The Journal of Neuroscience, August 24, 2016 • 36(34):8977– 8984 • 8977
Systems/Circuits
The Nucleus Reuniens of the Midline Thalamus Gates
Prefrontal-Hippocampal Modulation of Ventral Tegmental
Area Dopamine Neuron Activity
Eric C. Zimmerman and X Anthony A. Grace
Departments of Neuroscience, Psychiatry, and Psychology, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
The circuitry mediating top-down control of dopamine (DA) neurons in the ventral tegmental area (VTA) is exceedingly complex.
Characterizing these networks will be critical to our understanding of fundamental behaviors, such as motivation and reward processing,
as well as several disease states. Previous work suggests that the medial prefrontal cortex (mPFC) exerts a profound influence on VTA DA
neuron firing. Recently, our group reported that inhibition of the infralimbic subdivision of the medial prefrontal cortex (ilPFC) increases
the proportion of VTA DA neurons that are spontaneously active (i.e., “population activity”) and that this effect depends on activity in the
ventral subiculum of the hippocampus (vSub). However, there is no direct projection from the mPFC to the vSub. Anatomical evidence
suggests that communication between the two structures is mediated by the nucleus reuniens of the midline thalamus (RE). Here, we used
in vivo electrophysiological and behavioral approaches in rats to explore the role of the RE in the circuitry governing VTA DA neuron
firing. We show that pharmacological stimulation of the RE enhances VTA DA neuron population activity and amphetamine-induced
hyperlocomotion, a behavioral indicator of an over-responsive DA system. Furthermore, the effect of RE stimulation on population
activity is prevented if vSub is also inhibited. Finally, pharmacological inhibition of ilPFC enhances VTA DA neuron population activity,
but this effect does not occur if RE is also inhibited. These findings suggest that disruption of ilPFC–RE–vSub communication could lead
to a dysregulated, hyperdopaminergic state, and may play a role in psychiatric disorders.
Key words: dopamine; thalamus; VTA
Significance Statement
Dopamine (DA) neurons in the ventral tegmental area (VTA) are involved in a variety of fundamental brain functions. To
understand the neurobiological basis for these functions it is essential to identify regions controlling DA neuron activity. The
medial prefrontal cortex (mPFC) is emerging as a key regulator of DA neuron activity, but the circuitry by which it exerts its
influence remains poorly described. Here, we show that the nucleus reuniens of the midline thalamus gates mPFC control of VTA
DA neuron firing by the hippocampus. These data identify a unique role for this corticothalamic-hippocampal circuit, and suggest
that dysfunction in these regions likely influences the pathophysiology of psychiatric disorders.
Introduction
The afferent circuitry regulating the activity of dopamine (DA) neurons of the ventral midbrain, including the ventral tegmental area
(VTA), is exceedingly complex (Sesack and Grace, 2010; WatabeReceived April 27, 2016; revised July 1, 2016; accepted July 6, 2016.
Author contributions: E.C.Z. and A.A.G. designed research; E.C.Z. performed research; E.C.Z. analyzed data; E.C.Z.
and A.A.G. wrote the paper.
This work was supported by National Institutes of Health Grants F30MH109199 (E.C.Z.), and MH57440 (A.A.G.).
We thank all members of the Grace laboratory for their helpful advice and technical assistance.
E.C.Z. declares no competing financial interests. A.A.G. reports potential competing financial interests related to
associations with the following companies: Johnson & Johnson, Lundbeck, Pfizer, GlaxoSmithKline, Merck, Takeda,
Dainippon Sumitomo, Otsuka, Lilly, Roche, Asubio, Abbott, Autofony, Janssen, and Alkermes.
Correspondence should be addressed to Eric C. Zimmerman, Department of Neuroscience, A210 Langley Hall,
Pittsburgh, PA 15260. E-mail: ecz5@pitt.edu.
DOI:10.1523/JNEUROSCI.1402-16.2016
Copyright © 2016 the authors 0270-6474/16/368977-08$15.00/0
Uchida et al., 2012; Beier et al., 2015; Lerner et al., 2015). Although a
number of regions project directly to the VTA, numerous studies
have shown that there are complex multisynaptic networks that potently impact DA neuron activity states (Floresco et al., 2003; Butts et
al., 2011; Patton et al., 2013; Chang and Grace, 2014; Ferenczi et al.,
2016). These circuits provide multiple sites of dynamic regulation
and amplification of DA neuron output. Characterizing the mechanisms by which these afferent or “upstream” networks control DA
neuron firing, and therefore DA tone in VTA output regions, will be
fundamental to our understanding of complex behaviors, such as
motivation and reward processing, as well as several disease states.
We have previously characterized a circuit comprising the
ventral subiculum (vSub), the nucleus accumbens (NAc), and the
ventral pallidum (VP), which potently influences DA system responsivity (Floresco et al., 2001, 2003; Lodge and Grace, 2007;
8978 • J. Neurosci., August 24, 2016 • 36(34):8977– 8984
Valenti et al., 2011). Stimulation of the vSub, via the NAc and VP,
leads to disinhibition of VTA DA neurons and an increase in the
proportion of VTA DA neurons that are spontaneously active
(i.e., “population activity”). This effect is accompanied by DA
release in the NAc (Floresco et al., 2003) and an increase in
amphetamine-induced hyperlocomotion (White et al., 2006), a
behavioral correlate of DA system responsivity (Moore et al.,
2001; Lodge and Grace, 2007; Gill et al., 2011; Valenti et al., 2011;
Chang and Grace, 2013). Population activity is a key parameter of
DA neuron firing, because DA neurons can only exhibit rapid,
phasic, stimulus-driven burst firing (Grace and Bunney, 1984) if
they are spontaneously active (Lodge and Grace, 2006). Therefore, the vSub–NAc–VP circuit acts as a critical gain modulator of
behaviorally salient DA neuron outputs. In addition, this circuit
controlling population activity has been shown to play essential
roles in animal models of schizophrenia, depression, and drug
abuse (Lodge and Grace, 2007; Chang and Grace, 2014; Belujon et
al., 2016).
Hippocampal control of VTA DA neuron population activity
is in turn potently modulated by the medial prefrontal cortex
(mPFC). Specifically, attenuation of activity in the infralimbic
subdivision of the medial prefrontal cortex (ilPFC; but not the
prelimbic subdivision) increases population activity, and this effect is dependent on the vSub (Patton et al., 2013). However, no
direct projection exists between the ilPFC and the vSub (Laroche
et al., 2000; Vertes, 2004). The nucleus reuniens of the midline
thalamus (RE) forms an anatomical link between the mPFC and
the hippocampus, sending a potent, glutamatergic projection to
the hippocampus (Herkenham, 1978; Vertes et al., 2006) that
densely innervates the stratum lacunosum-moleculare of the
dorsal and ventral CA1, as well as the molecular layer of the dorsal
and ventral subiculum (Bokor et al., 2002; Vertes et al., 2006;
Hoover and Vertes, 2012). Electrophysiological studies show that
the RE excites CA1 neurons (Dolleman-Van der Weel et al.,
1997). Fibers from the RE also distribute throughout layers 1 and
5/6 of the mPFC, including dense projections to the ilPFC (Vertes
et al., 2006). In turn, the RE receives reciprocal connections from
the vSub and the ilPFC, forming a functional network (McKenna
and Vertes, 2004; Varela et al., 2014).
Given these rich interconnections between the ilPFC, RE, and
vSub, the RE could play a role in the afferent control of VTA DA
neuron activity. However, this possibility has not been evaluated
experimentally. In the current study, we use in vivo, circuit-based
electrophysiological and behavioral approaches in rats to explore
the role of RE in the descending cortical–subcortical circuitry
governing VTA DA neuron firing.
Materials and Methods
Animals. All experiments were performed in accordance with the guidelines outlined in the United States Public Health Service Guide for Care
and Use of Laboratory Animals and were approved by the Institutional
Animal Care and Use Committee of the University of Pittsburgh. All
experiments were performed in adult (⬎65 d old) male Sprague Dawley
rats (105 rats total; 300 – 450 g).
Electrophysiology. Animals were anesthetized with an initial dose of
chloral hydrate (Sigma-Aldrich; 400 mg/kg, i.p.) and were supplemented
periodically (intraperitoneally) to maintain suppression of the hindlimb
withdrawal reflex. Rats were then placed in a stereotaxic frame (Kopf)
and body temperature was maintained at 37°C with a temperaturecontrolled heating pad and rectal probe (Fintronics). In vivo, extracellular recordings were performed using single glass microelectrodes (WPI;
impedance 6 – 8 M⍀) filled with a 2% Chicago sky blue (Sigma-Aldrich)
solution in 2 M NaCl. This impedance ensures that we were able to clearly
resolve the waveform from a single cell with a very high signal-to-noise
Zimmerman and Grace • Nucleus Reuniens Control of Dopamine Neuron Firing
ratio and without contamination from neighboring cells. Following a
craniotomy, electrodes were lowered into the VTA in nine sequential,
vertical “tracks” at 0.2 mm intervals in a predetermined grid pattern in
the x–y plane via hydraulic micropositioner (Kopf). Tracks began at the
following coordinates from bregma/skull surface (in mm): anteroposterior (AP), ⫺5.3; mediolateral (ML), 0.6; dorsoventral (DV), ⫺6.5 to ⫺9,
according to the Paxinos and Watson brain atlas (Paxinos and Watson,
2013), and sampled a block of tissue from AP ⫺5.3 to ⫺5.7 and ML
0.6 –1.0, as previously described (Chang and Grace, 2014). Individual,
putative DA neurons were recorded with open filter settings (low pass, 10
Hz; high pass, 16 kHz) enabling identification using well established
criteria, including (1) slow (2–10 Hz), irregular, or bursting firing pattern; (2) long-duration (⬎2.2 ms) biphasic action potential with initial
segment-somatodendritic positive phase break; and (3) temporary inhibition of firing during tail or foot pinch (Grace and Bunney, 1983; Ungless and Grace, 2012). Three properties of identified DA neurons were
measured: (1) population activity, quantified as the average number of
spontaneously active DA neurons per electrode track, i.e., “cells/track”
(calculated for each rat); (2) average firing rate, and (3) percentage of
spikes occurring in bursts. Burst initiation was defined as the occurrence
of two spikes with an interspike interval of 80 ms, and burst termination
as the occurrence of an interspike interval of 160 ms, as previously described (Grace and Bunney, 1984). Each neuron was recorded for ⱖ3
min and, given the typical ⬃4 Hz firing rate of identified DA neurons
(Grace and Bunney, 1983; Ungless and Grace, 2012), this resulted in
⬃720 spikes being included in the analysis of firing properties for each
neuron.
Intracranial infusions in anesthetized rats. Local infusions were performed in anesthetized animals immediately before VTA DA neuron
recordings. Using the stereotaxic frame, a guide cannula (23 gauge) was
placed above the ilPFC, RE, and/or vSub at the following coordinates
from bregma/skull surface (in mm): ilPFC: AP, ⫹2.7; ML, 0.5; DV, ⫺3.5;
RE: AP, ⫺2.2; ML, 2.3; DV, ⫺7.2; 15° angle from vertical; vSub: AP,
⫺5.5; ML, 4.8; DV, ⫺7.1, according to the Paxinos and Watson brain
atlas (Paxinos and Watson, 2013). Subsequently, an infusion cannula (33
gauge) was inserted into the guide cannula, extending 1 mm beyond the
tip of the guide cannula. Pharmacological agents dissolved in Dulbecco’s
PBS (dPBS; Sigma-Aldrich) or dPBS vehicle only were administered
through the infusion cannula at a rate of 0.5 l/min. The guide cannula
was left in place for 3 min following infusions to allow for adequate
diffusion of drug. Drug doses were as follows for all experiments: ilPFC:
TTX, 1 M in 0.5 l; RE: NMDA, 0.75 g in 0.2 l; TTX, 1 M in 0.2 l; vSub:
TTX, 1 M in 0.5 l.
Chemical stimulation was deliberately used to enable stable, longduration neuronal excitation without the confounds associated with current spread, activation of fibers of passage, or potential lesions during
extended electrical or optical stimulation. All pharmacological agents
were injected at doses reported previously to induce specific behavioral
and/or neurochemical effects (Lodge and Grace, 2007; Valenti et al.,
2011; Patton et al., 2013). Rats received only one injection per region and
DA cell recordings were typically performed from 10 min to 2.5 h after
infusions.
Survival surgery and cannula implantation for behavioral studies. All
survival surgeries were performed under general anesthesia in a sterile
environment. Briefly, rats were anesthetized with isoflurane (induction:
5%; maintenance: 1–3% in oxygen) and placed in a stereotaxic apparatus
using blunt, atraumatic ear bars. Bilateral cannulae (23 gauge) were implanted in the RE [coordinates from bregma/skull surface (in mm): AP,
⫺2.2; ML, 2.3; DV, ⫺7.2; 15° angle from vertical] and fixed in place with
dental cement and anchor screws. Once the cement was dry, antibiotic
cream was applied to the wound edge (Neosporin), and the rat was
removed from the stereotaxic frame and monitored closely until conscious. Rats received postoperative analgesia (carpofen 5 mg/kg, i.p.,
once per day for 72 h and Tylenol syrup in softened rat chow 5% v/w
available ad libitum for 72 h) and were allowed to recover for ⱖ1 week
before behavioral experiments.
Amphetamine-induced hyperlocomotion. Following surgeries, rats were
housed in a reverse light/dark cycle room (lights on 7:00 P.M. to 7:00
A.M.) for ⱖ1 week before behavioral experiments. Baseline locomotor
Zimmerman and Grace • Nucleus Reuniens Control of Dopamine Neuron Firing
J. Neurosci., August 24, 2016 • 36(34):8977– 8984 • 8979
Figure 1. Activation of the RE produced an enhancement of VTA DA neuron population activity. A, Left, Representative image of electrode tracks (solid black arrows) and electrode tip (dashed arrow) in the
VTA. Right, Representation of histological placements of infusion cannulae into the RE (open circles). B, Activating the RE with NMDA enhanced the number of spontaneously active DA neurons firing in the VTA
(expressed as cells/track, green bar) compared with infusion of vehicle (VEH; blue bar). C, D, The average firing rate of spontaneously active DA cells was not affected by infusion of NMDA into the RE, but the
percentage of cells firing in bursts was increased. E, F, Distribution of firing rate and burst firing were not affected by infusion of NMDA into the RE (Kolmogorov–Smirnov test). *p ⬍ 0.05 (unpaired t test). VEH,
n ⫽ 8 rats; NMDA, n ⫽ 8 rats; VEH, n ⫽ 59 neurons; NMDA, n ⫽ 111 neurons. Data are represented as mean ⫾ SEM.
activity and amphetamine-induced hyperlocomotion were measured by
beam breaks in the x–y plane of an open-field arena (Coulborn) and
analyzed in 5 min epochs. Following measurement of baseline locomotor
activity, NMDA (0.75 g in 0.2 l) or dPBS vehicle was injected into the
RE via the previously implanted guide cannula, followed immediately
by D-amphetamine sulfate (0.75 mg/kg, i.p.). Amphetamine-induced hyperlocomotion was then measured for 60 min.
Histology. After electrophysiology, the recording site was marked via
electrophoretic ejection of Chicago sky blue dye from the tip of the recording electrode (⫺25 A constant current, 20 –30 min). All rats were
killed and decapitated. Their brains were removed, fixed for ⱖ48 h (8%
w/v paraformaldehyde in PBS), and cryoprotected (25% w/v sucrose in
PBS) until saturated. Brains were sectioned (60 m coronal sections),
mounted onto gelatin-chrom alum-coated slides, and stained with cresyl
violet for histochemical verification of electrode and/or acute/chronically implanted cannula placement. All histology was performed with
reference to a stereotaxic atlas (Paxinos and Watson, 2013). Only data
from animals with accurate placements in all regions were included in the
analysis.
Analysis. Electrophysiological analysis of DA neuron activity was performed using commercially available software (LabChart and NeuroExplorer). Locomotor behavior was recorded using TruScan software. All
data are represented as the mean ⫾ SEM, unless otherwise stated. All
statistics were calculated using the GraphPad Prism software program
(GraphPad Software).
Results
RE activation increases VTA DA neuron population activity
We showed previously that the vSub exerts a profound influence
on VTA DA neuron activity (Floresco et al., 2003; Lodge and
Grace, 2006, 2007; Patton et al., 2013). Given the dense, excitatory projections from the RE to the vSub (Dolleman-Van der
Weel et al., 1997; Bertram and Zhang, 1999; Vertes et al., 2006),
we tested whether RE activation would also affect VTA DA neuron activity. Following infusion of vehicle into the RE (dPBS; n ⫽
8 rats and 59 neurons; Fig. 1A), we found an average of 1.05 ⫾ 0.1
spontaneously active DA neurons per electrode track in the VTA
(i.e., “cells/track”), with an average firing rate of 4.57 ⫾ 0.3 Hz
and 27.82 ⫾ 3.4% of action potentials fired in bursts (Fig. 1B–F ),
all of which are consistent with previous findings in untreated
animals (Lodge and Grace, 2007; Chang and Grace, 2014; Gill et
al., 2014). In contrast, following NMDA infusion into the RE
(0.75 g in 0.2 l; n ⫽ 8 rats and 111 neurons), we observed a
nearly 63% increase in VTA DA neuron population activity over
controls (1.65 ⫾ 0.2 cells/track; unpaired t test, t(14) ⫽ 2.69, p ⫽
0.02), with no significant change in firing rate (4.61 ⫾ 0.2 Hz).
Infusion of NMDA into regions adjacent to the RE, including the
interanteromedial, ventromedial, and paraxiphoid nuclei of the
thalamus, had no effect on VTA DA neuron activity (data not
shown). We also observed a small but significant increase in burst
firing following NMDA infusion into the RE (36.61 ⫾ 2.6%,
unpaired t test, t(168) ⫽ 2.00, p ⫽ 0.047). These data show that RE
stimulation is sufficient to enhance VTA DA neuron population
activity.
RE activation increases amphetamine-induced
hyperlocomotion
Given that VTA DA neuron population activity correlates with
amphetamine-induced hyperlocomotion (Moore et al., 2001;
Lodge and Grace, 2007; Gill et al., 2011; Valenti et al., 2011;
Chang and Grace, 2013), we measured the locomotor response
to amphetamine following acute infusion of dPBS vehicle or
NMDA into the RE (Fig. 2A) in a separate group of awake, behaving rats. Rats that received an acute NMDA infusion (0.75 g
in 0.2 l; n ⫽ 8 rats) into the RE displayed a significantly enhanced locomotor response to D-amphetamine sulfate injection
(0.75 mg/kg, i.p.) compared with vehicle controls (n ⫽ 13 rats),
both when quantified as total distance traveled (Fig. 2B, unpaired
t test, t(19) ⫽ 2.22, p ⫽ 0.04) and ambulatory distance in discrete
time bins (Fig. 2C; two-way repeated-measures ANOVA, main
effect of treatment F(1,19) ⫽ 4.9, p ⫽ 0.03 and treatment ⫻ time
8980 • J. Neurosci., August 24, 2016 • 36(34):8977– 8984
Zimmerman and Grace • Nucleus Reuniens Control of Dopamine Neuron Firing
Figure 2. Activation of the RE increased amphetamine-induced hyperlocomotion. A, Representation of histological placements of infusion cannulae into the RE (open circles). D-amphetamine
sulfate (0.75 mg/kg, i.p.) was delivered immediately after acute microinjection of NMDA or dPBS vehicle (VEH) into the RE (both represented by dashed arrow). Baseline locomotor activity (0 –30
min) and amphetamine-induced hyperlocomotion (35–90 min) were then measured. B, Pharmacological activation of RE increased total distance traveled in the 60 min postinjection period
compared with controls (unpaired t test, *p ⬍ 0.05). C, Pharmacological activation of the RE increased ambulatory distance during the postinjection period (two-way repeated-measures ANOVA,
Holm–Sidak post hoc, *p ⬍ 0.05). VEH, n ⫽ 13 rats; NMDA n ⫽ 8 rats. Data are represented as mean ⫾ SEM.
interaction F(8,152) ⫽ 2.35, p ⫽ 0.04, Holm–Sidak post hoc).
Therefore, both VTA DA neuron population activity and the
locomotor response to amphetamine are increased by RE
activation.
0.95) or burst firing (31.32 ⫾ 3.6%, one-way ANOVA, F(3,237) ⫽
0.33, p ⫽ 0.80) were observed in this group compared with control
animals. These findings confirm that the vSub is necessary for RE
stimulation to increase VTA DA neuron population activity.
The effect of RE activation on VTA DA neuron population
activity requires the vSub
Given evidence that the vSub potently controls VTA DA neuron
activity (Floresco et al., 2003; Lodge and Grace, 2006, 2007; Patton et
al., 2013), we examined whether the enhancement of VTA DA neuron population activity following RE activation was dependent on
the vSub. This was tested by pharmacologically inactivating the vSub
by TTX infusion (1 M in 0.5 l of dPBS) during simultaneous
activation of the RE by NMDA infusion (0.75 g in 0.2 l; Fig. 3A).
Rats receiving either vehicle infusion in both regions, or vehicle in
the RE and TTX in the vSub, exhibited similar DA neuron firing
properties (dPBS/dPBS: 0.98 ⫾ 0.08 cells/track, 4.40 ⫾ 0.3 Hz,
36.30 ⫾ 4.1%, n ⫽ 8 rats and 40 neurons; dPBS/TTX: 0.90 ⫾ 0.2
cells/track, 4.46 ⫾ 0.3 Hz, 31.65 ⫾ 4.1%, n ⫽ 8 rats and 54 neurons;
Fig. 3B–F). These findings are consistent with previous data from
our group in untreated animals (Lodge and Grace, 2007; Patton et
al., 2013). In addition, vehicle infusion into the vSub did not attenuate the enhancement of VTA DA neuron population activity observed following NMDA infusion into the RE (1.56 ⫾ 0.2 cells/track,
4.33 ⫾ 0.2 Hz, 31.43 ⫾ 2.9%, n ⫽ 10 rats and 107 neurons), which
was significantly greater than in control animals (one-way ANOVA,
main effect of treatment F(3,28) ⫽ 5.7, p ⫽ 0.004, Tukey’s post hoc)
and consistent with our findings above (Fig. 1). In contrast, infusion
of TTX into the vSub completely abolished the effect of RE activation
on VTA DA neuron population activity: animals in this group (n ⫽
6 rats and 41 neurons) displayed 0.98 ⫾ 0.08 active cells/track on
average, which was not different from controls (one-way ANOVA,
95% CI of the difference: ⫺0.65 to 0.49). In addition, no changes in
firing rate (4.55 ⫾ 0.2 Hz; one-way ANOVA, F(3,238) ⫽ 0.12, p ⫽
ilPFC control of VTA DA neuron population activity requires
the RE
We have shown previously that excitation or inhibition of the ilPFC
bidirectionally modulates VTA DA neuron population activity. ilPFC inactivation induces an increase in population activity that does
not occur following simultaneous vSub inhibition (Patton et al.,
2013). Given that there is no direct projection from the ilPFC to the
vSub (Laroche et al., 2000; Vertes, 2004), we tested whether the RE
could be mediating the effect of ilPFC inhibition on VTA DA neuron
population activity. This was tested by pharmacologically inactivating the RE by TTX infusion (1 M in 0.2 l of dPBS) during simultaneous inactivation of the ilPFC by TTX infusion (1 M in 0.5 l of
dPBS; Fig. 4A). In rats receiving either vehicle in both regions, or
vehicle in the ilPFC and TTX in the RE, the number of spontaneously firing DA neurons per electrode track, firing rate, and burst
firing were comparable (dPBS/dPBS: 1.0 ⫾ 0.1 cells/track, 4.21 ⫾ 0.2
Hz, 28.5 ⫾ 3.3%, n ⫽ 10 rats and 65 neurons; dPBS/TTX: 1.0 ⫾ 0.2,
4.2 ⫾ 0.2 Hz, 30.4 ⫾ 3.4%, n ⫽ 11 rats and 68 neurons; Fig. 4B–F).
These findings are consistent with previous data from our group in
untreated animals (Patton et al., 2013). In addition, vehicle infusion
in the RE did not influence the increase in VTA DA neuron population activity observed following TTX infusion into the ilPFC (1.60 ⫾
0.2 cells/track, 4.67 ⫾ 0.2 Hz, 27.15 ⫾ 2.8%, n ⫽ 7 rats and 83
neurons), in which we observed a significant increase over controls
(one-way ANOVA, main effect of treatment F(3,32) ⫽ 6.7, p ⫽ 0.001,
Tukey’s post hoc). However, infusion of TTX into the RE completely
prevented the effect of ilPFC inhibition on VTA DA neuron population activity (n ⫽ 8 rats and 40 neurons; 0.58 ⫾ 0.08 cells/track),
making this group statistically indistinguishable from controls (one-
Zimmerman and Grace • Nucleus Reuniens Control of Dopamine Neuron Firing
J. Neurosci., August 24, 2016 • 36(34):8977– 8984 • 8981
Figure3. EnhancedVTADAneuronpopulationactivityfollowingREactivationwaspreventedbyinhibitionofthevSub.A,RepresentationofhistologicalplacementsofinfusioncannulaeintotheRE(left)and
thevSub(right;opencircles).TTXordPBSvehicle(VEH)wasinjectedintothevSub,followedimmediatelybyinjectionofNMDAordPBSvehicleintotheRE.VTADAneuronpopulationactivity,firingrate,andburst
firingwerethenmeasured.B,PharmacologicalactivationoftheREincreasedpopulationactivity,whileparallelinhibitionofthevSubpreventedthiseffect.C,D,ThefiringrateofspontaneouslyactiveDAcellsand
thepercentageofcellsfiringinburstswerenotaffectedbyanymanipulation.E,F,DistributionoffiringrateandburstfiringwerenotaffectedbyinfusionofNMDAintotheRE(Kruskal–Wallistest,firingrate,H⫽
1.70, p ⫽ 0.64; bursting, H ⫽ 2.15, p ⫽ 0.54). *p ⬍ 0.05, **p ⬍ 0.01 (one-way ANOVA, Tukey’s post hoc). x-axis in B–D is infusions in vSub/RE. n ⫽ 6 –10 rats/group; n ⫽ 40 –107 neurons/group. Data are
represented as mean ⫾ SEM.
Figure 4. EnhancedVTADAneuronpopulationactivityfollowingilPFCinactivationwaspreventedbyinhibitionoftheRE.A,RepresentationofhistologicalplacementsofinfusioncannulaeintotheilPFCand
theRE(opencircles).TTXordPBSvehicle(VEH)wasinjectedintotheRE,followedimmediatelybyinjectionofTTXordPBSvehicleintotheilPFC.VTADAneuronpopulationactivity,firingrate,andburstfiringwere
then measured. B, Pharmacological inhibition of ilPFC increased population activity, while parallel inhibition of the RE prevented this effect.C, D, The firing rate of spontaneously active DA cells was not affected
by any manipulation, while the percentage of cells firing in bursts was significantly enhanced in the TTX/TTX group. E, F, Distribution of firing rate and burst firing were not affected by infusion of NMDA into the
RE(Kruskal–Wallistest,firingrate,H⫽0.060,p⫽0.99;bursting,H⫽0.54,p⫽0.91).*p⬍0.05,***p⬍0.001(one-wayANOVA,Tukey’sposthoc).x-axisinB–DisinfusionsinRE/ilPFC.n⫽7–11rats/group;
n ⫽ 40 – 83 neurons/group. Data are represented as mean ⫾ SEM.
8982 • J. Neurosci., August 24, 2016 • 36(34):8977– 8984
Zimmerman and Grace • Nucleus Reuniens Control of Dopamine Neuron Firing
way ANOVA, 95% CI of the difference: ⫺0.16 to 0.97). In addition,
combined infusion of TTX into the ilPFC and the RE resulted in a
small but statistically significant enhancement of burst firing (oneway ANOVA, main effect of treatment F(3,251) ⫽ 2.7, p ⫽ 0.046,
Tukey’s post hoc). These findings confirm that activity in the RE is
necessary for the effect of ilPFC inhibition on increasing VTA DA
neuron population activity, and suggest that the RE acts as a necessary intermediary between the ilPFC and the vSub, playing a crucial
role in cortical modulation of VTA DA neuron activity.
Discussion
We present evidence demonstrating that the RE is a novel region
involved in control of VTA DA neuron population activity. Pharmacological stimulation of the RE enhances DA neuron population
activity, without affecting average firing rate, and mildly enhancing
burst firing. The same stimulation paradigm also enhances amphetamine-induced hyperlocomotion, which strongly correlates with
DA system responsivity (Moore et al., 2001; Lodge and Grace, 2007;
Gill et al., 2011; Valenti et al., 2011; Chang and Grace, 2013). Furthermore, we show that the effect of RE stimulation is prevented if
the vSub is also inhibited, suggesting that activity in the vSub is
necessary for the RE to drive VTA DA neuron firing. Finally, inactivation of the RE prevents the increase in VTA DA neuron population activity observed following ilPFC inhibition, suggesting that the
ilPFC potently regulates RE drive of VTA DA neuron activity.
We and other groups have shown previously that stimulation
of the vSub increases VTA DA neuron population activity and
amphetamine-induced hyperlocomotion, without affecting firing
rate or burst firing (Floresco et al., 2001, 2003; Hammad and Wagner, 2006; White et al., 2006). In addition, the RE has been shown to
drive activity in CA1 and subiculum via asymmetric synapses onto
the distal dendrites of pyramidal neurons (Wouterlood et al., 1990;
Dolleman-Van der Weel et al., 1997; Bertram and Zhang, 1999).
Therefore, the current findings suggest that stimulation of the RE
enhances population activity and amphetamine-induced hyperlocomotion via direct, as well as indirect (via CA1), excitation of the
vSub. Multiple previous studies have characterized the role of the
RE in mediating communication between the mPFC and the hippocampus in behaviors requiring intact spatial and working memory (Hembrook and Mair, 2011; Hembrook et al., 2012; Cassel et al.,
2013; Cholvin et al., 2013; Prasad et al., 2013; Duan et al., 2015; Ito et
al., 2015; Layfield et al., 2015; Prasad et al., 2016; i.e., predominantly
dorsal hippocampal functions). However, the present work is one of
the first to show that the RE modulates ventral hippocampal functions, which is consistent with the fact that projections from the
RE to the ventral hippocampus are the most dense of any RE efferents to the hippocampus (Herkenham, 1978; Hoover and Vertes,
2007; Varela et al., 2014).
We have shown previously that the ilPFC bidirectionally modulates VTA DA neuron population activity: inhibition of ilPFC
enhances VTA DA neuron population activity, and this effect is dependent on the vSub, whereas excitation of the ilPFC attenuates
VTA DA neuron population activity, and this effect depends on the
basolateral amygdala (Patton et al., 2013). However, there are multiple potential pathways between the ilPFC and the vSub that could
mediate the effect of ilPFC inhibition. The current findings provide
strong evidence that the RE constitutes the connection by which the
ilPFC can control vSub drive of VTA DA neuron activity. This finding is consistent with those of several studies characterizing monosynaptic interconnections between the ilPFC and the RE (Vertes,
2002; McKenna and Vertes, 2004; Hoover and Vertes, 2007). ilPFC
projections to the RE are likely glutamatergic, making it apparently
paradoxical that inhibition of the ilPFC could lead to drive of the
Figure 5. SchematicrepresentationoftheproposedpathwaysbywhichtheREcouldacttomodulate VTA DA neuron activity. A, In the baseline state, this circuitry is in balance, resulting in a typical
proportion of spontaneously active (green triangles) and quiescent (gray triangles) DA neurons in the
VTA (i.e., population activity). B, When the RE is stimulated with NMDA, this results in an increase in
vSub activity, and a subsequent enhancement of population activity (indicated by higher number of
greentriangles)viapreviouslydescribedsubcorticalcircuitry.C,WhentheilPFCisinhibited,thisresults
in an increase in population activity in an analogous fashion to what occurs following RE stimulation.
The manner in which ilPFC inhibition could result in enhanced activity of the RE has not been described, although one possibility would be via the inhibitory reticular nucleus of the thalamus (TRN).
vSub via the RE. However, there is mounting evidence that cortical
regions may influence excitability of thalamic circuits via feedforward inhibition mediated by the reticular nucleus of the thalamus
(Cornwall et al., 1990; Paz et al., 2011; Pratt and Morris, 2015; Wimmer et al., 2015). In this context, our findings would suggest that the
ilPFC provides a potent down-modulation of RE drive of the hippocampus via the reticular thalamus. The finding that inhibition of
the RE alone did not alter VTA DA neuron population activity does
not preclude this model, because the RE likely exhibits low levels of
activity at baseline in the anesthetized state. This model could explain the effects of ilPFC hyperactivity, which is proposed to play a
role in models of depression (Belujon and Grace, 2014; Chang and
Grace, 2014), in disrupting limbic emotional influences on memory
processes. It also implies that dysfunction in the ilPFC would remove
any feedforward inhibition provided by the reticular thalamus, leading to RE–vSub hyperexcitability.
It should be noted that, despite anatomical evidence for direct
connections, our manipulations are not projection-specific, and
Zimmerman and Grace • Nucleus Reuniens Control of Dopamine Neuron Firing
therefore we cannot completely rule out the involvement of intervening structures in these effects. However, we believe that the regions manipulated here form vital nodes in the circuit for several
reasons. First, while the RE receives a diverse array of afferent inputs
(McKenna and Vertes, 2004), its major projection targets are restricted primarily to the hippocampus and the mPFC (Vertes, 2002;
Vertes et al., 2006). Given that our NMDA infusions targeted the
portion of the RE that contains primarily neurons projecting to the
ventral hippocampus (i.e., the rostral portion; Vertes et al., 2006),
RE–mPFC projections are not likely involved in the responses observed. In addition, our data show a complete reversal of the effect of
RE stimulation on VTA DA neuron population activity with vSub
inhibition, supporting a direct RE–vSub action. Finally, although
projections from the mPFC to the entorhinal cortex (Vertes, 2004)
and from the mPFC to the VTA (Vertes, 2004) have been described
anatomically, these circuits are unlikely to be involved in these findings given that RE inhibition completely reversed the effect of ilPFC
inhibition on VTA DA neuron population activity. It should also be
noted that recordings were performed in anesthetized animals.
Nonetheless, whereas anesthesia is likely to impact baseline activity,
it should not qualitatively impact the effects of pathway activation.
The fact that our behavioral studies in awake animals were consistent
with our electrophysiological findings supports this contention.
Together, our findings support a model whereby inactivation of
the ilPFC leads to an increase in RE activity, driving the vSub and
enhancing VTA DA neuron population activity (Fig. 5). We propose
that the RE is a novel, key modulator of subcortical/limbic circuits
involved in the control of VTA DA neuron firing, and as such could
play a role in psychiatric illnesses involving dysfunction in these
circuits. In particular, abnormally high DA tone underlies the pathophysiology of psychosis across psychiatric illnesses (Heinz and
Schlagenhauf, 2010; Howes et al., 2012; Winton-Brown et al., 2014),
most notably in schizophrenia (Howes et al., 2012). In addition,
increased activity in the subiculum (Tregellas et al., 2007, 2014; Allen
et al., 2012) and thalamus (Silbersweig et al., 1995; Tregellas et al.,
2007) have been described in patients with schizophrenia. Our
group has modeled the hippocampal hyperexcitability observed in
schizophrenia using the methylazoxymethanol acetate (MAM) developmental disruption model, which exhibits enhanced VTA DA
neuron population activity and aberrantly high vSub activity
(Moore et al., 2006; Lodge and Grace, 2007; Modinos et al., 2015).
Given that stimulation of RE was sufficient to replicate the hyperdopaminergic state observed in MAM animals, thalamic projections to
the hippocampus could represent a key connection that perpetuates
the subcortical hyperexcitability and hyperdopaminergic state seen
in patients with schizophrenia. Indeed, this idea has been proposed,
but never tested experimentally (Lisman et al., 2010). In addition,
these findings are particularly interesting in light of recent studies
demonstrating reduced functional connectivity between the mPFC
and thalamus in patients with schizophrenia (Anticevic et al., 2014),
a measure that can differentiate psychotic and nonpsychotic patients
(Anticevic et al., 2015). This suggests that targeting thalamic regions
that are interconnected in humans with both the mPFC and the
hippocampus could represent an effective approach for developing
novel treatments for psychotic disorders, including schizophrenia.
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