Associative learning of food cues that link location in space to food availability guides feeding behavior in mammals. However, the function of specific neurons that are elements of the higher-order, cognitive circuitry controlling feeding behavior is largely unexplored. Here, we report that hippocampal dopamine 2 receptor (hD2R) neurons are specifically activated by food and that both acute and chronic modulation of their activity reduces food intake in mice. Upstream projections from the lateral entorhinal cortex (LEC) to the hippocampus activate hD2R cells and can also decrease food intake. Finally, activation of hD2R neurons interferes with the encoding of a spatial memory linking food to a specific location via projections from the hippocampus to the septal area. Altogether these data describe a previously unidentified LEC > hippocampus > septal higher-order circuit that regulates feeding behavior.

The hippocampus is also known to encode an engram of high valence, contextual experiences including footshocks delivered in a specific context (). As termed by Semon more than 100 years ago, an engram refers to a (presumed) physical, biological alteration in the brain that occurs after a specific experience, thus encoding a memory of that experience (). Semon suggested that changes in the activity or other aspects of the function of specific neuronal populations after high valence experiences leads to the formation of a memory of that experience (engram). An animal’s ability to associate food with a particular location, i.e., construct an engram of that experience, is critical for efficiently finding food and conserving energy. We hypothesized that the hippocampus might also respond to food similarly to the way it responds to cues associated with other experiences such as footshocks (). We thus set out to determine whether the presentation of a meal could activate specific hippocampal cells, so we could then evaluate whether those neurons play a role in feeding behavior and memory. Toward this end, we used PhosphoTrap to molecularly profile hippocampal neurons that were activated by food. This unbiased profiling method revealed that dopamine 2 receptor neurons in the hilar and CA3 region of the hippocampus (hD2R) are food-responsive and that the selective modulation of the activity of hD2R neurons reduced food intake. Consistent with the effect to decrease food intake, activation of these neurons also interfered with the encoding of a memory for the spatial location of food via specific projections to the septal area (SA). hD2R neurons are regulated by inputs from the lateral entorhinal cortex (LEC) thus identifying a neural circuit from LEC > hippocampus > septal that regulates food intake.

While this suggests that “cognitive” centers can regulate feeding behavior, knowledge of the neural mechanisms responsible for the modulation of feeding by the hippocampus is incomplete, and the role of specific neural populations in these processes has not been fully elucidated. Thus, the molecular identification of specific ensembles of neurons that respond to food would further enable a functional analysis of a top-down circuit that processes sensory information to modulate food intake.

The role of the hippocampus in episodic memory and spatial location in rodents and humans is well established, and lesions in this brain region can result in memory loss (). The hippocampus is divided into discrete anatomic regions that serve several functions including the specification of location in space and time and the memory of an unpleasant (aversive) experience (). Recently, several groups have also shown that hippocampal dysfunction can alter feeding behavior (). For example, impairments in a variety of complex mental representations due to poor memory () and deficits in the regulation of satiety have been reported in patients diagnosed with retrograde amnesia (). In addition, it has been shown that imagining food can induce physiological changes, such as the production of gastric juices and salivation () and that this imagined consumption can decrease food intake in humans through habituation (). Finally, a recent report has shown that a projection from CA1 to the nucleus accumbens is required for encoding a reward-place memory ().

What causes humans to begin and end a meal? A role for memory for what has been eaten, as evidenced by a study of multiple meal eating in amnesic patients.

Homeostatic control of energy balance is vital for the survival of all animals. Specific populations of neurons in the brain control food intake and energy balance by integrating a panoply of relevant sensory and hormonal signals, thus orchestrating an adaptive behavioral response (). Defined neural populations in several brain regions, including the hypothalamus, midbrain reward centers (), parabrachial nucleus, amygdala, prefrontal cortex, dorsal raphe nucleus, and others, have all been shown to control appetite (). In addition, mutations in genes that impact the function of some of these neuronal populations are associated with obesity or anorexia (). However, the neural processes by which these and other brain regions integrate relevant, previous experiences and environmental cues to regulate feeding and enabling an animal to efficiently locate food are largely unknown.

Finally, in order to assess whether the ability of hD2R neurons to diminish encoding of a memory is specific for food, we performed a different non-appetitive, spatial memory task or object location task ( Figure 6 D). In this experiment, animals are trained in a cage with two similar objects. Then after 24 h, the location of one of the objects was changed, and the animal’s ability to discriminate between the old and changed object is assessed by monitoring the amount of time the animals spend in the quadrant to which the object had been moved. Notably, activating hD2R neurons during training has no effect on the encoding of the spatial memory assaying in this task (test, discrimination index of hM3Dq mice was 0.63 ± 0.17, p < 0.05). These data show that the circuit we identified is specific for the modulation of food-place associations in mice.

Finally, we tested the role of the projection of hD2R neurons from hippocampus to SA on this memory task using optogenetics. An AAV expressing a cre-dependent ChR2- or Arch3.0-YFP was injected into the dorsal hippocampus of Drd2-cre mice, and optical fibers were placed above the SA ( Figures 6 B and 6C). We tested the role of this hD2R-SA projection by turning the lasers ON only during the training session (training; Figures 6 B and 6C). We then tested during the OFF epochs whether this affected the time animals spent in the quadrant of the chamber where the food had been previously located (test). We found that activation of hD2R-SA projections during the training reduced the time animals spent, during the test session, in the quadrant of the chamber where the food had been located ( Figure 6 C, test, cf. ChR2 and YFP, yellow bars and blue bars and discrimination index of 0.48 ± 0.19). However, inhibition of this projection during the training session did not affect discrimination during the test session ( Figure 6 B, test panel, cf. Arch3.0 and YFP, yellow bars and blue bars, p < 0.01 and discrimination index of 0.68 ± 0.16). It is important to note that, as mentioned previously, the training period (when food is available to fasted animals) lasts only 5 min and that food consumption is negligible (<0.01 g) during this period for both the control animals and those in which hD2R neurons were activated or inhibited during training. Thus, both groups consumed similarly minimal amounts of food and neither group received a reward. The data further suggest that hD2R activation disrupts this association, which is consistent with the reduced food intake and negative valence that is seen after hD2R activation ( Figure S13 ).

Consistent with this possibility, we found that chemogenetic activation of hD2R neurons during training in hM3Dq-expressing mice reduced the amount of time that trained mice spent in the quadrant that previously contained food ( Figure 6 , test, hM3Dq, cf. yellow bars with blue bars, no statistical significance and discrimination index of 0.42 ± 0.2 on average). Injection of CNO had no effect on the time spent in this quadrant during the training session when food was present ( Figure 6 , training), and injection of CNO in control or in hM4Di animals also had no effect on this task ( Figure 6 , test, YFP and hM4Di). Activating or inhibiting hD2R neurons also failed to induce anxiety behavior or alter locomotion as shown using an elevated plus maze ( Figure S15 A) and an open-field test ( Figure S15 B).

Our findings that the state of activation of hD2R neurons is altered by sensory cues associated with food and that these neurons can regulate food intake raised the possibility that these neurons might play a role in the encoding of a memory that links food to a specific context. To investigate this, we tested whether manipulating the activity of hD2R neurons during the encoding of a memory associating food and spatial location could modulate a subsequent behavior (indicative of an effect on the formation of that memory). In these studies, we employed the same novel task described earlier (see Figure 2 A). In this protocol, fasted animals are placed in a chamber (to which they have been habituated) with food present in one of the quadrants (training). When animals receiving food during this training interval are later placed in the same chamber but without food, they spend significantly more time in the quadrant where the food was previously located (see Figure 2 A). We thus asked whether modulation of the activity of hD2R neurons during the training period altered the time animals subsequently spent in the quadrant where the food had been previously placed ( Figure 6 ).

(D) Schematic drawing of object location task performed in cre-dependent DREADD- or YFP-injected Drd2-cre mice. Middle: the discrimination index during training. Right: discrimination index during testing. O1 and O2 depicts similar objects, OO depicts old object, and CO depicts changed object. Paired Student’s t test, ∗ p < 0.05 and ∗∗ p < 0.01; n = 10. Data are represented as mean ± SEM.

(C) Left: schematic drawing showing Drd2-cre mice that were injected in the dorsal hippocampus with AAV5-Ef1a-Dio-ChR2-YFP. Right: the schematic drawing of the food location task. Middle: discrimination index of YFP- or ChR2-injected mice during the training session. Right: discrimination index of YFP- or ChR2-injected mice during the test session. Paired Student’s t test, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; n = 8.

(B) Left: schematic drawing showing Drd2-cre mice that were injected in the dorsal hippocampus with AAV5-Ef1a-Dio-Arch3.0-YFP. Right: the schematic drawing of the food location task. Middle: discrimination index of YFP- or Arch3.0-injected mice during the training session. Right: discrimination index of YFP- or Arch3.0-injected mice during the test session. Paired Student’s t test, ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001; n = 7–10.

(A) Left: schematic representation of a food location task performed in DREADD- or YFP-injected mice. Middle: discrimination index of YFP-, hM4Di-, or hM3Dq-injected mice during the training session. Right: discrimination index of YFP-, hM4Di-, or hM3Dq-injected mice during the test session. Paired Student’s t test, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001; n = 10–15.

The function of the hD2R to SA projection was tested by injecting an AAV with cre-dependent expression of either ChR2-YFP or Arch3.0-YFP into the dorsal hippocampus of Drd2-cre mice and placing optical fibers above the SA, leading stimulation of axonal fibers there ( Figures 5 E–5J), which includes the MS but also a lesser number of hD2R fibers that project to the LSD and the diagonal band of broca (DBB). Drd2-cre mice injected with a cre-dependent AAV expressing YFP alone were used as controls. Arch3.0 and ChR2-expressing mice were habituated to patch cables, and food intake was measured using a 1-h OFF-ON-OFF feeding session ( Figures 5 C and 5D, respectively), in which food intake was measured after each 20-min session. Immunohistochemistry for YFP and electrophysiology confirmed an effect of ChR2 and Arch3.0 expression on hD2R neural activity ( Figure S12 A). We found that in fed mice, inhibiting the hD2R projections to the SA increased food intake ( Figure 5 C, red symbols, p < 0.05), while activating this circuit in fasted mice reduced food intake during the ON epochs ( Figure 5 D, red symbols, p < 0.05). No effect was observed during the OFF epochs or in control mice ( Figure 5 C and 5D, black symbols). These experiments used 10-Hz stimulation in ChR2-expressing mice, but a decreased food intake was also observed using lower frequencies (1 and 5 Hz; Figure S12 B, cf. blue bars with white bar). We also assayed the role of this hD2R-SA circuit using a real-time place preference (RTPP) task in ChR2-YFP-expressing Drd2-cre mice ( Figure S13 ). We found that, during the test, mice avoided the light-paired side of the RTPP chamber but only when fasted ( Figure S13 , p < 0.05). This shows that hD2R-SA stimulation is associated with negative valence, but this effect is only observed when the animals are hungry. Optogenetic activation of this projection failed to alter behavior in an open-field test in all experimental groups suggesting that this circuit does not affect anxiety or total locomotor activity ( Figure S14 ). During and after optogenetic activation of the neurons, the mice failed to show any abnormal behavior, and we did not see evidence for seizures, digging, or changes in motor activity or arousal (data not shown). These data suggest that hD2R neurons can reduce food intake in hungry animals via projections to the SA.

We further confirmed these septal projections by injecting a AAV-CAG-GFP, a retrograde viral tracer (), into the middle of the SA (which includes the MS and the LSD) of Drd2-mCherry mice ( Figure 5 B). We observed bilateral labeling of hD2R cells with GFP 2 weeks post-injection, confirming that hD2R neurons project to these septal regions ( Figure 5 C, overlay, on average 4+ neurons per hippocampus or 13.3% of D2Rneurons). Finally, we used a cre-dependent herpes virus (HSV-lsl-GFP) as an anterograde polysynaptic transneuronal tracer, to map the circuit from hD2R neurons to the SA ( Figure S10 Table S6 ). We found that a majority cells in the MS were GFP+ (an average of 13 cells/slice and a total of 80 neurons/mouse). We also found GFPcells in other nuclei, such as LSD and VDB/HDB (an average of 6 cells/slice in the LSD, 11 cells/slice in the VDB, and 3 cells/slice in the HDB). These data corroborate our previous experiments thus defining a synaptic connection between hD2R and septal neurons mostly to the MS, but also to the LSD and VDB/HDB. A full quantification of the GFP-expressing cells in the whole brain in this experiment is shown in Table S6 . To analyze the possible function of this projection, we injected Drd2-cre mice in the hilar region with an AAV containing activatory DREADDs (hM3Dq) and analyzed c-fos immunoreactivity in the SA (MS and LSD) after neural activation. An increase in c-fos expression in the LSD and MS was observed after CNO injection, while this change was not observed in control animals ( Figure S11 , p < 0.001 for LSD and p < 0.05 for MS).

In order to map the outputs of the hD2R neurons, we injected an AAV expressing a cre-dependent YFP into the hilar region of Drd2-cre mice ( Figures 5 A and S8 ) and analyzed YFP expression in brain sections. Images of Drd2-YFP brains revealed dense intrahippocampal projections of hD2R neurons, primarily originating from a hilar population, to the granule cell region of the DG ( Figure 5 A, first panel; Figure S8 , last two on the bottom). We also observed strong projections of hD2R neurons to the medial septum (MS) (see Figure 5 A, second panel; Figure S8 , first two panels) but also weaker projections to the dorsal lateral septum (LSD) (see Figure 5 A, third panel; Figure S8 , first two panels and fourth panel), and the horizontal and vertical limbs of the diagonal band of broca (HDB/VDB) (see Figure 5 A, last brain panel; Figure S8 , first and third panel). YFP expression was not seen in the entorhinal cortex or other cortical regions ( Table S4 ), suggesting that the LEC connection to hD2R cells is unidirectional. Labeling of hD2R neurons also revealed only a very weak projection to the hypothalamus ( Table S4 ) raising the possibility that the control of food intake by hD2R neurons is unlikely to be conveyed directly through the hypothalamus. In addition to mapping axonal projections, we also injected Drd2-cre mice with an AAV containing a cre-dependent synaptophysin-VenusGFP to map presynaptic terminals in the whole brain ( Figure S9 Table S5 ). A large number GFPterminals were found in MS but also in the LSD and DBB (both HDB and VDB), supporting our findings that hD2R neurons project to the SA (including the MS and LSD) and the basal forebrain (VDB/HDB).

(D) Left: schematic drawing showing Drd2-cre mice that were injected in the dorsal hippocampus with AAV5-Ef1a-Dio-ChR2-YFP. Right: schematic drawing of the 1-h OFF-ON-OFF feeding session and the measurement of food intake (g) during the 1-h feeding session. Two-way ANOVA with Bonferroni correction, Time × Group, F(2,20) = 8.13, p = 0.002; Control × Subject, ∗ p < 0.05, n = 6. Data are represented as mean ± SEM.

(C) Left: a schematic drawing showing Drd2-cre mice that were injected in the dorsal hippocampus with AAV5-Ef1a-Dio-Arch3.0-YFP Right: schematic drawing of the 1-h OFF-ON-OFF feeding session and the measurement of food intake (g) during the 1-h feeding session are shown. Two-way ANOVA with Bonferroni correction, Time × Group, F(2,20) = 9.81, p = 0.018; Control × Subject, ∗ p < 0.05, n = 6.

(B) Schematic representation of retrograde tracing using retroAAV-CAG-GFP injected in the septal area (SA) of Drd2-mCherry mice. Middle: images of GFP + cells that are also Drd2 + in the Drd2-mCherry mouse hippocampus and quantification of Drd2 + /GFP + cells per slice per hippocampus are depicted. Scale bars, 200 μm.

(A) Drd2-cre mice were injected with AAV5-Ef1a-DIO-YFP, and brain slices were stained with anti-GFP. Brain annotation of axonal projections were done using as reference the annotated areas in the stereotaxic atlas by: medial septum (MS), dorsal lateral septum (LSD), and horizontal limb of the DBB (HBB).

To test whether these neurons were able to modulate hD2R neurons activity, we injected the LEC with an AAV containing an activating DREADD under the control of a CamkIIa promoter and analyzed c-fos expression in Drd2-YFP neurons in the hilus. We found that an average of 6 c-fosneurons per hilus per slice or 20% of hD2R neurons per slice showed activation subsequent to the LEC activation ( Figure S7 D). We then tested whether LEC neurons can also modulate feeding behavior by injecting an AAV expressing ChR2 under the control of the CamkIIa promoter into the LEC of wild-type mice. Optical fibers where then implanted above the DG to assay the effect of activating projections from LEC to hippocampus ( Figures 4 G–4J, S7 B, and S7C). The food consumption of control and ChR2-injected mice was tested after local light exposure in a 1-h OFF-ON-OFF protocol in which food intake was measured after each session ( Figure 4 H). Optical stimulation of LEC-DG projections decreased food consumption in fasted mice during the ON epochs, but not during the laser OFF epochs or in control mice ( Figure 4 I, red symbols, p < 0.05). These data show that CamkIIaLEC neurons activate hD2R neurons and reduce food intake.

The hippocampus is known to receive inputs from several brain regions that modulate its function (). To identify brain regions that project to hD2R neurons, we injected, pseudorabies virus (PRV), a retrograde tracer, expressing a cre-dependent tdTomato reporter into the dorsal hippocampus of Drd2-cre mice (PRV-lsl-tdTomato; Figures 4 A–4D;). This virus propagates retrogradely and serially labels presynaptic neurons (). Using this method, we found that hD2R neurons receive inputs from contralateral neurons of the dorsal hippocampus ( Figure 4 C, on average 28 tdTomatocells per hilus and DG, n = 3; Table S3 ) with dense inputs from neurons in the superficial and middle layers of the LEC as well as a sparse population of neurons in the perirhinal and ectorhinal cortices (164 tdTomatoneurons per LEC, n = 3, Figure 4 D; Table S3 ). To confirm this, we injected an anterograde tracing herpes simplex virus (HSV) () into the LEC of Drd2-YFP mice and found co-localization of YFP and HSV-tdTomato ( Figures 4 E and 4F, on average 17 tdTomato/YFPneurons per hippocampus or 56.6% of hD2Rneurons, n = 3). Finally, we used a cre-dependent rabies virus as a cell-specific monosynaptic tracer to confirm that neurons in the LEC project to hD2R neurons ( Figure S7 A, on average of 14 YFPneurons per slice in the LEC, n = 4).

Acute inhibition of hD2R neurons increased food intake ( Figure 3 B, red bars, p < 0.01 in 8 h and p = 0.05 in 24 h versus controls) as did chronic inhibition of these neurons ( Figure 3 C, left, p < 0.001 in day 4 and p < 0.01 in day 5). In contrast, chemogenetic activation of hD2R neurons decreased food intake acutely ( Figure 3 B, right, p < 0.01 in 8 h and p < 0.001 in 24 h versus controls), and chronically ( Figure 3 C, right; p < 0.001 in day 5 and in day 6). Saline injection to DREADD-expressing Drd2-cre mice or CNO injection in mCherry-expressing Drd2-cre controls did not significantly alter food intake ( Figures S6 H and S6I and 3B and 3 C, black bars and symbols, respectively). Optogenetic modulation of hD2R neuronal activity in a 20-min feeding session also showed the same effects as seen in the chemogenetic study ( Figures S6 J–S6L, p < 0.05). These data show that hD2R neurons respond to sensory appetitive cues and modulate food intake in mice.

We tested the function of hD2R neurons by bilaterally injecting adeno-associated viruses (AAVs) containing either a cre-dependent activating DREADDs (hM3Dq; Figure 3 , blue bars) or an inhibitory DREADD (hM4Di; red bars) into the hilar region of the hippocampus of Drd2-cre mice. Food intake was measured acutely ( Figure 3 B, 24 h, acute) and chronically (8 days, chronic) after an i.p. injection of either CNO or saline. hD2R neurons have a baseline firing rate of 1–2 Hz, and in control studies we found that application of CNO in hD2R neurons expressing hM3Dq induced neuronal depolarization and increased their firing rate to 2–8 Hz, while hD2R neurons expressing hM4Di were hyperpolarized by CNO and fired at a rate of <1 Hz ( Figures S6 A–S6D). Mice receiving the inhibitory hM4Di showed decreased c-fos expression in hD2R neurons after CNO injection ( Figures S6 E and S6F), while CNO increased c-fos in mice in hD2R neurons transduced with the activating hM3Dq DREADD ( Figure S6 G). We next tested the effect of these treatments on food intake.

(C) Left: chronic food intake (g) after 3 days of saline injection, 3 days of CNO injection (i.p; 1 mg/kg), and 2 days of saline injection in hM4Di-injected mice (red lines; n = 11) and mCherry-injected mice (black lines; n = 11). Two-way ANOVA with Bonferroni correction, Time × Group, F(7, 133) = 2.87, p < 0.01; Control × Subject, ∗∗∗ p < 0.001 and ∗∗ p < 0.01. Right: chronic food intake (g) after 3 days of saline injection, 3 days of 12/12 h CNO injection (i.p; 1 mg/kg), and 2 days of saline injection in hM3Dq-injected mice (blue lines; n = 20) and mCherry-injected mice (black lines; n = 12). Two-way ANOVA with Bonferroni correction, Time × Group, F(7, 210) = 2.04, p = 0.05; Control × Subject, ∗∗∗ p < 0.001. Data are represented as mean ± SEM.

(B) Left: acute cumulative food intake (g) after a single injection of CNO (i.p; 1 mg/kg) in hM4Di-injected mice (red bars; n = 11) and mCherry-injected mice (black bars; n = 15). Two-way ANOVA with Bonferroni correction, Time × Group, F(4,96) = 4.80, p < 0.001; Control × Subject, ∗∗ p < 0.01. Acute (right) cumulative food intake (g) after a single injection of CNO (i.p; 1 mg/kg) in hM3Dq-injected mice (blue bars; n = 10) and mCherry-injected mice (black bars; n = 10). Two-way ANOVA with Bonferroni correction, Time × Group, F(4,68) = 2.57, p < 0.05 Control × Subject, ∗∗ p < 0.01 and ∗∗∗ p < 0.001.

As mentioned, in the comparisons between the Context (control) and Context+Food (experimental), the animals were provided with food for only 5 min ( Figure 2 A). The limited food intake during this brief interval is not sufficient to induce satiety (or reward) in fasted mice, and we thus considered the possibility that hD2R neurons might be activated by a sensory cue associated with food rather than by the caloric intake. We next tested whether these neurons are regulated by visual or olfactory stimuli emanating from the food in the cups. To test a possible effect of smell, we placed fasted mice into a chamber (to which they had been habituated) with either an empty, clean glass jar (control) or an empty glass jar that had been previously filled with chow (smell). After the chow is removed, there is a residual odor allowing us to assess the role of olfactory cues. To test for visual cues, we enclosed food pellets in a tightly sealed glass jar (to block release of odorants) ( Figure S5 B). Mice are able to discern familiar shapes and visual cues (), and this enabled us to test whether the animals are able to respond to the (familiar) shape of pellets from their home cage. In this manner, we quantitated c-fos immunoreactivity in hD2R-positive cells in response to the olfactory and visual stimuli (Drd2-YFP; Figure S5 B). While the presence of an empty, clean jar failed to induce c-fos expression in hD2R neurons ( Figure S5 B, middle, control), the sight of food significantly induced c-fos expression in hD2R neurons ( Figure S5 B; middle, sight, p < 0.05). Although not significant, a positive trend was observed in mice exposed to the smell of food ( Figure S5 B, middle, smell, p = 0.3). These data suggest that hD2R neurons can be activated by visual cues and possibly olfactory cues associated with the presence of food. These data prompted us to evaluate whether these neurons could in turn modulate food intake and possibly modulate the association of sensory cues to food location.

Prior reports have shown that hD2R neurons are glutamatergic and thus might represent a subset of the CamkIIaneurons that we showed regulate food intake (). We confirmed this by co-injecting an AAV5-Ef1a-DIO-YFP and AAV5-CamkIIa-mCherry in the dorsal hippocampus of Drd2-cre mice and analyzing the co-localization of Drd2-YFP and mCherry (CamkIIa) in the injected area ( Figure S4 A). Because our previous data showed that Drd2 mRNA is significantly enriched in mice exposed to food compared to a novel environment (see Figure 2 I), we assayed the levels of c-fos expression in hD2R neurons in fed and fasted animals ( Figure S4 B). We found that c-fos expression in hD2R neurons is increased in fed relative to fasted mice. Since overnight fasting has been shown to alter water intake and cortisol levels in mice, we also analyzed c-fos expression in hD2R neurons in response to water deprivation and a single corticosterone injection (530 nM; Figure S5 A;). Although no significant effect was observed, there was a trend suggesting that corticosterone can activate hD2R neurons. We also observed that the hD2R-positive neuronal population in the hilar region is composed of both hilar mossy cells and CA3c cells (), and the activity of both cell types are modulated by nutritional changes.

The activation of Drd2neurons in the Context+Food group was further confirmed using dual immunohistochemistry for pS6 expression in a Drd2-cre mouse injected in the hilar region with a virus carrying a cre-dependent mCherry reporter (Drd2-mCherry). Co-localization of Drd2-mCherry and pS6 in the hippocampus was significantly increased in the Context+Food group compared to the Context group ( Figure 2 J; two-way ANOVA, p < 0.01), while differential expression of pS6 in Context+Food versus Context was not seen in mCherry-negative cells ( Figure 2 J; non-Drd2; two-way ANOVA, p = 0.38). This shows that Drd2 neurons are activated by the presence of food in the Context+Food group. We thus hypothesized that hippocampal Drd2neurons (hD2R) neurons might regulate food intake.

Homogenates of dorsal hippocampi were prepared from Context and Context+Food mice and immunoprecipitated (IP) with a phosphoS6-specific antibody, and the precipitated polysomal RNA was sequenced. The enrichment of each gene was calculated as the number of reads in the precipitated RNA relative to the total (IP/input [INP]; Figure 2 E). As expected, we found enrichment for activity-related genes (arc, fosB, and cfos; Figure S3 C) and a depletion of glial markers (gfap, aif1, and mal; Figure 2 F) in both samples. We next analyzed the RNA sequencing (RNA-seq) data to identify genes that were significantly enriched ∼2-fold or greater in the precipitated polysomes from hippocampal homogenates prepared from the Context+Food group versus the Context group (q value <0.05, Figures 2 G; Tables S1 and S2 ). We found enrichment for several markers for hilar neurons in the hippocampus including Drd2 (∼4.5 average fold; Figures S3 D, 2 G, and 2H, p = 0.005 and q value = 0.01). Drd2 was significantly enriched in the Context+Food experimental group (IP versus input; q value = 0.01) but not in animals in the context group (IP versus input; q value = 0.48) and naive (IP versus input; q value = 0.24) animals ( Figure 2 I, 4.5 average fold in Context+Food versus 1.5 average fold in Context versus 1.7 average fold in naive, p < 0.01). These results were confirmed using TaqMan analysis of Drd2 RNA in the pS6 IP samples from Context+Food animals (∼4-fold; Figure S3 E).

In order to identify molecular markers for neurons in the hippocampus whose activity had been altered by food presented in a specific context, we used PhosphoTrap, a method that enables molecular profiling of gene expression based on a change in the state of activation of specific neural populations (). This method uses an antibody to the phosphorylated S6 ribosomal protein to precipitate polysomes, and we began by performing immunohistochemistry for c-fos and pS6 in the control (Context) and experimental groups (Context+Food) as well as in naive, fasted mice that had not been placed in the chamber with the food cups ( Figures 2 B–2D and S3 A;). When compared to mice in the naive and Context groups, the Context+Food group showed increased numbers of hippocampal pS6-positive cells in CA3 and the hilar region ( Figures 2 B–2D and S3 A). While the background level of pS6 expression is higher in the hippocampus than in other regions, we observed that in the hilus nearly all of the c-foscells in Context+Food group also expressed pS6(87.8% of cells are c-fosand ps6 Figure S3 B) enabling the use of anti-pS6 to precipitate polysomes from these neurons (). The availability of molecular markers enables functional studies of the role of the specific neural subpopulations that are identified. The method makes use of the fact that the level of phosphorylation of the S6 ribosomal protein is highly correlated with the state of activation of neurons. Thus, after an acute or chronic stimulus RNAs that are enriched after precipitation of polysomes with a phospho-specific antibody to S6 mark cells that were activated (). Similarly, RNAs that are depleted after polysome precipitation mark cells that have been inhibited.

In order to test whether exposing fasted mice to food in a familiar context was capable of creating a memory, the experimental group was then subjected to the following test ( Figure 2 A, food location test). After training (i.e., placing fasted animals in a cage for 5 min with two cups, one of which contained food), the animals were fasted for 24 h and again placed in the familiar chamber for 5 min but now with two empty cups (test session). The amount of time spent in the quadrant where the food had been previously placed during training was then monitored. We found that, during the test period, mice spent a greater portion of their time in the quadrant that had previously contained the food-containing cup with a discrimination index of 0.64 ± 0.1 compared to the quadrant that previously contained the empty cup ( Figure 2 A, test, hatched yellow bars, p < 0.01). These data suggest that the mice had learned to associate a specific location with food and that a memory of this association had been established. We also monitored the time interval ( Figure S2 B, red lines) for this memory to disappear. We found that when the test was performed 72 h after the training (or longer), animals no longer spent a greater period of time in the quadrant where the food had been previously placed. Because our previous data indicated that state of activation of neurons in the hippocampus is altered by changes in nutrition and that glutamatergic neurons in the hippocampus can regulate food intake, we next set out to identify a specific hippocampal subpopulation(s) that play a role in these processes.

The hippocampus plays a key role in short-term memory and helps to construct a representation of an experience or a memory (). We hypothesized that the appetitive characteristics of food (appearance, taste, odor, or calories) might activate hippocampal neurons and construct a memory of that meal. We evaluated this possibility by devising a behavioral task in which food is associated with a specific spatial location in a familiar context ( Figure 2 A). In this novel paradigm, groups of experimental and control mice were exposed to a novel environment for 5 min (habituation). They were then returned to their home cage and fasted. After the fast, control mice were returned to the previous environment, and empty food cups were placed on opposite sides of the chamber. This group is referred to as “Context.” In the experimental group, mice were fasted overnight and placed in the environment (to which they were habituated), but, in this case, food was now placed in one of the two food cups in a consistent location while the other remained empty. This group is referred to as “Context+Food.” As expected, fasted mice placed into an environment with the empty food cups (Context) spent equal amounts of time in the quadrants containing the two cups. In contrast, in the experimental group given food (Context+Food), mice spent a significantly greater amount of time in the quadrant where the food-containing cup was placed, resulting in a discrimination index of 0.63 ± 0.1 ( Figure 2 A, right, Context+Food, blue bar, p < 0.05). The discrimination index is the time spent in the quadrant where the food cup was located divided by the sum of the time spent in the two quadrants in which the two cups were previously placed. The discrimination index (DI) used here has been previously employed in other memory tasks () and accurately reflects the performance of the task as animals spend little time in the two quadrants without cups ( Figure S2 A, white bars). In this test, food was provided for only 5 min, and the mice consumed a negligible amount of food during this interval (<0.01 g of food consumed). Thus, in this paradigm the mice were not satiated at the end of the training period, and the very limited amount of food consumption was likely to be insufficient to convey a reward.

(J) Schematic representation of the exposure of mice to food in a context (left). After, brains of Drd2-mCherry mice were stained for pS6 (right; green), and Drd2 + and non-Drd2 cells per slice per hippocampus were counted for each group; two-way ANOVA with Bonferroni correction, Drd2 Groups × Context Exposure, F(1,20) = 4.95, p = 0.03; Drd2 + Context × Drd2 + Context+Food, ∗∗ p < 0.01; n = 6. Scale bars, 125 μm. Data are represented as mean ± SEM,

(G) Plot depicting the average IP value and average input values (log2) of all genes analyzed. Enriched genes (>2-fold; red) and depleted genes (<2-fold; blue) are shown, and activation or target gene markers are shown: Drd2, Egr4, Vgf, and Hbb-b1 (n = 3). All genes depicted have q values <0.05 as calculated by Cufflinks.

(F) Average fold of change (IP/INP Log2) of Hbb-b1, Rbfox3, Gfap, Aif1, and Mal genes in Context (black bars) and Context+Food (white bars) (n = 3). Hbb-b1 is used as a protocol control (see STAR Methods ).

(B–D) Images showing c-fos (red) and pS6 (green) expression in the dorsal hippocampus of (B) naive mice, (C) Context mice, and (D) Context+Food mice. Overlay is shown in the second right panel, and overlay zoom of the hilar region is shown in the first right panel. Scale bars, 200 μm and zoom 125 μm.

(A) Left: schematic representation of food exposure in a context. Briefly, mice were habituated to an empty arena and fasted overnight. After 24 h, mice were exposed to empty food cups (Context; yellow) or to one food cup containing mouse chow pellets (blue, Context+Food). An additional day (test) was added to the Context+Food task enabling us to test memory acquisition and retrieval (food location test). Right: discrimination index after 5-min exploration time of empty (yellow bars), food-containing cups (blue bars), and cups that previously contained food but now were empty (yellow hatched bars). Paired Student’s t test, ∗∗ p < 0.01, ∗ p < 0.05; n = 5.

To determine whether hippocampal neurons are activated after food deprivation, we assayed the levels of the immediate early gene c-fos in the hippocampus of fasted and fed mice ( Figures 1 A and 1B ). After an overnight fast, several hippocampal areas, including CA1, CA3, dentate gyrus (DG), and the hilus, showed significantly lower levels of c-fos immunoreactivity compared to fed mice ( Figure 1 B). Based on this finding, we next evaluated whether manipulating the activity of hippocampal cells in mice could influence food intake ( Figures 1 C–1E). We targeted the inhibitory Gi- (hM4Di) or excitatory Gq-coupled (hM3Dq) designer receptor exclusively activated by designer drugs (DREADDs) to glutamatergic neurons in the hippocampus by injecting viruses expressing these constructs under the control of the CamkIIa promoter in wild-type mice ( Figure 1 C). As controls, we used mice bilaterally injected with a mCherry-expressing virus into the hippocampus ( Figure 1 C, right). We found that a single clozapine N-oxide (CNO) injection significantly increased food intake for 24 h in mice expressing the CamkIIa-hM4Di inhibitory DREADD in the hippocampus ( Figures 1 D, acute; and S1 B). Conversely, acute activation of hippocampal neurons expressing the activating CamkIIa-hM3Dq DREADD by CNO acutely decreased food intake ( Figure 1 D, right, acute; and Figure S1 C). Extended treatment of animals expressing the CamkIIa-hM4Di inhibitory DREADD in hippocampus with CNO for 3 days also led to a significant increase of food intake ( Figure 1 E, left, chronic). Chronic activation of these cells trended toward a decrease in food intake ( Figure 1 E, right, chronic). However, while significant differences between the groups treated with CNO for 3 days was seen using two-sample t tests ( Figure 1 E, right at days 4–6), these differences did not survive a post hoc Bonferroni adjustment. Saline injections in DREADD-expressing mice as well as CNO injection in mCherry controls did not alter food intake ( Figures 1 D and 1E, black bars and symbols). Overall, these data suggested that a glutamatergic subpopulation in the dorsal hippocampus can modulate food intake in mice.

(E) Left: chronic food intake (g) after 3 days of saline injection, 3 days of CNO injection (i.p; 1 mg/kg), and 2 days of saline injection in hM4Di-injected mice (red lines; n = 8) and mCherry-injected mice (black lines; n = 8). Two-way ANOVA with Bonferroni correction, Time × Group, F(7,105) = 11.14, p < 0.01; Control × Subject, ∗∗ p < 0.01. Right: chronic food intake (g) after 3 days of saline injection, 3 days of CNO injection (i.p; 1 mg/kg, 12/12 h), and 2 days of saline injection in hM3Dq-injected mice (blue lines; n = 9) and mCherry-injected mice (black lines; n = 8). Two-way ANOVA with Bonferroni correction, Time × Group, F(7,98) = 1.49, p < 0.18; Control × Subject; non-significant (ns). Data are represented as mean ± SEM.

(D) Left: acute cumulative food intake (g) after a single injection of CNO (i.p; 1 mg/kg) in hM4Di-injected mice (red bars; n = 8) and mCherry-injected mice (black bars; n = 8). Two-way ANOVA with Bonferroni correction, Time × Group, F(1,78) = 16.63, p < 0.0001; Control × Subject, ∗ p < 0.05 and ∗∗∗ p < 0.001. Right: acute cumulative food intake (g) after a single (light-blue bars) or double (dark-blue bars) injection of CNO (i.p; 1 mg/kg) in hM3Dq-injected mice (light- and dark-blue bars; n = 8) and mCherry-injected mice (black bars; n = 8). Two-way ANOVA with Dunnett correction, Time × Group, F(1,101) = 11.14, p < 0.0001; Control × Subject, ∗ p < 0.05 and ∗∗∗ p < 0.001.

Discussion

Higgs, 2016 Higgs S. Cognitive processing of food rewards. Mela, 2006 Mela D.J. Eating for pleasure or just wanting to eat? Reconsidering sensory hedonic responses as a driver of obesity. Feeding is a complex behavior in which a panoply of sensory inputs is processed to generate an adaptive behavioral response. The successful generation of an adaptive feeding response requires that an animal learn which sensory, spatial, and interoceptive cues are associated with food availability. Thus, a combination of stimuli that characterize food as safe or unsafe, rewarding or neutral, as well as its spatial location is required for the encoding of lasting memories of previous experiences of successful foraging (). However, the identity of neurons in top-down circuits that modulate feeding behavior through processing of these inputs are still largely unknown, as are the neural pathways through which they act. Because the hippocampus is known to play a key role in episodic memory and learning, we set out to establish the role of specific hippocampal neurons and their neural connections in food intake.

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Friedman J.M. Molecular profiling of activated neurons by phosphorylated ribosome capture. After showing that the activity of hippocampal neurons is sensitive to changes in nutritional state, we used PhosphoTrap, an unbiased RNA profiling method, to identify hippocampal neurons that are regulated by food (). We found that neurons in the hippocampus that express the dopamine 2 receptor (hD2R) are specifically activated by food and that activation of these neurons suppresses food intake even in mice that had been previously fasted. We further show that activation of these neurons diminishes an animal’s ability to associate food, but not an object, with a particular location. Finally, we show that hD2R neurons receive inputs from the LEC and send output to the SA to regulate food intake in mice.

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Tonegawa S. Creating a false memory in the hippocampus. Parent, 2016 Parent M.B. Cognitive control of meal onset and meal size: role of dorsal hippocampal-dependent episodic memory. The hippocampus is subdivided into several molecularly defined areas and is involved in diverse behaviors. Hippocampal cells are necessary for encoding time, location in space and episodic memories (). Thus, a specific ensemble of cells in the hippocampus can be activated by specific experiences (e.g., footshocks received in a specific context), creating an engram, or memory trace of that experience (). Indeed, it was shown that the cell in the hippocampus that encodes negative experiences (e.g., footshocks) can modulate the activity of other brain areas, such as the amygdala (). While previous pharmacological experiments have suggested that the hippocampus can also regulate food intake in rodents (), neither the identity of the neural populations responsible for this effect have been determined nor had the role of hippocampus in the acquisition of memories associating location with food.

+ population in the CA3c layer ( Scharfman, 1991 Scharfman H.E. Dentate hilar cells with dendrites in the molecular layer have lower thresholds for synaptic activation by perforant path than granule cells. We found that most D2R neurons in the hippocampus are localized in the hilar region, with a much sparser D2Rpopulation in the CA3c layer (). The activity of hD2R neurons are altered by sensory attributes of food primarily by visual and, to a lesser extent, olfactory cues ( Figure S5 B). We further found that hD2R neurons are silenced by fasting, and that their activation reduces food intake ( Figure 3 ). Activation of hippocampal D2R neurons decreases place preference raising the possibility that these neurons induce avoidance and possibly influence motivation. Thus, it is possible that hD2R neurons in the hippocampus decrease food intake by sensing food cues, thus altering the motivation to eat. Possible effects on motivation have not been tested and will be a subject for future investigation.

Sweeney and Yang, 2015 Sweeney P.

Yang Y. An excitatory ventral hippocampus to lateral septum circuit that suppresses feeding. In addition to hD2R neurons, we also found differences in c-fos expression in other hippocampal regions in fed versus fasted mice outside the hilar region, including CA3 and DG ( Figure 1 A). This suggested that other neural populations also respond to food. This is consistent with our finding that activation of glutamatergic CamkIIa-expressing cells in the hippocampus show a different temporal response and effect size than those seen after hD2R activation ( Figures 1 C–1E). These data suggest that while hD2R neurons represent a functionally important subset of hippocampal cells involved in feeding behavior, it is possible that other cell populations in the hippocampus also play a role to regulate food intake, such as ventral hippocampal vGlut2 cells (). Further studies will be required to identify and establish the function of these other populations.

+ LEC neurons are glutamatergic, further studies will be necessary to determine whether or not this is a direct effect. The entorhinal cortex is known to convey sensory information from gustatory, olfactory, and visual cortices ( Li et al., 2017 Li Y.

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et al. Identification of a brainstem circuit controlling feeding. Activity mapping using c-fos as a marker for neural activation showed that hD2R neurons can be activated by inputs from glutamatergic CamkIIa-expressing neurons in the LEC. While a direct excitatory effect is consistent with the fact that the CamkIIaLEC neurons are glutamatergic, further studies will be necessary to determine whether or not this is a direct effect. The entorhinal cortex is known to convey sensory information from gustatory, olfactory, and visual cortices (), and it too shows changes in its activity in response to changes in metabolic state (). As mentioned, we found that within the first minutes of exposure to food, hD2R neurons are activated by olfactory and visual stimuli raising the possibility that a population of CamkIIa-expressing neurons in the LEC might convey relevant sensory cues reflecting the availability of food to the hD2R neurons. Further studies will also be required to determine the molecular identity of these putative LEC neurons and whether they convey sensory and/or interoceptive cues to regulate feeding behavior.

Etter and Krezel, 2014 Etter G.

Krezel W. Dopamine D2 receptor controls hilar mossy cells excitability. While the neurons we identified express D2R, it is unclear whether dopamine signaling influences this circuit we identified. The dopamine receptor 2 expressed in striatum is a Gi-coupled receptor and thus dopamine signaling should decrease neuronal activity. However, in the hippocampus an application of a D2R agonist has been shown to increase mossy cells excitability (). This suggests that the signaling mechanisms downstream of the D2R receptor in hippocampal cells are different than those in striatum. hD2R neurons spontaneously fire in fed animals ( Figures S6 A–S6D) and are relatively silent during fasting periods ( Figure S4 B). Although it is not known what role dopamine plays to modulate the activation of the hD2R neurons, the presence of dopamine receptors on the hD2R neurons raise the possibility that they may also receive inputs from reward centers or possibly other populations of dopaminergic neurons.

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et al. A cholinergic basal forebrain feeding circuit modulates appetite suppression. Sweeney and Yang, 2016 Sweeney P.

Yang Y. An inhibitory septum to lateral hypothalamus circuit that suppresses feeding. Knox and Keller, 2016 Knox D.

Keller S.M. Cholinergic neuronal lesions in the medial septum and vertical limb of the diagonal bands of Broca induce contextual fear memory generalization and impair acquisition of fear extinction. We also characterized the functional outputs of hD2R neurons and found out that hD2R neurons decrease food intake by activating cells in the SA ( Figure 5 ). hD2R cells project to the posterior part of dorsal part of the LSD, MS, VDB, and HDB ( Figures 5 A and S8 ). Our studies using different viral approaches suggest that the MS receives the majority of hD2R projections. Both the MS and VDB/HDB have a large number of cholinergic neurons that have been reported to project to the paraventricular nucleus and the lateral hypothalamic area (LHA) to regulate feeding (). The MS and VDB/HDB have also been implicated in learning and memory (). While the evidence suggests that the MS is the major projection affecting food intake and memory processing observed in our study, the LSD and VDB/HDB may also contribute as well as fibers of passage that could be activated during the optogenetic studies. Future studies activating these specific projections will seek to identify and test the function of the specific populations in the SA and VDB/HDB that are activated by hD2R neurons.

Trouche et al., 2019 Trouche S.

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Dupret D. A hippocampus-accumbens tripartite neuronal motif guides appetitive memory in space. The ability to encode a robust memory denoting the location of food has important adaptive value because a stronger memory would increase the chances of successful foraging and minimize the loss of energy associated with inefficient foraging. Our data suggest the novel finding that the activation of hD2R neurons is associated with both a decrease of food intake and the strength of this food-place memory. This further suggests that, while hD2R neurons diminish this association, other pathways must reinforce it. Indeed, a recent report has shown that a dCA1-Nac pathway plays a role in encoding a food-place memory (). This raises the intriguing possibility that, similar to hypothalamus and other brain regions, paired populations in hippocampus exert opposing effects on food intake as well as the encoding of a food-place memory. Consistent with this possibility, we find projections of dCA1 to hD2R neurons ( Figure S7 A) raising the possibility that there might be cross talk between these two circuits.

Senzai and Buzsáki, 2017 Senzai Y.

Buzsáki G. Physiological properties and behavioral correlates of hippocampal granule cells and mossy cells. Danielson et al., 2017 Danielson N.B.

Turi G.F.

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Losonczy A. In vivo imaging of dentate gyrus mossy cells in behaving mice. While our data indicate that hD2R neurons and their projections play a role in encoding food-place associations, the mechanisms used by hD2R neurons to mediate this association need to be determined. While it is possible that hD2R cells receive both nutritional and spatial information, the effects observed after activating these cells could also reflect an alteration in the animal’s motivational state and influence learning or reward preference in the food location task. Another possibility is that hD2R cells activation influences spatial navigation as it has been shown that hilar mossy cells that also express D2R can encode multiple place fields () and undergo robust remapping in response to contextual manipulation (). One interesting possibility is that these hippocampal ensembles form a network that can couple the regulation of food intake to a food-place memory as part of a system that enables animals to embark on a particular behavior. However, in our study, we investigated whether manipulating hD2R activity influences the encoding of a memory in a food location test, and we have not tested manipulating activity during testing. Thus, possible changes in motivation might influence memory recall, and these possible effects on motivation and memory recall will be the subject of future studies.

In summary, we show that hD2R neurons are an important cellular component of an LEC > hD2R > septal circuit regulating food intake. hD2R cells sense nutritional state and regulate feeding behavior as well as a memory associating food with a specific location. A fuller understanding of the neural mechanisms by which high-order brain areas and cognitive processing control feeding behavior represent an important area for further inquiry. Our study provides an important starting point for this line of investigation.