Interaction between hypothalamic dorsomedial and the determines intensity of anticipatory behavior

Guadalupe Acosta-Galvana,1, Chun-Xia Yia,1, Jan van der Vlietb,1, Jack H. Jhamandasc, Pertti Panulad, Manuel Angeles- Castellanose, María del Carmen Basualdoa, Carolina Escobare, and Ruud M. Buijsa,2

aDepartamento de Biología Celular y Fisiología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, DF 04510, Mexico; eDepartamento de Anatomía, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, DF 04510, Mexico; bTytgat Institute Academisch Medisch Centrum, 1105 BK, Amsterdam, The Netherlands; cDepartment of Medicine (Neurology), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 2S2; and dNeuroscience Center, Institute of Biomedicine, University of Helsinki, 00014, Helsinki, Finland

Edited by William J. Schwartz, University of Massachusets Medical School, Worcester, MA, and accepted by the Editorial Board February 24, 2011 (received for review October 18, 2010) Food anticipatory behavior (FAA) is induced by limiting access to and redundant neuronal network in which even the participation food for a few hours daily. Animals anticipate this scheduled meal of genes does not seem essential (12, 13). In the present event even without the suprachiasmatic nucleus (SCN), the bi- study, we therefore examined the hypothalamic circuitry that is ological clock. Consequently, a food-entrained oscillator has been involved in FAA by using a combination of behavior with neu- proposed to be responsible for meal estimation. Recent ronal tracing, neuronal activity markers, and specific lesioning studies suggested the dorsomedial (DMH) as the techniques. Because daytime neuronal activity of the SCN and site for this food-entrained oscillator, which has led to considerable light-induced neuronal activity of the SCN are known to inhibit controversy in the literature. Herein we demonstrate by means of locomotor activity in nocturnal rodents (14, 15), we hypothesized c-Fos immunohistochemistry that the neuronal activity of the that during food restriction, to allow FAA during the light phase, suprachiasmatic nucleus (SCN), which signals the rest phase in the activity and influence of the SCN should be reduced. Because nocturnal animals, is reduced when animals anticipate the sched- the DMH is one of the structures with the most c-Fos activity uled food and, simultaneously, neuronal activity within the DMH during FAA (5, 8), we explored the contribution of the DMH– increases. Using retrograde tracing and confocal analysis, we show SCN interaction toward FAA in the rat as nocturnal rodent. We that inhibition of SCN neuronal activity is the consequence of show that the DMH has GABAergic projections to the SCN that activation of GABA-containing in the DMH that project to are active during FAA and reduce the activity of the SCN. We the SCN. Next, we show that DMH lesions result in a loss or show that a lesion of the DMH decreases that inhibition and diminution of FAA, simultaneous with increased activity in the SCN. leads to a highly active SCN, leading to reduction or disap- A subsequent lesion of the SCN restored FAA. We conclude that in pearance of FAA. The disappearance of FAA following a lesion intact animals, FAA may only occur when the DMH inhibits the of the DMH and its reappearance in the same animal following activity of the SCN, thus permitting locomotor activity. As a result, a lesion of the SCN supports the notion that it is indeed the SCN FAA originates from a neuronal network comprising an interaction that inhibits locomotor activity, thereby preventing FAA during between the DMH and SCN. Moreover, this study shows that the the rest phase in DMH-lesioned animals. DMH–SCN interaction may serve as an intrahypothalamic system to Consequently the present results show that FAA is modulated gate activity instead of rest overriding circadian predetermined by the interaction of the SCN and DMH and suggest that FAA is temporal patterns. generated by a neuronal network, of which the SCN and the DMH are two components. hysiology and behavior of all mammals is organized in an al- Pternating pattern of rest and activity cycles, whereby the en- Results and Discussion dogenous and light-induced daily neuronal activity of the DMH–SCN Interaction. We used rats as our experimental animal suprachiasmatic nucleus (SCN) signals rest in nocturnal rodents and initially explored neuronal activation during FAA by means and activity in diurnal primates, such as (1, 2). Restricting of c-Fos immunohistochemistry in the hypothalamus. We used c- food access to a short and predictable episode during the rest Fos as a neuronal marker to investigate which areas are phase changes this behavioral pattern, such that an animal activated during FAA in a restricted food protocol. C-Fos is becomes active and for up to several hours anticipates the up- generally accepted as a marker for neuronal activity based on the coming feeding event. This food anticipatory activity (FAA) is early experiments of Morgan and Curran (16, 17); the limitation even exhibited without the known circadian oscillator, the SCN of this technique is that certain neuronal systems may not show (3), and thus may rely on a different circadian pacemaker. c-Fos resulting from their activation. We observed, in agreement In search of the location of this so-called “food entrained with earlier studies (18, 19), that together with increased c-Fos in oscillator” (FEO), two recent studies have claimed that its po- sition is within the dorsomedial nucleus of the hypothalamus (DMH) (4, 5). The designation of the DMH as master clock for Author contributions: C.E. and R.M.B. designed research; G.A.-G., C.-X.Y., J.v.d.V., M.A.-C., M.d.C.B., and R.M.B. performed research; J.H.J. and P.P. contributed new reagents/ana- food entrainment is, however, controversial because some lytic tools; G.A.-G., J.v.d.V., and C.E. analyzed data; and G.A.-G., C.-X.Y., C.E., and R.M.B. groups reported unimpaired FAA despite large lesions of the wrote the paper. DMH (6, 7); others have shown that lesions of the DMH disturb The authors declare no conflict of interest. and diminish the intensity of FAA (6). The possible participation This article is a PNAS Direct Submission. W.J.S. is a guest editor invited by the Editorial of other brain structures in FAA is evident from studies that Board. demonstrate modulation of neuronal activity and induction of 1G.A.-G., C.-X.Y., and J.v.d.V. contributed equally to this paper. clock-gene rhythmicity in hypothalamic and limbic structures by 2To whom correspondence should be addressed. E-mail: [email protected].

– NEUROSCIENCE feeding schedules (8 11). Thus, it has been suggested that FAA This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. depends on a multioscillatory system comprised of a complex 1073/pnas.1015551108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1015551108 PNAS | April 5, 2011 | vol. 108 | no. 14 | 5813–5818 Downloaded by guest on September 24, 2021 the ventral DMH, neurons in the ventral part of the SCN de- from the (Fig. 1G). Both observations suggest that the creased their activity (Fig. S1). Following our hypothesis that at projections of the DMH to the SCN may interact with light input least one brain structure that is involved in food anticipation that is arriving from the retina and may modulate the activity of needs to interact with the SCN to inhibit its activity, we placed the SCN in that area. the neuronal tracer cholera toxin subunit B (CtB) into the SCN (n = 24). These injections, especially when placed in the ventral DMH Inhibits SCN Neuronal Activity During FAA. In view of the SCN (n =4)(Fig. S2A), revealed retrograde-labeled neurons in projections of the RFRP neurons to the ventral SCN (Fig. 1D), several hypothalamic areas, including the DMH (Fig. 1A). We and in view of the evidence that many neurons of the DMH observed that the CtB-positive neurons were present in the same contain the inhibitory amino acid GABA (23), we examined area of the DMH that showed c-Fos activation during food re- whether the RFRP projections from the DMH to the SCN may striction (Fig. 1 A and B). The next observation was that the contain GABA. Therefore, the presence of the enzyme for the distribution of the neurons within the DMH projecting to the synthesis of GABA, glutamate decarboxylase (GAD), was ex- SCN was similar to those neurons in the DMH that express Arg- amined in RFRP-positive fibers terminating in the SCN. Con- Phe-Amide-related (Fig. 1C), one of the five active focal microscopic analysis revealed that RFRP fibers terminating mammalian restricted-food amide (RFRP). This ob- in the SCN contained GAD, indicating an inhibitory role of this servation was further investigated using injection of the tracer DMH input to the SCN (Fig. 1 H and I). CtB in the SCN, resulting in retrograde-labeled neurons in the To investigate the relevance of this DMH–SCN interaction for DMH containing RFRP (Fig. 1D). Because RFRP peptides are FAA in 30 naive animals, we first established a baseline of FAA postulated to have a feeding-related functionality (20–22), we by a restricted-food protocol. Because naive animals show a large examined and demonstrated by means of double-labeling im- variation in FAA, we only chose 24 animals that showed clear munofluorescence and confocal microscopical analysis that some evidence of FAA. We divided these animals into three groups neurons with c-Fos labeling during FAA are also positive for (n = 24): (i) a control group that received a (sham) lesion of the RFRP (Fig. 1E). In addition, neurons were observed that dem- DMH; (ii) a group that received a bilateral electrolytic lesion of onstrated both c-Fos and CtB, which indicates that restricted the DMH; and (iii) a group that received bilateral kainic acid food activated neurons of the DMH projection to the SCN (Fig. lesions of DMH. After 20 d of the second restricted-food pro- S2B). Further confocal microscopy analysis showed that RFRP cess, rats were killed at the moment of food arrival to analyze fibers terminate exclusively in the ventral SCN. RFRP fibers c-Fos immuno-reactivity in the brain. Because c-Fos protein target vasoactive-intestinal peptide (VIP) and -releasing takes between 60 and 90 min to be expressed after the activation peptide neurons that are known to receive retinal input (Fig. 1F). of a (24), this time point reflects the neuronal activation neurons did not receive any input from RFRP during FAA, at least 60 min before expected food arrival. fibers. This observation was confirmed by labeling projections To compare the SCN neuronal activity in ad libitum-fed animals from the retina to the SCN, via injecting CtB in the vitreous of versus restricted food animals, we chose animals with the most the . This experiment showed that the distribution of RFRP accurate DMH lesion: four with kainic acid and four with thermic fibers in the SCN was observed coincident with the fibers arriving lesions versus four sham animals with the strongest anticipation.

Fig. 1. RFRP neurons in the DMH project to the retinor- ecipient region of the SCN, contain the enzyme for the in- hibitory GABA (GAD), and are activated during food anticipation. (A) Retrograde-labeled neurons in the ventral area of the DMH after an injection of CtB into the SCN. These neurons projecting to the SCN are present in the same ventral area of the DMH that is activated during food anticipation. The distribution of RFRP neurons in the ventral area of the DMH in A coincides with the area expressing c-Fos during FAA in B and coincides with the location of RFRP neu- rons in the DMH (C). (D) Retrograde-labeled neurons in the DMH after an injection of CtB (red) into the SCN, double- labeled for RFRP (green). (E) Confocal image of RFRP (green) -labeled neurons colocalizing with c-Fos (red) after food re- striction. (F) RFRP (green) innervation of the SCN in the area of VIP (red) cellbodies; G shows that this RFRP (green) input is closely associated with retinal fibers (red). H and I illustrate that in the SCN these RFRP (green) fibers colocalize with GAD (red), as is illustrated by the yellow color of green RFRP and red GAD fibers. The arrow indicates the same area in H and I. (Scale bars: A and B, 100 μm; C–F,50μm; G and H,25μm; I,10μm.)

5814 | www.pnas.org/cgi/doi/10.1073/pnas.1015551108 Acosta-Galvan et al. Downloaded by guest on September 24, 2021 The size and site of the lesions was documented and the successful obtain reduced FAA with electrolytic lesions than with kainic acid DMH lesions are presented in Fig. S3. Invariably, lesions that led lesions; interestingly, the lesion of the DMH needed to be small to to diminished or no anticipation covered the ventral and peri- let the anticipation disappear, which in our hands was easier by ventricular area of the DMH bilaterally, but lesions that did not a kainic acid lesion then by an electrolytic lesion. This finding could affect FAA were unilateral, were outside the DMH area, or cov- be because of the fact that an electrolytic lesion also destroys fibers ered a larger area than the DMH itself (Figs. S3 and S4). The four of passage, for example of the SCN to nearby target structures, and animals from each lesion group with the most accurate DMH that the kainic acid lesion does not kill all neurons in the injection lesions are presented in Fig. 2 and Fig. S3. Well-lesioned animals site, as became clear of the c-Fos activation of some sparse neurons did not show a significant FAA compared with their ad libitum located in the lesion site (Fig. S6). The differential effects of both activity, but sham-lesioned animals showed increased FAA before lesion strategies may explain why such controversy in the literature food arrival compared with their own ad libitum activity (P < exists concerning the effect of the lesion of the DMH with respect 0.001) (Fig. S5). In addition, only the sham group showed a de- to FAA (25, 26). In general, it can be stated that in studies dem- creased c-Fos activity in the SCN compared with intact ad libitum onstrating little or no diminishment on FAA after lesions of the animals (Fig. 2 and Fig. S5). In general, it was more difficult to DMH, the lesions encompassed the DMH completely, as well as

Fig. 2. Diminished food anticipatory behavior after DMH lesion concurs with high neuronal activity in the SCN. (Top)Two double-plotted actograms of locomotor activity illustrate the initial baseline food anticipation (food pre- sented in the time of the translucent rectangle) and the subsequent loss of this anticipatory activity after an electrolytic (Left) or neurotoxic (Right) lesion of the DMH. The activity 2 to 3 h preceding the translucent rectangle is taken as “food anticipatory activity.” (Middle) The neu- ronal activity by means of c-Fos staining in the SCN 5 h after light onset in an ad libitum control and in a sham restricted-food (RF) animal at the moment of food arrival. The DMH-lesioned animals electrolytic (RF-DMH-X EL) or kainic (RF- DMH-X KA) of the respective actograms above show as high c-Fos expression in the SCN of the ad libitum-fed animal. Only the sham-lesioned food-anticipating animal (RF-antic) showed less c-Fos in the SCN. The quantification of the number of c-Fos–positive neurons in the SCN and the quantification of the FAA for the 2 h before expected food arrival is given in Fig. S5. (Bottom) Waveform analysis of activity during the last 5 d of a restricted-food protocol of the two groups of DMH-X animals illustrating their 24-h activity in 10-min averages. The anticipa- tory activity to food (as percentage of total 24-h activity) after the lesion is diminished signifi- cantly. The vertical bar indicates the time (ZT5) of food delivery (5 h after light onset) and the horizontal black bar the dark (see Fig. S5

for the quantitative analysis of FAA). (Scale NEUROSCIENCE bars, 100 μm.)

Acosta-Galvan et al. PNAS | April 5, 2011 | vol. 108 | no. 14 | 5815 Downloaded by guest on September 24, 2021 parts of neighboring structures, thus often resulting in nearly ar- a restricted-food condition. DMH-X rats showed decreased or loss rhythmic animals (6, 7, 25). This loss of rhythmicity suggests that of FAA. Subsequently, the same animals received bilateral elec- with such large lesions, the SCN is unable to impose its rhythmicity trolytic lesions of the SCN (SCN-X). After recovery, the rats were to areas such as the perifornical area, the ventro lateral hypo- again submitted to a period of restricted food. The subsequent , and the thalamus (27, 28). Large lesions may either SCN-X completely reversed the DMH-X pattern and resulted in destroy these areas or disrupt the fibers of the SCN passing to these animals (n = 5) that, despite their two hypothalamic lesions, areas, and consequently in this condition the SCN cannot prevent strongly and significantly anticipated the scheduled meal time (Fig. locomotor activity during the daytime. The latter may also explain 3 and analysis in Fig. S9). The results also showed that the SCN why with kainic acid lesions in the DMH, it was easier to obtain does not need to be completely lesioned; a partial lesion that results a diminished FAA than with thermic lesions, and why in our hands in a twofold increase of daytime activity was sufficient to reinstate small lesions were doing better than bigger ones. When we ex- the initial baseline FAA. This phenomenon cannot be explained by amined the c-Fos staining in the SCN during the restricted-food a possible recovery of function of the DMH because animals with protocol, we observed that in the animals perfused at the time of double neurotoxic lesions of the DMH area show very little c-Fos, food arrival, the c-Fos immunopositive profiles (which reflects the which is similar to single DMH-lesioned animals when killed at the situation at least 1 h earlier) in both groups of DMH-X animals moment of expected food arrival (Fig. S6). Moreover, surgery or (n =2× 4) was as high as that in animals under ad libitum feed- retraining also does not explain the loss and reappearance of FAA ing conditions (n = 4). In contrast, only the SCN of sham food- because sham-lesioned animals or animals with lesions outside the anticipating animals (n = 4) showed a significant reduction of SCN did not show this loss and subsequent gain of function. (See SI c-Fos (Fig.2 and Fig S5) compared with all other animal groups Materials and Methods and Figs. S10–S12 for more details.) (P < 0.01). Furthermore, the same animals that endured a DMH lesion also showed a pronounced diminishment of RFRP inner- Hunt for the FEO. Since 1972 it has been recognized that the or- vation in the SCN (Fig. S7). These results support the hypothesis ganization of circadian activity in locomotor, hormonal, or au- that lesioning the DMH results in removal of RFRP/GABA neu- tonomic rhythms is the exclusive domain of the SCN because, rons and diminishes the inhibitory input to the SCN. after lesions confined to the SCN, all these overt rhythms dis- appeared (29, 30). When it was discovered that by giving food to FAA Is Lost After Lesion of the DMH but Reappears After a an animal once a day during its rest phase, the animal could Subsequent Lesion of the SCN. The fact that after a DMH lesion anticipate this event without the SCN (3, 31), a similar clock, the FAA disappears or is significantly diminished has led to the con- FEO, was proposed (32). Ever since then, scientists have tried to clusion that the DMH may harbor the FEO (4). However, the ascribe the location and function of such a FEO to a single present demonstration of an inhibitory input from the DMH to structure in the brain by lesioning different areas and examining the SCN suggests that the DMH may inhibit neuronal activity of the effect on FAA (33). Recently, following the observation of the SCN to permit FAA to occur. This emerging hypothesis was a high activation of c-Fos in the DMH (8), it was suggested that examined in a series of experiments, of which the first part is pre- the FEO resided in the DMH (4). Present results demonstrate sented in the SI Materials and Methods and in Fig S8. In the fol- that both the SCN and DMH are relevant but not indispensable lowing experiments the intensity of FAA was first established in for FAA, and that the DMH and SCN are just two of possibly intact rats under restricted-food conditions; this was followed by many structures that modulate FAA. We propose that the FEO a bilateral lesion of the DMH (DMH-X) with kainic acid. Rats consists of a network of interacting brain structures that is ini- were allowed to recover for 3 wk and were then tested again under tially driven by information from the periphery (Fig. S13).

Fig. 3. Loss and gain of food anticipation in the same animals bearing, first, a lesion of the DMH, followed by a lesion of the SCN. Both double-plotted actograms depict the activity of animals showing normal antici- patory activity just before the onset of meal delivery (translucent rectangle). This anticipatory activity is nearly completely lost after a kainic acid DMH lesion and returns when the same animal receives an addi- tional SCN lesion. The waveform analysis illustrates the average activity of the same five animals in intact conditions under restricted-food conditions (green line) (the moment of food delivery depicted by the black vertical bar), and illustrates the loss of FAA after DMH-X (blue line) and its return after a successive SCN- X (red line). The right part of the figure shows the injection site of the kainic acid and the section of the hypothalamus showing the lesion of the SCN. The right actogram shows the result of a partial (>70%) lesioned SCN animal that still recovers FAA. See Fig. S9 for the quantification of the activity 3 h before food arrival. (Scale bar, 200 μm.)

5816 | www.pnas.org/cgi/doi/10.1073/pnas.1015551108 Acosta-Galvan et al. Downloaded by guest on September 24, 2021 DMH: Hypothalamic Center of Circadian and Metabolic Integration. explanation as to why in early studies lesioning the ventro medial Early tracing studies examining the output of the DMH have shown hypothalamus resulted in diminished FAA, which recovered in that its projections are mainly limited to hypothalamic structures time (44, 45). We have not studied the effect of DMH lesion for (34). This observation, together with data showing that the DMH periods longer than 6 wk, but it is possible that over time, the receives an important input from the nucleus of the , strength of other inhibitory inputs to the SCN [e.g., from the ar- arcuate, and SCN (35–37), supports the view that the DMH is cuate nucleus (46)] may increase and thus result in recovery of essential for the distribution of metabolic and circadian signals FAA consequent to an increase in inhibitory input to the SCN. within the hypothalamus. The present results emphasize the im- Because we have observed a similar mechanism for arcuate–SCN portant role of the DMH because they show that, under normal interaction (47), we would like to propose that this is a general conditions, the DMH has the capacity to “silence” the SCN to al- feature for hypothalamic structures, allowing them to impose low locomotor activity at a moment of the day when such behavior adaptive changes depending on the requirements of the body. is normally prevented by the activity of the SCN. This finding In conclusion, the present study shows that the FEO may explains why DMH lesions could result in an absence of FAA. depend on an oscillating neuronal network that consists of dif- ferent cell groups in different locations in the brain with or SCN and FEO Multioscillatory Structures. There is no doubt that without the active participation of clock genes (12, 42, 48) (Fig. FAA stems from activity within the brain, although the initiation S13). The present study does not identify all of the structures of this activity may arise from peripheral oscillating or stimulating that may participate in food entrainment, but it is likely that in processes (38–40). All in all, the appearance of c-Fos or PERIOD addition to a number of core structures, such as the SCN, DMH, immunoreactivity in many brain areas after food restriction and the , nucleus of the solitary tract, and para- the failure to prevent FAA by lesioning different brain areas (13) brachial nuleus (33), a large number of other brain structures suggests that the organization of the oscillatory nature of FAA may participate and provide input to this system. Moreover, this should be within a system of interacting brain structures. Perhaps study shows that the DMH–SCN interaction may serve as an the most surprising outcome of the present study is that although intrahypothalamic system modulating behavioral activation at the DMH was seen as an excellent candidate for distributing the hours when the SCN signals and rest. signal of food entrainment, the present results show that even after DMH-SCN lesions, FAA is not diminished, and in some Materials and Methods cases, even enhanced. The present experimental data clearly See SI Materials and Methods for details on animal handling and experi- demonstrated that other structures are involved and are sufficient mental procedures. to induce FAA. In intact animals, the interaction between the SCN and the DMH may be essential for the expression of FAA, as Experiment 1. Analysis of c-Fos and RPRF in the DMH after FAA; relation with the is apparent following lesions of the DMH. These lesions, the SCN. One week before the food restriction, animals (24) received a stereotaxic resulting reduction of FAA, and the concomitant increased c-Fos injection of CtB aimed to the SCN. At day 14 of the food-restriction protocol, expression in the SCN indicate that indeed the DMH inhibits the animals were killed at the moment the food would normally have been presented and the were examined for the localization of c-Fos, CtB, SCN to allow locomotor activity. Consequently, the earlier con- and RPRF. Four animals had a successful injection into the ventral SCN. clusion that the SCN does not play a role in FAA needs to be reconsidered (see also ref. 41). The present study demonstrates Experiment 2. Analysis of the RPRF innervation of the SCN. Animals (n =5)re- that the SCN will normally prevent FAA. In fact, the SCN par- ceived an injection of CtB in the vitreous of the eye under brief ether an- ticipates by shaping a window for FAA, as is evident from animals esthesia. One week after this injection, animals were perfused with with SCN lesions that show longer and more intense FAA than paraformaldehyde and the SCN examined for RPRF and CtB to examine the intact animals (41). The observation that FAA reappears after relationship between RPRF innervation and the input of the retina. Other both DMH and SCN are lesioned cannot be explained as a re- intact animals (n = 5) were perfused at ZT4 and analyzed simultaneously for covery of function in the DMH, because in the kainic acid lesioned- RPRF, VIP, gastrin-releasing peptide, and vasopressin using different fluo- DMH, induced decrease in c-Fos was visible in rats showing FAA rescent markers to evaluate which cell bodies in the SCN received RPRF in- put. In addition, antibodies to RPRF and GAD (Santa Cruz) were used to after SCN lesion. visualize terminations of both . Under high magnification The arcuate nucleus is essential for metabolic integration and by confocal microscopy, sections of the SCN of these animals were examined projects to many regions inside and outside the hypothalamus; it to identify possible colocalization of RPRF with GAD. shows entrainment during food restriction and still shows c-Fos after DMH and SCN lesion. Thus, the arcuate nucleus seems Experiment 3. Food restriction in DMH-lesioned animals followed by analysis of a possible candidate within the network of structures for driving neuronal activity in the SCN. After the first food-restriction protocol, 24 animals FAA (40). Other possible candidates are the , were allowed food ad libitum for 3 d, after which 16 animals received a ste- the paraventricular nucleus of the thalamus, and the cerebellum reotaxic lesion aimed to the DMH either with kainic acid or with electrolytic (9, 42). Clearly, more research needs to be done to define the lesioning.Eightanimalsreceived a shaminjectioninto theDMH.Thereafter,the nature of this food-entrained network. Recently it has been shown animals were allowed to recover for at least 2 wk before the second restricted- that the oscillatory property of the SCN is possibly a network feeding protocol was initiated. On the last day of this protocol, animals were intracardially perfused under deep Nembutal anesthesia at the moment they property rather than the property of single neurons (43). We may normally received food. Brains were used for histological analysis and immu- therefore need to view in a similar manner the FEO as a neuronal nohistochemical staining for c-Fos in the SCN and DMH. network: its oscillatory capacity is neither derived nor dependent c-Fos count. From each group, four animals were selected with high antici- (as we have shown here) on one structure, but rather the in- pation (Sham) or with the most accurate DMH lesion (electrolytic and kainic teraction between the participating structures shapes, different DMH-lesioned groups). Site and size of the lesions was established. See aspects of FAA. We propose that the oscillations in this network SI Materials and Methods and Figs. S3 and S4 for more details. Expression of are initiated, driven, and maintained by restricted-food schedules; c-Fos protein was explored in the region of the DMH and in the SCN. To without the continuous reinforcement of food restriction, the os- quantify c-Fos–positive cells in the SCN, three representative sections (rostral, cillation cannot be sustained for more than several days, probably middle, and caudal) were selected. Images of selected sections were obtained at a 200× magnification using a computerized image system (Image-Pro plus because other inputs into the FEO system, for example the SCN 5.1; Media Cibernetic) attached to a Zeiss light microscope. Cells positive for c- and light-dark cycle, disturb and prevent the materialization of the Fos were counted bilaterally in the ventral and total SCN in the three selected oscillation. This finding could serve to explain why FAA is altered sections with the image-processing program ImageJ (National Institutes of

following lesions in some parts of the brain but maintained with Health). To minimize the number of false-positives, background optic density NEUROSCIENCE lesions elsewhere (5). These observations may also provide an was established for each section in a nearby region lacking c-Fos; stained nuclei

Acosta-Galvan et al. PNAS | April 5, 2011 | vol. 108 | no. 14 | 5817 Downloaded by guest on September 24, 2021 that reached or surpassed 2× the background optic density were considered sion (12) were considered, as well DMH-lesioned, and were selected to positive and were included, whereas cells under this staining threshold were continue for a bilateral lesion of the SCN. After interrupting the second food discarded. A single examiner, who was blinded to treatment conditions, per- restriction, animals bearing a DMH lesion received a stereotaxic bilateral formed all counts. The mean of the number of c-Fos nuclei in the three sections electrolytic lesion (1 min, 0.2 μ Amp) aimed at the SCN. Thereafter, rats were was taken as the number of c-Fos in the SCN of that animal. allowed to recover for at least 3 wk, during which the success of their SCN lesion was examined by inspection of the actograms. When animals had Experiment 4. Food restriction in DMH- and SCN-lesioned animals. For this initial more than 30% of their 24-h activity in the light period they were consid- experiment, a group of 24 animals received a kainic acid lesion or an electric lesion of the DMH, followed after recovery by a restricted-food protocol. ered SCN-lesioned. On the last day of this protocol, at the moment of food Thereafter, the SCN was electrically lesioned, followed again by a restricted- expectancy, animals were intracardially perfused under deep Nembutal food protocol. The results are presented in Fig. S8. However, because we anesthesia. Thereafter, the brains were used for histological analysis. The realized that already intact animals may anticipate with a different intensity, results of these animals are presented in Fig. 3 and Fig. S9. we decided to first determine a baseline FAA for the following experiments. Thus, in 24 intact animals a baseline FAA was first established; thereafter, in ACKNOWLEDGMENTS. This work is supported by the Consejo Nacional de 20 animals that showed clear FAA, the DMH was lesioned using kainic acid. Ciencia y Tecnologia, Grants 79797 and 82462, and Grants DGAPA-PAPIIT- Only those animals that showed a clear diminished FAA after the DMH le- UNAM IN-215038 and IN-203907.

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