than controls which nor- ments. Specifically, with DBS the patient regained the mally would be interpreted as a hippocampal-dependent (or ability to follow verbal commands, purposively manipulate memory associated) deficit. The authors, however, viewed objects, intelligibly communicate, and orally consume food this as a non-mnemonic (or prefrontal-associated impair- (Schiff et al. 2007). Regarding possible mechanisms for ment) in that the lesioned rats initially swam to the correct these effects, Shah and Schiff (2010) suggested that the quadrant but quickly abandoned this behavior, favoring one central thalamus is instrumental in the transfer of arousal- of ‘search over all the pool’ for the missing platform. This related information to the forebrain which is critical for rapid switch in strategy was described as inflexible (or an maintaining requisite levels of cortical activation for impulsive) adaptation to an environmental change—or a effective cognitive functioning. In effect, DBS of intact PFC deficit. regions of the midline thalamus serves to ‘reactivate’ pre- By contrast with the foregoing, subsequent reports have viously dormant regions of cortex to restore levels of described hippocampal-dependent deficits with RE lesions consciousness necessary for purposeful behavior. (Davoodi et al. 2009, 2011; Hembrook and Mair 2011). In summary, the present results show that RE strongly Davoodi et al. (2009, 2011) initially showed that the targets the hippocampus and the mPFC, with separate reversible suppression of RE with tetracaine significantly populations favoring one or the other site, and that a rel- impaired performance on reference and working memory atively significant percentage of RE neurons project to both tasks on the water maze (WM), and subsequently that structures via axon collaterals. RE is thus critically posi- inactivating RE prior to, or immediately after, the acqui- tioned to influence limbic forebrain structures, particularly sition of a passive avoidance task disrupted performance on the HF and the mPFC, and the functions associated with this task when tested 24 h later. them. Supporting this, Hembrook and Mair (2011) recently demonstrated that rats with lesions of RE (and the dorsally Acknowledgments This research was supported by National Sci- adjacent rhomboid nucleus) exhibited significant deficits in ence Foundation grant IOS 0820639 to RPV. 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 References lesions would have a much greater disruptive effect on tasks involving both the hippocampus and the PFC (delay Bertram EH, Zhang DX (1999) Thalamic excitation of hippocampal CA1 neurons: a comparison with the effects of CA3 stimulation. 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