extfieldsofstudy: Medicine Biology

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Myc and ChREBP transcription factors cooperatively regulate normal and neoplastic hepatocyte proliferation in mice

Analogous to the c-Myc (Myc)/Max family of bHLH-ZIP transcription factors, there exists a parallel regulatory network of structurally and functionally related proteins with Myc-like functions. Two related Myc-like paralogs, termed MondoA and MondoB/carbohydrate response element–binding protein (ChREBP), up-regulate gene expression in heterodimeric association with the bHLH-ZIP Max-like factor Mlx. Myc is necessary to support liver cancer growth, but not for normal hepatocyte proliferation. Here, we investigated ChREBP's role in these processes and its relationship to Myc. Unlike Myc loss, ChREBP loss conferred a proliferative disadvantage to normal murine hepatocytes, as did the combined loss of ChREBP and Myc. Moreover, hepatoblastomas (HBs) originating in myc−/−, chrebp−/−, or myc−/−/chrebp−/− backgrounds grew significantly more slowly. Metabolic studies on livers and HBs in all three genetic backgrounds revealed marked differences in oxidative phosphorylation, fatty acid β-oxidation (FAO), and pyruvate dehydrogenase activity. RNA-Seq of livers and HBs suggested seven distinct mechanisms of Myc–ChREBP target gene regulation. Gene ontology analysis indicated that many transcripts deregulated in the chrebp−/− background encode enzymes functioning in glycolysis, the TCA cycle, and β- and ω-FAO, whereas those dysregulated in the myc−/− background encode enzymes functioning in glycolysis, glutaminolysis, and sterol biosynthesis. In the myc−/−/chrebp−/− background, additional deregulated transcripts included those involved in peroxisomal β- and α-FAO. Finally, we observed that Myc and ChREBP cooperatively up-regulated virtually all ribosomal protein genes. Our findings define the individual and cooperative proliferative, metabolic, and transcriptional roles for the “Extended Myc Network” under both normal and neoplastic conditions.

The c-Myc (Myc) oncoprotein is deregulated in many human tumors while also playing roles in normal proliferation, differentiation, and metabolism (1)(2)(3)(4)(5). Myc coordinates the transcription of thousands of protein-coding genes, rRNAs and tRNAs, microRNAs, and long-noncoding RNAs (6 -15). However, its role in maintaining growth and survival of normal or neoplastic cells depends on the timing and level of its expression and its cellular context. For example, whole-body depletion of Myc in the embryo is lethal but is compatible with postnatal survival (16 -18). Indeed, total-body dominant-negative inhibition of endogenous Myc in older mice is associated with mild and reversible intestinal epithelial and hematopoietic hypoplasia and long-term survival (19), yet these mice demonstrate regression of Ras oncogene-induced lung adenocarcinomas, thereby demonstrating the differential Myc dependence of normal and transformed tissues. Myc hypomorphism is associated with smaller organs resulting from a reduction in cell number but not size (18).
The consequences of Myc knockout in the liver have been controversial, particularly with regard to its role in regeneration following partial hepatectomy (PH) 3 (20 -23). We recently reexamined this in a novel mouse model of Type I hereditary tyrosinemia in which the replacement of diseased hepatocytes by transplanted nontyrosinemic hepatocytes can be monitored over several months (24 -26). Using competitive transplants, we demonstrated that WT and myc Ϫ/Ϫ (Myc-KO) hepatocyte populations possessed similar long-term repopulation poten-This work was supported by National Institutes of Health RO1 Grants DK103645 (to A. W. D.) and CA174713 (to E. V. P.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Figs. S1-S11 and Tables S1-S11.

Raw and processed original data have been deposited in the National Center for
Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) under accession number GSE114634. 1  cro ARTICLE tial. In contrast, hepatoblastoma (HB) development driven by mutant forms of ␤-catenin and yes-associated protein (YAP) was markedly delayed in Myc-KO livers (13,27), indicating that Myc plays distinct roles in normal and neoplastic proliferation. Alternatively, Myc-KO hepatocytes could be rescued by factors that functionally substitute for Myc. Indeed, Myc-KO HBs upregulated expression of the weakly transforming Myc paralog L-Myc (13,28,29).
Myc, L-Myc, and another paralog, N-Myc, are members of a group of related bHLH-ZIP transcription factors that bind canonical E-box elements (CA(C/T)GTG) and regulate gene expression. The central member of this group is Max, whose dimerization with Myc is necessary for E-box binding. In contrast, heterodimers formed between Max and the four bHLH-ZIP members of the Mxd family compete with Myc-Max heterodimers for DNA binding and transcriptional modulation (30,31). Hereafter, we refer to these proteins as the "Myc-Max-Mxd" family.
Following the discovery of the Myc-Max-Mxd family, it was appreciated that a related bHLH-ZIP family with Myc-like functions exists. Analogously, this also consists of a central, Max-like bHLH-ZIP protein, Mlx; two closely related Myc-like factors termed MondoA/Mlxip and MondoB/ChREBP/Mlxipl (hereafter ChREBP) that positively regulate gene expression; and a negative regulator termed Mnt (32)(33)(34). We refer to this as the "ChREBP-Mlx-Mnt" family and to the two families collectively as the "Extended Myc Network" (Fig. S1A). Cross-talk occurs by virtue of the fact that Mxd1 and Mxd4 also interact with Mnt. The canonical binding site for the ChREBP-Mlx-Mnt family, termed the carbohydrate response element (ChoRE), consists of two Myc-like E-box elements separated by 5 nucleotides (CAYGYGnnnnnCRCRTG) (35)(36)(37)(38). Whereas the two families regulate common genes (14,39,40), those under ChREBP-Mlx-Mnt family control comprise a subset of those regulated by the Myc-Max-Mxd family, particularly those involved in carbohydrate and lipid metabolism (14,32,34,36,(41)(42)(43)(44)(45). Moreover, unlike nuclearly localized Myc proteins, MondoA and ChREBP shuttle between the nucleus and cytoplasm in response to their binding of glucose 6-phosphate, thereby suggesting that they serve as nutrient sensors and regulate target gene expression in accord with metabolic status (34,40,46,47).
We have now compared both the repopulation and tumorigenic potential of Myc-KO (or MKO) hepatocytes with those lacking ChREBP (ChREBP-KO or CKO) and both Myc and ChREBP (Double-KO or DKO). We chose for study ChREBP rather than the related MondoA because, even though both are widely distributed, ChREBP expression is more prominent in liver and appears to play a more important role (32,48,49). Our findings demonstrate that, in contrast to Myc, ChREBP's absence leads to a profound proliferative disadvantage in competitive hepatocyte repopulation assays and that Double-KO hepatocytes are even more impaired. HB growth rates were also markedly slowed, although tumor initiation remained unaffected. A combination of metabolic, biochemical, and transcriptional profiling better defined the abnormal phenotypes. Our findings indicate that the inhibition of normal and neoplastic proliferation resulting from Myc and/or ChREBP loss originates from multiple and in some cases overlapping and cooperative effects on key metabolic processes, primarily centered around carbohydrate and lipid metabolism and protein synthesis.

Myc-KO, ChREBP-KO, and Double-KO livers accumulate neutral lipid
Livers from young Myc-KO mice appear relatively normal but gradually accumulate neutral lipids (25). We confirmed this by demonstrating more prominent vacuolization in hematoxylin and eosin-stained Myc-KO livers (Fig. S1B). ChREBP-KO livers from comparably aged mice were even more abnormal, with lipids tending to be particularly prominent in subcapsular and periendothelial regions. Similar changes were seen in Double-KO livers, with certain areas being more noticeably affected. Hepatic triglyceride content largely reflected these findings (Fig. 1C). Thus, ChREBP appears to play a more important role than Myc in maintaining hepatic triglyceride content.
In contrast, HBs were largely devoid of neutral lipids, although occasional areas of vacuolization were observed. However, the adjacent regions of nontransformed parenchyma largely retained the patterns of neutral lipid staining described above (Fig. S1B). Tumor triglyceride content was also uniformly low (Fig. S1B).

ChREBP-KO and Double-KO hepatocytes function poorly in repopulation assays
Liver repopulation by WT and Myc-KO hepatocytes occurs at similar rates (25). To assess this, we employed the hereditary tyrosinemia model described above. Donor mice lack the enzyme fumaryl acetoacetate hydrolase (FAH) (50) and succumb to the progressive accumulation of toxic tyrosine catabolites unless rescued with the drug 2-(2-nitro-4-trifluoromethyl-benzoyl)-1,3-cyclohexanedione (NTBC), which blocks the upstream enzyme p-hydroxyphenylpyruvate dioxygenase. Mice can also be rescued by the intrasplenic injection of fah ϩ/ϩ hepatocytes (25,26,51,52). During iterative periods of NTBC discontinuation, donor cells gradually replace their fah Ϫ/Ϫ counterparts and, upon reaching an equilibrium state, comprise as much as 80% of the recipient's hepatocytes. This assay is superior to PH for assessing donor hepatocyte proliferative potential in that it imposes greater replicative demand that can reveal subtle differences in repopulation potential not appreciable with the PH model. Additionally, whereas 30 -40% of post-PH hepatocytes do not replicate and about an equal fraction undergo hypertrophy rather than actual division (53,54), repopulation following transplant assesses only proliferation.
We initially performed transplants using single populations of hepatocytes and noted the time needed to achieve NTBC independence, as defined by the ability of recipients to discontinue the drug and maintain Ͼ80% of their pretransplant weight (25). Consistent with previous findings, we found this time to be 9.5 Ϯ 0.3 and 10.1 Ϯ 0.4 weeks following transplantation with WT and Myc-KO hepatocytes, respectively (not significant as determined by Student's two-tailed t test) (25). In contrast, transplantation with ChREBP-KO hepatocytes required 11.8 Ϯ 0.4 weeks to achieve NTBC independence (p ϭ Myc and ChREBP cooperation in hepatocyte proliferation 5.18 ϫ 10 Ϫ3 , compared with WT). Double-KO hepatocytes required 18.9 Ϯ 1.0 weeks (p ϭ 1.40 ϫ 10 Ϫ8 , compared with WT). Additionally, the time to NTBC independence was significantly prolonged in Double-KO mice versus ChREBP-KO mice (p ϭ 1.91 ϫ 10 Ϫ5 ). Thus, both ChREBP-KO and Double-KO hepatocytes are replication-impaired.
We next performed competitive repopulation assays with ChREBP-KO ϩ WT or Double-KO ϩ WT hepatocytes ( Fig.  1A) (25,51). Upon attaining NTBC independence, a TaqManbased approach was used to quantify total donor and recipient populations (Fig. 1). In both cases, ϳ75% of hepatocytes were found to be of donor origin (Fig. 1C). Further investigation of the ChREBP-KO ϩ WT transplants showed that ChREBP-KO hepatocytes comprised 61.5 and 27.7% of the pre-and posttransplant donor population, respectively ( Fig. 1 (D and E), p ϭ 5.2 ϫ 10 Ϫ10 ). Even more striking were the results obtained with Double-KO ϩ WT transplants, where 52.8% of the pretransplant input donor population but only 7.5% of the posttransplant hepatocytes were Double-KO cells (p ϭ 5.2 ϫ 10 Ϫ10 ). The difference in repopulation efficiencies between ChREBP-KO and Double-KO was highly significant (p ϭ 5.0 ϫ 10 Ϫ6 ). Thus, both ChREBP-KO and even more so Double-KO hepatocytes demonstrated a profound competitive disadvantage.  The values for C and E were obtained from reconstitution experiments using eight different known ratios of each of the two DNA populations being quantified as described previously (51). Correlation coefficients in each case were Ͼ0.99 (not shown).

Growth inhibition of Myc-KO, ChREBP-KO, and Double-KO HBs
We next examined HB tumorigenesis in response to liver expression of oncogenic forms of ␤-catenin and YAP (13,27,51). WT animals succumbed with a mean survival of ϳ13 weeks ( Fig. 2A) (13,27,51), whereas survival in all knockout groups was delayed, and tumor sizes were somewhat smaller (Fig. 2B). Proliferating cell nuclear antigen and phosphohistone H3 levels as markers of proliferation (55,56) were indistinguishable among the tumor groups (Fig. 2C). Thus, ChREBP loss, or combined Myc ϩ ChREBP loss, affects tumor growth rates but not initiation.
We examined expression of other members of the Extended Myc Network as well as other Myc target genes capable of restoring myc Ϫ/Ϫ cell growth, including myct1, hmga1, and shmt (57-60) (Fig. 2D). Aside from confirming that the three knockout tissues lacked expression of their respective fulllength transcripts, we showed variable degrees of change in the livers and tumors of mice with regard to a number of these transcripts, including those for L-Myc, N-Myc, Mxd3, Mnt, Myct1, and Shmt.
Members of the Extended Myc Network possess varying degrees of structural and functional redundancy (14,28,36,(62)(63)(64)(65), suggesting that they might be able to rescue the proliferative defects of KO hepatocytes. As expected, Myc-KO livers and tumors did not express Myc, ChREBP-KO livers and tumors did not express ChREBP, and Double-KO livers and tumors did not express either protein ( Fig. 2E and Fig. S2). Regardless of genotype, all livers and tumors expressed low or undetectable levels of N-Myc. L-Myc levels were variably elevated in all tumors. Similarly, livers expressed low levels of MondoA, which was increased modestly in all tumors. ChREBP was expressed at similar levels in WT and Myc-KO livers and tumors. From these studies and the fact that L-Myc and MondoA are about equally up-regulated in all tumors regardless of genotype, we conclude that neither of these is likely to be expressed at levels sufficient to support non-

Myc and ChREBP cooperation in hepatocyte proliferation
transformed hepatocyte proliferation. The poor correlation between transcript and protein levels for some of these factors ( Fig. 2 (D and E) and Fig. S2) indicates that much of their control was post-transcriptional.

Metabolic alterations in livers and tumors
WT and Myc-KO liver and tumor metabolism differ in several critical ways that impact TCA cycle function, FAO, and levels of critical substrates, such as ATP and acetyl-CoA (AcCoA) (13,25). Because of ChREBP's known influence on carbohydrate and lipid metabolism and its cooperation with Myc in regulating these activities (40, 41, 44, 45, 49, 66 -69), we reevaluated some of these behaviors in our four liver and HB cohorts.
Oxygen consumption rates (OCRs) of WT and Myc-KO livers were similar, as reported previously (Fig. 3A) (13). In contrast, ChREBP-KO liver OCRs were lower than those of WT or Myc-KO livers. This discrepancy was corrected in Double-KO livers, suggesting that, in this largely quiescent population, Myc and ChREBP affected readout oppositely. These differences were largely attributable to alterations in Complex II function, which accounts for the majority of Oxphos in liver (Fig. 3A) (13,25,51). Complex I and Complex II responses were distinguished by measuring OCR before and after the addition of the Complex I inhibitor rotenone. B, reduction of mtDNA content in HBs is independent of tumor growth rates and the presence of Myc and/or ChREBP. Two different sets of TaqMan probes (Set 1 and Set 2) were used to independently amplify different regions of the mitochondrial genome. C, ATP levels in livers and tumors. D, AcCoA levels in livers and tumors. E, PDH activity in fresh livers and tumors. PDH was assessed by measuring the release of 14 CO 2 from 14 C-labeled pyruvate (13,51). F, FAO rates in fresh livers and tumors. FAO was assessed by measuring the release of water-soluble products from 3 H-labeled palmitate-BSA (13,51). G, representative Coomassie Bluestained nondenaturing gels of ETC complexes from livers (L) and tumors (T) isolated from the indicated groups. The panel shows two sets of results. H, typical appearances of developed nondenaturing gels following in situ enzymatic assays for the ETC complexes I, III, IV, and V. I, quantification of ETC complex activities. Four sets of gels similar to those shown in H were scanned and normalized to the activity of Complex IV, which showed little variation among the different tissues (13,25,105). J, complex II activities were assayed in vitro on tissue lysates because the in situ assay is unreliable. All results were normalized to the mean activity of each complex measured in WT livers. Error bars, S.D.

Myc and ChREBP cooperation in hepatocyte proliferation
Also, as shown previously, WT and Myc-KO tumor OCRs were significantly lower than their corresponding normal livers, indicating greater reliance on glycolysis (13,24,51). In contrast, both ChREBP-KO tumors and Double-KO tumors were indistinguishable from their corresponding livers, and this was also reflected in their or in vitro activities The reduction of WT and Myc-KO tumor OCRs was an expected consequence of the Warburg effect, which diverts glycolytic substrates away from mitochondria and into anabolic pathways that abet tumor proliferation (70 -73). We recently reported that, like many human cancers (74), both HBs and Myc-induced hepatocellular carcinomas have a reduced mitochondrial DNA (mtDNA) content relative to livers, thus providing a potential physical basis for their reduced Oxphos and the Warburg effect (13,75). Indeed, this was true for all four sets of HBs (Fig. 3B). Our results indicate that certain ETC functions are under differential control by ChREBP and Myc but that neither is responsible for the maintaining mtDNA.
Despite their reduced mtDNA content, WT and Myc-KO HBs had higher ATP levels than their corresponding livers (13). We confirmed this and further showed that these persisted in ChREBP-KO and Double-KO HBs (Fig. 3C). This correlated with a higher AcCoA content in WT HBs but not in Myc-KO, ChREBP-KO, or Double-KO tumors (Fig. 3D). Thus, the impaired growth of knockout tumors cannot be attributed to ATP deficiencies but could be due to an AcCoA deficit.
Increased pyruvate dehydrogenase (PDH) activity accompanies HB and hepatocellular carcinoma tumorigenesis and may reflect a reduced reliance on FAO and/or an attempt to maximize the conversion of limiting amounts of pyruvate to AcCoA (76). We confirmed this but also showed that Myc-KO, ChREBP-KO, and Double-KO livers and tumors had progressively larger reductions in PDH activity. Most strikingly, PDH activity in Double-KO tumors, although still elevated relative to its corresponding liver, was not significantly higher than the activity in WT livers (Fig. 3E).
The relatively high FAO rate of normal liver is Myc-dependent and is reduced by ϳ50% in its absence. A further ϳ50% decline in FAO rates then occurs in both WT and Myc-KO HBs, with the latter reflecting the already reduced activity in Myc-KO livers (13). FAO rates were equally dependent on ChREBP, and Double-KO livers and HBs had the lowest FAO rates as well as the lowest PDH activities among their respective groups (Fig. 3F). These results reveal that FAO is positively regulated by the combined actions of Myc and ChREBP. Tumors and their corresponding livers therefore progressively suppress PDH and FAO activities that normally function reciprocally and serve as major sources of ATP and AcCoA.
Myc's loss is associated with significant ETC dysfunction in both livers and HBs (Fig. 3A) (13,25). To determine how ChREBP and Myc ϩ ChREBP affected these processes, we resolved ETC complexes using blue native gel electrophoresis (Fig. 3G) and performed functional assays for all complexes. As seen in Fig. 3 (H-J), the only consistent abnormality was a loss of Complex V (ATP synthase) activity in Double-KO livers that was lower than the corresponding activity in Double-KO tumors or other liver or tumor groups.

Transcriptional profiling of livers
We next conducted RNA-Seq on five tissues from each group and identified distinct transcript expression profiles. The relationships of these to one another and the ways in which Myc and ChREBP may potentially interact to differentially affect target gene expression are depicted in Fig. 4 (A-C).

Myc and ChREBP cooperation in hepatocyte proliferation
genes plus three additional down-regulated Cyp450 genes and Kdm5d (Table S3) red) and down (green) arrows signify general up-or down-regulation of genes belonging to a given pathway. Pathways without arrows contain dysregulated genes but could not be assigned directionality with any degree of confidence. An analysis for livers is not shown, as the number of dysregulated genes in each Venn diagram group were generally too low to reliably predict pathway directionality. cent to MYC, often co-amplifies with it, and stabilizes the Myc protein (Table S4) (85). 29 genes were dysregulated following the loss of ChREBP or Myc, but not both (Shared dependent response; Fig. 4 (B-D), Group 6). Of these, 25 were up-regulated and four were downregulated. Among these were the estrogen sulfotransferase Sult1e1 (up-regulated 11-fold) and three MHC Class II genes (Table S5). Members of this group were too few in number to permit any further functional classification using gene ontology analysis.
494 genes were dysregulated in Double-KO livers compared with normal liver and included genes contained within Groups 1, 2, 3, and 7 (281 up-regulated and 213 down-regulated; Fig. 4, B-D). Similar to Myc-KO livers, and more so ChREBP-KO livers, pathway analysis revealed roles for many of these genes in the metabolism of steroids and other lipids (Fig. S3C and http://prochownik.pitt.edu/chrebp/). Dysregulated genes falling into groups 1-3 have been discussed above. Because 232 of these were only dysregulated in Double-KO livers, we classified them as showing a "cooperative response" indicating that loss of either ChREBP or Myc fully compensated for the other's absence (Fig. 4 (B-D), Group 7). Members included 16 down-regulated ribosomal protein (RP) genes (average down-regulation ϭ 1.43-fold) and the gene encoding acetoacetyl-CoA synthetase (Aacs, down-regulated 2.63-fold) (Table S6). 66 genes were dysregulated in all three liver groups (Dual-dependent response; Fig. 4 (B-D), Group 1 and Table S8). Of these, 38 were up-regulated, and 28 were down-regulated. The latter included genes encoding the leptin receptor (Lepr; downregulated 4-fold) and the rate-limiting enzyme in cholesterol synthesis, HMG-CoA reductase (Hmgcr; down-regulated 1.6fold) (Table S7).
Common to both ChREBP-KO and Double-KO tumors and strictly ChREBP-dependent response (Group 2) were 590 upregulated and 557 down-regulated transcripts (Fig. 4 (A, B, E, and F), Groups 1 and 2). These involved pathways regulating Oxphos and mitochondrial function, including 20 down-regulated NADH:ubiquinone oxidase genes and four ATP synthase genes (Fig. S5A). Genes involved in Wnt/␤-catenin signaling were also among this group (Fig. S5B), as were those encoding the mitochondrially localized members of the sirtuin family, sirt3-5 (Fig. S5C). Sirtuins function as lysine deacylase, ADPribosyltransferase, and desuccinylase proteins, and Sirt1 and Sirt3 also serve as tumor suppressors (86,87). Additionally, NRF2-mediated signaling was predicted to be decreased due to altered expression of 21 genes in this pathway (Fig. S5D). NRF2 is a leucine zipper transcription factor whose own targets include numerous genes involved in cytoprotective and antioxidant responses (88,89). Other notable genes in this group include Igf2 (up-regulated 5.6-fold) and the previously noted Pklr (down-regulated 3.5-fold).
1,657 genes were differentially expressed in Myc-KO tumors compared with WT tumors (816 up-regulated and 841 downregulated), with many of the involved pathways being the same as described in ChREBP-KO tumors (Fig. 4, E and F). These included various growth and cancer signaling pathways, metabolic and lipid metabolism pathways, and intracellular signaling (Fig. S6).
The subset of 248 genes dysregulated in both Myc-KO and Double-KO tumors consisted of 121 up-regulated and 127 down-regulated genes (Myc-dependent response; Fig. 4E, Group 3). Notably down-regulated were 12 genes encoding ribosomal proteins and two encoding eukaryotic initiation factors (Fig. S7A). Elevated expression of seven Cyp450 genes was also observed, as well as variable dysregulation of several genes involved in RXR signaling and lipid metabolism, including three phospholipases, carnitine palmitoyltransferase 1B (Cpt1b; down-regulated 2.8-fold) and others (Fig. S7B). Other notable genes in this group included the previously discussed Pvt1 (up-regulated 1.6-fold) and N-Myc (Mycn; up-regulated 5.7-fold).
Compared with WT tumors, 183 genes (101 up-regulated and 82 down-regulated) were differentially expressed when either ChREBP or Myc, but not both, was knocked out (Shared dependent response; Fig. 4E, Group 6). This group was largely concerned with immune signaling, including pathways involving granulocyte adhesion, cellular movement, phagocytosis, and T-cell function.
Double-KO tumors differentially expressed 3,604 transcripts compared with WT tumors (1,769 up-regulated and 1,835 down-regulated). These most commonly functioned in growth and cancer signaling, lipid metabolism and RXR signaling, the immune response, and intracellular signaling pathways (Fig.  S8). Dysregulated genes belonging to Groups 1, 2, and 3 ( Fig.  4A) have been discussed previously. 1,443 genes were only dys-

Myc and ChREBP cooperation in hepatocyte proliferation
regulated in Double-KO tumors and comprised the largest of the seven groups (Cooperative response, Group 7). 734 were up-regulated, and 709 were down-regulated (Fig. 4, E and F). Analysis of this subset was notable for the predicted downregulation of the RhoA signaling pathway (Fig. S9A) and revealed genes involved in nucleotide synthesis and salvage pathways. Additionally, up-regulation of Kdm5d (3.5-fold) and eight cytochromes P450 (average up-regulation ϭ 2.4-fold) was observed, as well as the down-regulation of two additional CYP450 genes (Fig. S9B).
The 766 genes deregulated in all experimental groups comprised the "dual-dependent response" (Fig. 4, A, D, E, and F,  Group 1). This contained 324 up-regulated and 442 down-regulated genes and was most notable for 64 strongly down-regulated RP genes and six eukaryotic initiation factors, which were most prominent in the Double-KO group (Fig. S9C). This included the down-regulation of 10 genes involved in glycolysis and gluconeogenesis (Fig. S9D) and numerous others involved in granulocyte adhesion and diapedesis (Fig. S9E).
To obtain an approximation of the fraction of transcripts that were products of direct Myc target genes, we queried the ChiP Atlas database with the above 1,657 transcripts whose expression differed between WT and Myc-KO tumors (q Ͻ 0.05). 71.2% of these were contained in this database, indicating that a substantial proportion of our differentially expressed transcript population originates from direct Myc target genes.

Myc and ChREBP cooperatively regulate ribosomal protein transcript (RPT) expression
As a group, genes encoding RPTs were up-regulated 2.2-fold in WT tumors relative to WT livers, but only 1.3-fold in Myc-KO and ChREBP-KO tumors and not at all in Double-KO tumors (Fig. 5 (A and B), p Ͻ 0.0001). Myc-KO tumors downregulated 73 of 80 RPTs relative to WT tumors, with the average expression of these being 52% of WT tumors. Similarly, 62 RPTs were down-regulated in ChREBP-KO tumors (average expression ϭ 62% of WT). Double-KO tumors not only downregulated a larger number of RPTs (79 of 80) but did so to a greater degree (average expression ϭ 36% of WT) (Fig. 5B). This indicated that Myc and ChREBP, either individually or together, regulate virtually all RPTs in tumors and, to a lesser extent, in livers.
RPT expression patterns can distinguish tumor types and normal tissues in ways that are independent of absolute RPT levels (90,91). Indeed, principal component analysis (PCA) showed that liver and tumor RPT expression patterns were distinct (Fig. 5C). Additionally, RPT patterns of knockout livers were distinct from those of WT livers, indicating that any knockout leads to a signature pattern of RPT expression (Fig.  5D). Whereas RPT expression patterns of all three KO liver groups displayed significant overlap, their cognate tumor groups clustered separately. Thus, RPT expression patterns from normal and transformed tissues are distinct and readily discernible from one another. Myc and ChREBP, therefore, in addition to dictating the absolute levels of tumor RPTs (and to a lesser extent liver RPTs), also regulate their expression patterns.
Finally, RP gene proximal promoters were analyzed for the presence of putative canonical E-boxes and ChoREs. Although 60 RP gene promoters contained at least one E-box within the regions analyzed, putative ChoRE sequences were found only in the promoters of Rps12 and Rpl13a (Fig. S10D). The lack of conserved E-boxes in 25% of RP genes plus the virtual absence of ChoREs argues that a significant amount of Myc regulation and virtually all of the ChREBP regulation of the RP gene family are likely to be indirect. However, it remains possible that less conserved E-boxes and ChoREs located in these promoters or outside the regions examined are involved in Myc-and/or ChREBP-dependent regulation.

Metabolic pathway dysregulation in livers and cancers mediated by the losses of Myc and ChREBP
The loss of ChREBP in livers resulted in an overall downregulation of transcripts encoding fatty acid elongation genes and HMG-CoA reductase, which encodes the rate-limiting enzyme in cholesterol synthesis (Fig. 6A). Myc-KO livers similarly down-regulated HMGCR transcripts but did not demonstrate strong down-regulation of those encoding enzymes involved in fatty acid elongation as did ChREBP-KO livers (Fig.  6B). Double-KO livers demonstrated down-regulation of transcripts involved in fatty acid elongation and cholesterol synthesis and a small number of genes involved in various other metabolic processes (Fig. 6C).
ChREBP-KO tumors strongly down-regulated the genes involved in glycolysis, the pentose phosphate shunt, the TCA cycle, Oxphos, and ␤and -oxidation. -Oxidation is an alternative nonenergy-generating cytosolic and microsomal FAO pathway that becomes activated in response to inefficient ␤-FAO and is mediated largely by Cyp450 members and alcohol and aldehyde dehydrogenases (Fig. 7A) (82,92). Myc-KO tumors also down-regulated transcripts for glycolysis and HMGCR and up-regulated those for glutamine metabolism and fatty acid elongation (Fig. 7B). Double-KO tumors also downregulated many glycolytic genes and mitochondrial ␤-oxidation and Oxphos genes and up-regulated numerous genes involved in fatty acid elongation and peroxisomal ␣and ␤-FAO (Fig. 7C).
Additional evidence supporting cooperation between Myc and ChREBP in the regulation of lipid metabolism was the finding that 26 of the 102 known murine Cyp450 genes in livers (25%) and 51 of 102 in tumors (50%) were deregulated in response to knockout of ChREBP and/or Myc (Tables S9 and  S10). In the latter case, 34 (56%) were members of the Cyp2, Cyp3, or Cyp4 families, which play particularly relevant roles in steroid and eicosanoid metabolism (93)(94)(95). In keeping with previous findings that Cyp450 genes are Myc targets both in untransformed hepatocytes and hepatoblastomas (13, 25), we found 35 Cyp450 genes to be exclusive targets of Myc, 15 to be exclusive targets of ChREBP, and six to be deregulated

Myc and ChREBP cooperation in hepatocyte proliferation
only when both Myc and ChREBP were eliminated. These results indicate that, as a gene family with important roles in fatty acid, sterol metabolism, and eicosanoid metabolism, Cyp450 genes are subject to extensive regulation by Myc and/or ChREBP.
Finally, to validate RNA-Seq results, we selected a small number of representative transcripts from the various families discussed above and performed real-time qRT-PCR analyses (Table S11). We found these results to be in excellent overall agreement with those obtained by RNA-Seq (Fig. S11).   The order of transcripts is based on the most to the least abundant in WT livers (WL). C, PCA of RPT relative expression in livers and tumors. Livers and tumors cluster distinctly from one another, indicating that each group possesses distinct patterns of RPT expression. D, PCA of RPT expression performed separately in livers and tumors. WT livers cluster distinctly from knockout livers, but none of the knockout liver groups can be distinguished from one another. All tumor groups cluster distinctly from one another, however, indicating that knockout of Myc, ChREBP, or both leads to distinct expression patterns of RPTs.

Discussion
Cross-talk among select members of the Extended Myc Network presumably underlies the responsiveness of its members to growth and differentiation signals and helps to coordinate the timing and robustness of these responses with nutrient availability, proliferative needs, and metabolic preparedness (14,32,34,36,46,47,63,96).
The hepatocyte-specific loss of Myc is associated with mild and age-dependent neutral lipid accumulation (25). Even more neutral lipids accumulate in the livers of ChREBP-KO and Double-KO mice (Fig. S1, B and C). Similar observations have been made in other cell types following Myc or N-Myc inhibition (97)(98)(99). This likely represents a generalized loss of coordination between exogenous lipid uptake, FAO, and de novo lipid biosynthesis with the rate of the former exceeding the rate of the latter two. Our finding that all KO livers were defective in FAO, coupled with previous observations that Myc inhibition enhances exogenous fatty acid uptake (13,(97)(98)(99), is consistent with this idea. Mitochondrial dysfunction in KO livers and tumors (Fig. 3, A, E, and F), together with the inability to properly up-regulate Oxphos-related genes (Fig. S5A), is consistent with other abnormalities in Myc-KO livers and HBs (13) and in cell lines with MondoA knockdown (63).
We previously found there to be no discernible differences in the rates at which WT and Myc-KO hepatocytes repopulate the fah Ϫ/Ϫ liver despite the latter's lipid processing defects (25,26,50). In contrast, ChREBP-KO and Double-KO hepatocytes, with more severe lipid abnormalities, were even more impaired (Fig. 1, D and E) (37). This suggests that ChREBP-regulated genes are just as important for sustained hepatocyte proliferation as Myc-regulated genes, if not more so. We cannot currently state with certainty whether this represents a direct effect on proliferation or an indirect one resulting, for example, from an inimical intracellular environment generated by accumulated lipids. In addition, the collaboration between the losses of Myc and ChREBP to produce a more severely impaired proliferative defect where none existed for the knockout of Myc alone suggests that this represents a form of synthetic lethality (100).
Despite differences in hepatocyte proliferative rates (Fig. 1E) (25), tumor growth rates were equally impaired (Fig. 2, A and B) (13). Tumors may rely more on the products of certain Myc and/or ChREBP target genes because of their more rapid growth. The pronounced AcCoA deficit in all KO tumors and their failure to increase RPT expression might be two such examples (Figs. 3D and 5 (A and B)). Additionally, Myc and ChREBP target genes are distinct in hepatocytes and tumors and could perhaps account for differential effects on tumor growth (Fig. 4) (13). Finally, ␤-catenin target gene products other than Myc could alter tumor cell sensitivity to Myc and/or ChREBP in ways that are distinct from those of hepatocytes. For example, Myc and ChREBP regulate smaller subsets of RPTs in livers than in tumors and do so less robustly (Fig. 5, A and B) (90). The defective RPT response of hepatocytes might not be fully appreciated until they are subject to the more demanding proliferative signals associated with transformation. Normal and transformed hepatocytes also have distinct metabolic properties, which may be direct or indirect consequences of Myc, ChREBP, and ␤-catenin expression (Fig. 3, A, E, and F).
WT and all KO tumor groups up-regulated L-Myc equally, which could potentially rescue the Myc-KO phenotypes if not the ChREBP and Double-KO phenotypes (25). L-Myc serves this function in myc Ϫ/Ϫ fibroblasts, although its transcriptional activation potential and transforming activity are markedly weaker than Myc's (28,29,101). Similarly, elevated MondoA in tumors might partially rescue ChREBP-KO tumor growth. MondoA enhances N-Myc transcriptional activity by altering chromatin conformation on some metabolic target gene promoters and by preventing Mlx's heterodimerization with Mxd and Mnt (60,90,91). Whether this occurs with L-Myc as well, however, is unknown.
MondoA and ChREBP target genes have been identified in a variety of cell lines, and ChREBP-bound genes have been cataloged in liver and white adipose tissue (37,40,43,63,67,69). However, it remains unclear how many of the nearly 6,000 ChREBP-binding sites actually alter gene expression (37). The gene expression profiles of our three KO liver and tumor groups suggest that at least seven distinct mechanisms account for these transcriptional responses (Fig. 4) and underscore the interplay between Myc and ChREBP.
In addition to the gene targets mentioned above, we unexpectedly found that ChREBP also controls RP genes, particularly in collaboration with Myc ( Fig. 5 and Figs. S9C and S10). Whereas numerous canonical E-boxes were identified in the proximal promoters of these genes, ChoRE elements were detected in only two (Fig. S10D). This suggests either that ChREBP regulation of most RP genes is indirect, that some ChoREs are more degenerate than those we defined as bona fide, that ChoREs reside outside the proximal promoter regions we analyzed, and/or that ChREBP binds to E-boxes as well as ChoREs. Regardless of the nature of the regulation, ChREBP's cooperation with Myc can be quite strong, as seen by the complete unresponsiveness of RP genes in tumors. This suggests that Myc and ChREBP are the major determinants of RPT expression in normal and neoplastic stimuli and cannot be replaced by other factors. It is difficult to escape the conclusion that a major proliferative defect in hepatocytes and livers lacking Myc and/or ChREBP is their inability to match translational rates with proliferative demands (102)(103)(104).
Given the variability in E-box binding affinities for Myc and a likely similar variability for ChREBP (105)(106)(107)(108), it is not surprising that the patterns of RPT expression in different tumor types    Fig. 6. B, transcriptional changes in metabolic pathways in Myc-KO tumors compared with WT tumors. C, transcriptional changes in metabolic pathways in Double-KO tumors compared with WT tumors. vary (Fig. 5, C and D). These likely reflect both the relative intrinsic affinities of binding sites within their gene promoters and the levels of Myc, ChREBP, and other members of the Extended Myc Network. Conceivably, the presumed resulting changes in ribosomal subunit composition can impact the efficiency with which different mRNAs are selected and translated, thereby achieving a distinct level of translational control independent of the transcriptional control imparted by Myc and ChREBP themselves (109).

Animals, hepatocyte transplants, and tumorigenesis studies
All animal work was performed in compliance with the Public Health Service Policy on Humane Care and Use of Laboratory Animal Research (DLAR) Guide for Care and Use of Laboratory Animals. All procedures were reviewed and approved by the University of Pittsburgh's institutional animal care and use committee. Mice were maintained in a pathogen-free facility under standard conditions with free access to food and water. UBI-GFP/B6-EGFP mice were purchased from Jackson Laboratories (Bar Harbor, ME). These mice, which express enhanced GFP in all tissues under the control of the ubiquitin C promoter, were used as a source of WT hepatocytes for all transplant studies. Mice bearing a conditional, hepatocyte-specific deletion of myc (Myc-KO mice) were generated by crossing c-Myc flox/flox (WT) mice (2,25) with Alb-Cre recombinasepositive mice (25) and were genotyped as described previously (25). chrebp Ϫ/Ϫ knockout (ChREBP-KO) mice (B6.129S6-Mlxipl tm1Kuy /J mice, strain 010537) were purchased from Jackson Laboratories and were genotyped as recommended by the vendor.
Immunocompromised FRG-NOD fah Ϫ/Ϫ mice (Yecuris, Inc., Tualatin, OR) were used as hepatocyte transplant recipients. They were maintained on 8 mg/liter NTBC (Yecuris) in their drinking water. Hepatocyte isolation was performed using a two-step collagenase perfusion method (24,25,51). For competitive transplant studies, WT and KO hepatocytes (Ͼ80% viability) were combined in approximately equal numbers, and 3.0 ϫ 10 5 were injected intrasplenically into 6 -8-week-old recipient FRG-NOD mice. Four days after transplantation, NTBC was discontinued and then reinstated when mice lost 20% of their body weight. NTBC cycling was continued until body weights stabilized. Mice were then maintained off NTBC for 4 -5 additional weeks to ensure maximal hepatic replacement and body weight normalization. Hepatocytes were then isolated as described above (24,25,51), washed twice in PBS, and snap-frozen for subsequent PCR-based analysis.
In some cases, mice were reconstituted noncompetitively using only Myc-KO, ChREBP-KO, or Double-KO donor hepatocytes. In these instances, the time to NTBC independence was noted, and entire livers were removed for histological assessment.
Determining the extent to which recipient fah Ϫ/Ϫ mice were reconstituted with the total donor population used a two-step TaqMan-based assay. PCRs were performed with a Kapa Probe Fast qPCR kit (Kapa Biosystems, Wilmington, MA) in a volume of 12 l with 10 ng of genomic DNA as described previously (51). Conditions for amplification were as follows: 95°C for 10 s, 40 cycles at 95°C for 15 s, 60°C for 60 s. Repopulation was calculated with primers that amplified the Pol2neo cassette inserted in exon 5 of fah Ϫ/Ϫ mice (forward, 5Ј-GGGAGGAT-TGGGAAGACAATAG-3Ј; reverse, 5Ј-ATTCTCCTTGCC-TCTGAACATAA-3Ј). The product was detected and quantified in real time with the probe, 5Ј/56-FAM/CTTCTG-AGG/ZEN/CGGAAAGAA/3IABkFQ/-3Ј. A standard curve with known amounts of fah Ϫ/Ϫ and fah ϩ/ϩ hepatocyte DNA was generated to allow the fractional contribution of each transplanted sample to be determined. To determine the contributions of Myc-KO and ChREBP-KO hepatocytes relative to WT hepatocytes or to one another, we used the approach depicted in Fig. 1A.

Induction of HBs
Tumorigenesis was induced in 6 -8-week-old mice using the hydrodynamic tail vein injection-mediated delivery of Sleeping Beauty plasmids (13,27,51). Mice received 10 g each of plasmids encoding a mutant, patient-derived form of human ␤-catenin (⌬90) and YAP (S27A) along with 2 g of a vector encoding Sleeping Beauty transposase (13,51). Plasmids were purified using Qiagen tip-500 columns (Qiagen, Inc., Valencia, CA) and were delivered in a total volume of 2 ml in 0.9% NaCl solution over ϳ5-9 s. Mice were evaluated three times weekly and euthanized when tumors reached ϳ2 cm in maximal diameter. Tumor weights and survival were recorded in all instances.

Metabolic studies
Livers or HBs were removed, immediately placed in a sterile tissue culture plate, and maintained on ice while pieces were apportioned for each of the assays described below or immediately snap-frozen in liquid nitrogen. Additional tissues were used for the isolation of mitochondria (25), which were snapfrozen in liquid nitrogen and stored at Ϫ80 ºC. Remaining tissues were apportioned in small aliquots, snap-frozen, and stored at Ϫ80º C for studies not requiring fresh tissue.
For FAO assays, ϳ50 mg of fresh tissue was minced in 350 l of SET buffer at 4°C (25,51,75) and disrupted by five passes in a Potter-Elvehjem homogenizer; 5 l of the homogenate was added to 195 l of FAO reaction buffer containing 100 mM sucrose, 10 mM Tris-HCl, pH 7.4, 0.2 mM EDTA, 80 mM KCl, 5 mM KH 2 PO 4 , 1 mM MgCl 2 , 0.1 mM malate, 0.05 mM CoA, 2 mM L-carnitine, 2 mM ATP, 1 mM DTT, 0.3% fatty acid-free BSA, pH 8.0, and 0.5 Ci/ml 3 H-labeled palmitate conjugated to BSA (PerkinElmer Life Sciences). Reactions were incubated at 37°C for 2 h and stopped with the addition of 40 l of 1 M KOH. They were further incubated at 60°C for 1 h to hydrolyze newly synthesized acyl-carnitine esters. 40 l of 4 M perchloric acid was added, and incubations were continued on ice for an additional 60 min. After organic extraction, water-soluble 3 H-labeled products were measured by scintillation counting.
PDH complex activity was determined as described previously (25,51,75). Briefly, 50 -100 mg of fresh tissue was minced in ice-cold glutamine-, glucose-, and pyruvate-free Dulbecco'smodified minimal essential medium and gently homogenized by several passages through an 18-gauge needle. 0.5-ml aliquots were placed into 0.5 ml of 2ϫ assay buffer containing 0.075 Ci

Myc and ChREBP cooperation in hepatocyte proliferation
of [1-14 C]pyruvate (PerkinElmer Life Sciences). Reactions were incubated at 37°C for 40 min with continuous shaking and terminated by injecting 0.5 ml of 4.0 M perchloric acid through the rubber stopper, which contained a central hanging basket (Kimble-Chase Life Science and Research Products, Rockwood, TN) into which had been placed a 25-mm glass microfiber filter (Whatman/GE Healthcare) saturated with 0.5 M KOH. 14 CO 2 was collected for 40 min at 37°C onto the filters and quantified by scintillation counting.
Respirometry was performed as described previously (25,51,75). Briefly, ϳ50 mg of fresh tissue was disrupted in ice-cold MiR05 buffer. ϳ300 g of the lysate was used to determine OCRs using an Oroboros Oxygraph 2k instrument (Oroboros Instruments, Inc., Innsbruck, Austria). Each reaction contained cytochrome c (10 M) (25), malate (2.0 mM), pyruvate (5 mM), ADP (5 mM), and glutamate (10 mM) to initiate electron flow via Complex I, followed by the addition of succinate to a final concentration of (10 mM) to measure the combined activities of ETC Complexes I ϩ II. Finally, rotenone was added (0.5 M final concentration) to inhibit Complex I and to allow for Complex II quantification. Activities were normalized to total protein content.
To quantify mtDNA content, two probe sets were used to amplify unique regions of mtDNA from livers and tumors using a TaqMan-based approach as described previously (25,51,75). The amplified segments included 101 bp of the cytochrome c oxidase I gene (probe set 1) and 90 bp of the D-loop region (probe set 2). Results were normalized to the product of a third TaqMan reaction that amplified a 73-bp segment of the nuclear apolipoprotein B gene. All reactions contained 10 ng of total DNA and were performed simultaneously on a CFX96 Touch TM real-time PCR detection system (Bio-Rad) using the following conditions: 95°C for 10 s, 40 cycles at 95°C for 15 s, and 60°C for 60 s.
ATP assays were performed on previously snap-frozen tissues using the ATP Lite detection system (PerkinElmer Life Sciences). AcCoA assays were also performed on frozen tissues using an assay kit (MAK039, Sigma-Aldrich). Both assays were performed according to the suppliers' directions. Triglyceride content was measured as described previously (110).
Purified mitochondria were used in nondenaturing blue native gel electrophoresis to separate each of the multisubunit complexes of the electron transport chain (ETC), followed by Coomassie Blue staining (25,51,75). In situ assays for ETC Complexes I, III, IV, and V were performed as described previously, whereas Complex II (succinate dehydrogenase) assays were performed on separately prepared lysates (13,25,111). Enzymatic activity for each sample was quantified by scanning the bands corresponding to enzymatic activity in gels using a Fluorchem M system (Protein Simple, San Jose, CA), and relative activity was compared with that Complex IV, which showed the least sample-to-sample or group-to-group variability.

Western blotting
This was performed as described (13,51,75). Frozen tissues were disrupted in lysis buffer containing protease and phosphatase inhibitors, sonicated, boiled, and stored in small aliquots at Ϫ80°C. The antibodies used, their dilutions, and the vendors from which they were purchased are provided in Table S1.

Transcriptional profiling of livers and HBs
All procedures were performed as described previously (13,75). Briefly, RNA purification from five representative tissues of each group under study was performed using Qiagen RNeasy columns (Qiagen, Inc., Valencia, CA) followed by DNase digestion. Final sample integrity was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Forster City, CA), and only samples with RNA Integrity Number (RIN) values Ͼ8 were processed further. RNA-Seq was performed at the Children's Hospital of Pittsburgh of UPMC Core Genomics Facility. Single-end single-indexed sequencing (ϳ75 bp) was performed with an Illumina NextSeq 500 sequencer (Illumina, Inc., San Diego, CA). Gene expression was calculated using Cufflinks and expressed as fragments per kilobase million (FPKM). Gene expression counts were normalized across samples and statistically compared between experimental groups using Cuffdiff, with a false discovery rate-adjusted p value (or q value) cutoff of 0.05. Raw and processed original data have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (61) and are accessible through GEO (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?accϭ GSE114634) at accession number GSE114634. Ingenuity Pathway Analysis (IPA, Qiagen) was used to categorize differentially expressed genes into pathways, with pathway significance was adjusted for false discovery using Bonferonni-Hochberg correction and a p value cutoff of 0.05. For each pathway, the following were recorded for display: overall significance (p value), ratios of dysregulated genes compared with all possible genes associated with that pathway (ratio), and predicted pathway activation or inhibition (Z-score, where applicable). Not all IPA pathways included the ability to estimate a Z-score. Significant pathways were then grouped into "superpathways" according to function using IPA's internal category scheme. These and their component transcripts can be accessed in an interactive format at https://prochownik.pitt.edu/chrebp/.
For select transcripts (Fig. S11), the same RNAs used for the above-described RNA-Seq experiments were used in qRT-PCRs to validate RNA-Seq results. Each qRT-PCR (20 l) was performed with a Power SYBR Green RNA-to-C T TM 1-Step kit (Applied Biosystems, Inc., Waltham, MA) according to the manufacturer's instructions. Real-time reactions were monitored on a StepOne Plus Real PCR system (Applied Biosystem). Each reaction was performed in duplicate with Ͻ10% variation between samples. A list of PCR primers is shown in Table S11.


extfieldsofstudy: Psychology Medicine

text:
Classical Conditioning: Classical Yet Modern

This manuscript is part of a special issue to commemorate professor Paul Eelen, who passed away on August 21, 2016. Paul was a clinically oriented scientist, for whom learning principles (Pavlovian or operant) were more than salivary responses and lever presses. His expertise in learning psychology and his enthusiasm to translate this knowledge to clinical practice inspired many inside and outside academia. Several of his original writings were in the Dutch language. Instead of editing a special issue with contributions of colleagues and friends, we decided to translate a selection of his manuscripts to English to allow wide access to his original insights and opinions. Even though the manuscripts were written more than two decades ago, their content is surprisingly contemporary. The present manuscript was originally published as part of a Liber Amicorum for Paul Eelen’s own supervisor, prof. Joseph Nuttin. In this chapter, Paul Eelen presents a modern view on Pavlovian learning. It appeared in 1980, at the heyday of cognitive psychology which initially dismissed conditioning. Paul Eelen’s perseverance in presenting learning principles as key to study human behaviour has proven correct and ahead of time. First published as: Eelen, P. (1980). Klassieke conditionering: Klassiek en toch modern. In Liber Amicorum, Prof. J. R. Nuttin, Gedrag, dynamische relatie en betekeniswereld (pp. 321–343). Leuven: Universitaire Pers Leuven.

Even though ever more complex areas of research have found their way into psychology, "Pavlov's dog" continues to fascinate many researchers. What causes the enduring fascination with conditioning research? Does such research even have psychological significance? Would it not be better if it remained a study field for physiologists, as it originally was? The answers to these questions are partly determined by one's conceptualization of classical conditioning. Most people are by now sufficiently familiar with its schematic representation: a conditioned stimulus (CS) elicits a conditioned response (CR), provided this stimulus has repeatedly been presented together with an unconditioned stimulus (US) that "inherently" elicits an unconditioned response (UR). Several limiting conditions qualify this schematic depiction. The CS must be "neutral" vis-à-vis the US. In other words, it cannot spontaneously elicit a response that is identical to the UR. The US must "inherently" Eelen, P. (2018). Classical Conditioning: Classical Yet Modern. Psychologica Belgica,58(1),DOI: https://doi.org/10.5334/pb.451 Jan De Houwer and Tom Beckers acted as editors for this translation. The original text has been edited slightly, in part on the basis of comments of an independent reviewer on an initial version of the translation. Parts of the section on taste aversion have been deleted. A full length version of the translation can be found at https://doi. org/10.17605/OSF.IO/VP5CU Eelen: Classical Conditioning 197 elicit a well-defined response, which is why stimuli that are biologically significant for the studied organism are typically used (for some theorists, this became a necessary condition for conditioning to take place). The resulting CR must be an autonomous response that is part of the reaction pattern that the US evokes. This schematic depiction also provides insight into the necessary (and sufficient) conditions for conditioning to occur: both stimuli have to occur simultaneously. Finally, this schematic representation already implies what is learned: learning is equated with the modified reaction pattern vis-à-vis the CS. To put it simply, the dog learns to salivate at the sound of the bell. This schematic depiction and the limiting conditions it implies constitute a strong simplification of the original phenomenon. After all, Pavlov's interest in conditioning originated from his observation that the dog started to salivate when it heard and saw the man who brought the food. This rather complex event -someone who brings food -was ultimately reduced to a little lamp or an auditory signal predicting food. The "food" event of seeing a meat chunk in a bowl was reduced to the injection of meat powder directly into the animal's mouth. The dog's overall reaction pattern upon hearing the man who brings the foodand anyone who has a dog will be familiar with this pattern -was ultimately reduced to droplets of saliva (the reductive nature of this response was already highlighted by Zener, 1937). Moreover, the autonomous reaction that held Pavlov's primary interest as a physiologist was initially not viewed as a core index of learning the relation between two events, but was subsequently seen as an almost integral part of the definition of classical conditioning (Gormezano & Kehoe, 1975). We can probably all concur with Rescorla and Holland's related observation that "if conditioning were confined to what some have called "spit and twitches", it would lose much of its psychological interest" (Rescorla & Holland, 1976, p. 184).
This strong reduction of the original events is probably characteristic of every type of operationalisation. This is justified in and of itself: operationalisations that reduce a phenomenon to its essence are vital for obtaining fundamental knowledge about the necessary and sufficient conditions that determine the occurrence of that phenomenon. But the danger exists that the question behind a concrete operationalisation is simply forgotten after a while. Moreover, there is a real danger that general statements and laws are formulated that are strongly connected to the concrete operationalisation. Something along those lines certainly happened in the study and appreciation of classical conditioning. The aim of this contribution, then, is to shed light on a number of recent trends in classical conditioning studies that might justify the title of this contribution. First, I summarize the most important findings that call for a broader framing of classical conditioning research. This is followed by a comprehensive discussion of one particular form of learning, that is, taste aversion that results from relations between the taste of food or drink on the one hand, and artificially induced nausea on the other hand. This phenomenon is sometimes referred to as the Garcia effect. The topic of taste aversion is discussed not because it is an almost prototypical example of classical conditioning, but because it contributed substantially to the questioning of important assumptions about conditioning. A number of authors have even called this the beginning of a "paradigmatic revolution" (Rozin, 1977;Bolles, 1975). The final part is somewhat speculative in nature: using the preceding observations as a starting point, it argues that a nontrivial similarity exists between recent theories in classical conditioning studies and those in a literature that at first glance appears to bear little relation to it, that is, attribution theories in social psychology.

Classical conditioning: learning associations between two events
Every existing organism must in some way or another be sensitive to both meaningful as well as more coincidental relations between events in the environment, especially when such relations concern biologically significant events. At the same time, it would be maladaptive for an organism if the mere coincident occurrence of two events would be a sufficient condition for the organism to establish a connection between the two. Nevertheless, a coincident occurrence has often been considered a sufficient condition for learning a relation. When doubts were expressed about this idea, they concerned the nature of either one of both events (does one of the events need to have reinforcement value) rather than the nature of the relation itself (i.e., co-occurrence). What follows will demonstrate that every organism can process a wider range of informational relations than the mere joint occurrence of events. In describing this broad range, we aim to list general facts rather than to go deep into possible explanations.

The role of contingency
Instead of using terms indicating co-occurrence, relations can also be expressed in terms of correlation or contingency. What is emphasised in this case is not the temporal relation between two events but their logical relation. Applied to the situation of Pavlov's dog, this means that a perfect positive correlation is introduced between the CS and the US in the experimental context. In other words, the conditional probability that the US is presented, given that the CS has been presented, equals 1; the probability that the US is presented in the absence of the CS equals 0. This is symbolically expressed as ρ(US/CS) = 1.0 and ρ(US/°CS) = 0. This can probably be further illustrated using what Seligman, Maier and Solomon called "The Pavlovian contingency space" (cf. Figure 1).
Given that the essence of the classical conditioning procedure lies in the experimenter's full control over the two stimuli that are presented, this paradigm lends itself superbly to a study of the effects of variations in the correlational strength of CS-US relations. Rescorla (1968) was one of the first to study this issue systematically. Over several experiments (Rescorla, 1975), he demonstrated that animals are sensitive to variations in contingency, ranging from a perfect positive correlation to a perfect negative correlation (respectively below and above the diagonal in Figure 1.) In this way, he succeeded in translating Pavlov's two most important findingsexcitatory and inhibitory conditioning -into contingency terms. Excitatory conditioning occurs whenever the animal learns that the CS and US tend to go together, in other words when ρ(US/CS) > ρ(US/°CS). A large number of behavioural indices then allow one to determine that the animal is behaving as if it "expects" the US when the CS is presented. Inhibitory conditioning occurs when the animal learns that the US and the CS tend not to go together, ρ(US/CS) < ρ(US/°CS). In this case, when the CS is presented, the animal will behave in a manner that is opposite to how it would behave in excitatory conditioning. When the CS and US are "randomly" presented, with no relation between both stimuli in other words, or ρ(US/CS) = ρ(US/°CS), it is observed that the CS does not acquire a new significance for the animal; in other words, x-axis represents the conditional probability that the unconditioned stimulus (US) occurs together with the conditioned stimulus (CS). The y-axis represents the probability that the US occurs without the CS. There is no contingency between both stimuli on the diagonal line where both probabilities are equal (after Seligman, Maier and Solomon, 1971).
the CS does not elicit a differential reaction. This nonetheless represents a form of active learning: learning that there is no relation is not synonymous to not learning (Mackintosh, 1973;Seligman, 1969). Let us illustrate this rather abstract formulation for what is known as "fear conditioning", which is usually operationalised through the administration of an electrical shock as a US and an external stimulus (e.g., a visual signal) as a CS. When, within the context of the experiment, the probability of a shock increases after the presentation of a given visual signal, this stimulus acquires a signalling function for the shock: the animal will behave "anxiously" when the CS is presented. If, however, the chance that a shock is administered is lower after the visual stimulus than in the absence of that stimulus, the animal will behave in a fairly "relaxed" fashion when the CS is administered. When the visual signal and the shock are "randomly" presented, the visual signal does not acquire a special meaning. Instead, the context as a whole becomes "fear-inducing" to the animal (Seligman, 1968).
The need for a contingent relation already indicates that mere co-occurrence is not a sufficient condition for an organism to learn the relation between two events. Some way or another, the organism is sensitive to the predictive value of stimuli and the covariance of events in its environment. What follows will illustrate that even a perfect contingency does not constitute a sufficient condition.

Latent inhibition
Lubov (1973) coined the term "latent inhibition" to describe the following observation: when a stimulus is repeatedly presented by itself (i.e., without a US) in a particular context and when it is subsequently always followed by a US, it is difficult to obtain conditioning. It is not all too clear whether one should invoke non-associative or associative principles to explain this phenomenon. On the one hand, it could be argued that the organism no longer is attentive to the stimulus and, as it were, no longer even notices the stimulus because it has repeatedly been presented in the past. On the other hand, it could be argued that the organism has learned that the stimulus is irrelevant because the stimulus has repeatedly been presented on its own and that it afterwards struggles to realise that it is precisely this stimulus that should be considered the signal for an important event (Mackintosh, 1975). There is a certain similarity between latent inhibition and what occurs when a US is administered repeatedly before it is preceded by a CS. Here too, the results show that it is difficult to make this CS acquire the function of a signal for the US. For instance, when a series of shocks are administered in a non-contingent fashion and every shock is afterwards preceded by a tone in that same context, it takes a long time for the organism to learn this tone-shock relation. Again, the explanation for such data can be sought in non-associative or associative principles (Randich & Lolordo, 1979).

Overshadowing
When two stimuli are presented together and consistently followed by a US, often only one of those stimuli will acquire the function of a signal for the US. Pavlov (1927) already discussed this phenomenon extensively and related it to the difference in "saliency" of the stimuli (as determined by the modality and intensity of the stimuli). Formal classical conditioning models have built in this " saliency" as a parameter -either as a fixed value (Rescorla & Wagner, 1972) or as a fluctuating value in accordance with the relation to the US (Mackintosh, 1975).

Relative information value
Suppose that stimulus A and B are presented together and followed by an electric shock. In group I, stimulus B is also presented separately but not followed by a shock in between the A+B presentations. In group II, B is also presented separately every now and then, but here it is followed by a shock. In group III, only A+B trials are presented. The question is what happens to the signal value of stimulus A. A and a shock are after all paired Eelen: Classical Conditioning 200 an equal number of times in all groups. The relative information value of A, however, varies between groups because B is presented separately in groups I and II. In group I, A becomes the best predictor for a shock. B is a better predictor in group II, while the information value of both stimuli is equal in group III. When A is now separately tested in the three groups, conditioned responding varies in accordance with the manipulated information value (Wagner, 1969).

Blocking
No phenomenon has probably made a larger contribution to clarifying the complexity of conditioning than blocking. It would be impossible to comprehensively list the relevant literature. We will therefore limit ourselves to a description of the basic phenomenon. Kamin (1969) was the first to bring this phenomenon to light in his "overshadowing" studies. Stimulus A (e.g., a visual signal) is frequently followed by an electric shock. When the conditioning is complete, stimulus B (e.g., an auditory signal) is presented together with stimulus A, and both are followed by a shock. B does not acquire a signal value even though there is a perfect correlation between B and a shock from this moment onward. This is evident from the fact that when B is presented on its own, it does not elicit a response. It is as if the previous conditioning of A is blocking the conditioning of B, hence the term. There are indications that the animal does notice stimulus B, but that it learns as it were that B presents irrelevant or at least redundant information about the US (Mackintosh, 1978).

Conclusion
The above information clearly indicates that mere stimulus co-occurrence is not a sufficient condition for an organism to relate two events. The discussion below will demonstrate that it is also not a necessary condition, which again offers a different perspective on classical conditioning. Instead of an automatic process that plays out in a passive organism, the organism emerges as an active information-processing system. It is probably possible to relate all the phenomena that were discussed above to the role of contingency. But the question remains what mechanism can be invoked for explaining the role of contingency. Some do not hesitate to postulate that the animal has a cognitive representation of the contingency space (Alloy & Seligman, 1979). Others have drawn more cautious conclusions. As Rescorla notes: "Most of us are not comfortable with the notion that organisms take in large blocks of time, count up numbers of US events, and somehow arrive at probability estimates … It is tempting to think of simple "tricks" that the organism could use to perform in this apparently rational fashion" (Rescorla, 1969, p. 84-85).
In other words, being influenced by a correlational relation does not ipso facto imply that the organism concerned has any understanding of this correlation. It is therefore remarkable that Rescorla, who perhaps highlighted the role of contingency more than anyone else, succeeded in developing a theory in which the learning of relations can be traced back to the co-occurrence of two events after all (Rescorla & Wagner, 1972). At the level of formalisation, this theory remains purely descriptive. We would like to note, however, the psychological intuition on which it was built (Rescorla, 1969). The notion of "expectation discrepancy" is central here. As soon as something (important) happens unexpectedly -in other words, it was not predicted -it is as if the animal starts searching for a predictor for this event. Expectation discrepancy appears to be a necessary condition for a stimulus to be interpreted as the signal for this unexpected event. No new learning occurs when either the context (see latent inhibition) or other signals (see blocking) had already predicted the event. This "expectation discrepancy" also explains inhibitory conditioning: when an event that an organism expects to occur in a particular context does not occur, a stimulus that is correlated with this expectation discrepancy may acquire an inhibitory function. Note that this theory emphasizes the role of the environment and the organism's prior history. We deliberately use metaphors like "to start searching for a predictor", "to interpret an event" etc. It is as if the facts can only be described in such terms. Such language becomes even more imperative when describing taste aversion.

Taste aversion: The Garcia effect
A short article by Garcia and Koelling published in Psychonomic Science in 1966 was the starting point of the literature on what is now known as the Garcia effect. At the time, there was nothing to suggest that the article would become a classic. Quite the contrary, the article had been rejected by a more renowned journal, which the then editor would later express his regrets about. As is often the case with "classics", the article was indeed rather weak at the methodological level, but it contained fairly far-reaching theoretical implications. Today, these are referred to as the "Garcia effect", "the message of Garcia" and "the paradigmatic revolution". At least 600 articles that were more or less inspired by the Garcia effect have been published since then. This exceptional level of attention does not guarantee scientific relevance in itself. Garcia's findings may have originally been called into question due to their methodological shortcomings, but the extensive attention has at the least ensured sufficient subsequent independent replications of Garcia's experiments. The phenomenon is real. The debate about its reach and interpretation, however, remains active today. We first discuss the meaning of "the message of Garcia" by describing a couple of typical experiments. We subsequently reflect on the varying attempts that have been made to interpret this phenomenon.

"The message of Garcia"
What the message of Garcia essentially revolves around is probably best illustrated with an anecdote recounted by Seligman (Seligman & Hager, 1972). After he was served "filet mignon with béarnaise sauce" during a dinner, he became unwell at night. This nausea later proved to be a harbinger of a flu attack. But Seligman had already ascribed it to the béarnaise sauce, and since then he cannot suffer the look, let alone the taste of this sauce. This anecdote raises several questions. Why did he "ascribe" his becoming sick to the béarnaise sauce? Why not to the filet, the dessert or the drinks? Why not to the restaurant or the other guests? Why did his aversion to béarnaise sauce not disappear when it later turned out that the flu was a far more likely cause? Why did béarnaise sauce taste so bad since then? It turns out that answering these questions becomes difficult when this event is translated into a conditioning paradigm, with flavour as the CS and becoming sick as the US (or UR). The most noticeable departures from the normal rules are the extended time period between the CS and US and the difficulty of the extinction, even after the adjusted interpretation. The nature of the US moreover appeared to determine the selection of the CS and, finally, a process that is qualitatively different appears to be at stake here: the béarnaise sauce is avoided not because it is seen as a predictor of nausea, but because it acquires an intrinsically bad flavour.
Garcia's studies evoke similar questions. The discussion of a typical study will illustrate this further. Garcia and Koelling (1966) deprived caged rats of water for the duration of the experiment. Every day, the rats were placed in individual test cages that contained a drink tube. After an adjustment period in which clean water was offered, the learning phase began. The water was replaced by a saline solution. Every time the drink tube opening was touched, a visual and auditory stimulus were presented so that every drinking attempt was paired with a "brightnoisy-tasty" constellation of stimuli. In the first group, drinking coincided with a period of radiation (X-rays). 1 In the second group, lithium chloride was used as the saline solution; this has a poisonous effect but the rats cannot distinguish it from a non-poisonous saline solution (Nachman, 1963). In a third group, an electric shock was administered two seconds after drinking. This learning phase was spread over several days in all the groups. On non-conditioning days, the test cage contained only normal water, the drinking of which was not paired with the abovementioned constellation of stimuli. This was followed by a test phase in which either the audiovisual stimulus or the flavour (saline solution) without the audiovisual stimulus was presented during the drinking of clean water. In the X-ray and lithium groups, there was a clear suppression of drinking with the flavour test but not with the audiovisual test, while precisely the opposite occurred in the shock group. In other words, there appears to be an interaction between the nature of the discriminative stimulus and the drinking consequences.
At first glance, several findings regarding conditioned taste aversion indeed contradicted the basic rules of conditioning. First and foremost, there was a clear parametric difference with more typical conditioning preparations: the time interval between the taste CS and administration of the aversive US (induced sickness) was typically much larger. "Records" of 24 hours of difference were set (Etscorn & Stephens, 1973)! In the experiment described above, an interaction moreover exists between the nature of the CS and the nature of the US, which is probably the finding that has prompted the most discussion: It is not possible to learn an association between whichever two things. Finally, we already noted that we are seemingly dealing with a qualitatively different phenomenon.

Theoretical reflections
The different attempts to explain flavour aversion can be separated into two main orientations. A first orientation refers to the biological nature of every organism. Through the course of natural selection, every organism has come to be equipped with specific learning mechanisms that, depending on the organism's adaptation, show specific characteristics as a function of the different challenges the animal faces in its environment. For instance, it is indeed vitally important for an animal to learn the association between certain food attributes and certain metabolic effects. A second orientation attempts to reconcile the properties of flavour aversion with the more general fundamental rules that govern the learning of relations between two events. It does not deny that parametric and perhaps qualitative differences clearly exist between learned taste aversion and the more conventional conditioning findings. But these differences supposedly originate from the particular characteristics of the used stimuli. Insofar that these characteristics can be described, their influence can be assessed through experiments -independent of the flavour aversion phenomenon.
It is indeed remarkable that all the factors that influence learning of an association between two events (cf. below) also have an influence on learned flavour aversion. First, there is the impact of contingency. Inducing a flavour aversion requires a positive correlation: "random" administration of a flavour and US does not have an effect, and a negative correlation between a flavour and the US results in a preference for this flavour (Best, 1975). Latent inhibition is also possible: flavour aversion is slow to develop when the animal is made to taste a certain flavour repeatedly before it is paired with an aversive substance (Domjan, 1972;Elkins, 1973). It is equally clear that when the animal is first repeatedly made ill in a way that is noncontingent to ingestion of a particular food, the animal subsequently no longer ascribes this becoming sick to the flavour of the food (Braveman, 1977). "Blocking" finally has also been demonstrated; a learned aversion to a particular flavour can "block" learning of aversion to a different flavour (Revusky, 1971). It is important to note a study by Rudy, Iwens and Best (1977) in this regard. They first induced a contingency between an external stimulus (black cage) and nausea. When the flavour of saccharine was subsequently involved in this contingency, the animal no longer ascribed the nausea to the flavour. This study is important in two regards. First, it demonstrates that associations between external stimuli and "nausea" can indeed be learned as long as an external stimulus is used that is fairly salient and that can compete with a flavour stimulus in terms of "novelty". In addition, the results of this experiment certainly do not appear to correspond to what one would expect from a "preparedness" view. If learning of a flavour-nausea contingency is truly "prepared", it does not seem very plausible that learning of this contingency can be fairly easily "blocked" by a pre-induced artificial or at least unprepared contingency.
These findings indicate that the Garcia effect is not as extraordinary as it appears to be at first glance and that it can in fact be integrated into the more general findings about association learning (Logue, 1979). The particular characteristics of the Garcia effect, however, have urged reflection on the more conventional procedures from a different perspective.
Consider, for instance, the parametric difference between flavour aversion and setups that are more conventional in terms of the time lapse between the CS and US (or between the discriminative stimulus and reinforcement). The hypothesis of an afterflavour during nausea was of course the most simple one, but it was emphatically rejected empirically (Revusky & Garcia, 1970). It is therefore almost certain that the Garcia effect is due to a memory phenomenon. When the rat becomes sick, he "remembers" the type of food that may have caused this. Revusky (1971Revusky ( , 1977 integrates these findings into what he describes as a more general associative interference theory. This theory inspired Lett (1973Lett ( , 1974Lett ( , 1975Lett ( , 1977 to demonstrate that a rat is capable of bridging a fairly large time interval between a discriminative stimulus and reinforcementand this with more conventional procedures. For this to happen, however, the situation must be designed so that the animal is urged to again "call to mind" the discriminative stimulus during reinforcement. It is remarkable that the Garcia effect, which is so deeply rooted in the biological singularity of the organism, is an illustration of the animal's cognitive capabilities and that it has helped integrate recent findings in the psychology of memory into conditioning studies (Best & Gemberling, 1977;Wagner, 1978).
In addition, there is the interaction between the nature of the CS and the US. This interaction is in fact only exceptional when one merely considers the external characteristics of a relationship between two events (contiguity, contingency). It becomes more comprehensible when one assumes that other factors also exist that influence the learning of relations, such as similarity, spatial factors, etc. This insight was probably best articulated by Testa (1975Testa ( , 1976; he related learned flavour aversion to the more general question of how the animal perceives causal relations in its natural environment. He argues for factors to be integrated into the study of conditioning that had already been previously underlined by gestalt psychologists in relation to perception. We find a similar plea in Revusky (1977) and Rescorla and Cunningham (1979).

Conditioning and attribution
After demonstrating how an organism can process complex relations, we have presented a discussion of learned flavour aversion because the latter highlights a central problem: what pushes the animal to selectively attribute certain effects to the ingestion of food or drink when both events are so far removed in time? Rather than viewing this simply as an innate mechanism, it was argued that this phenomenon should be integrated as much as possible into what we know about the learning of associations between events. What then is the meaning of all of this? It seems that there is a fundamental similarity between these findings in the conditioning literature and attribution theory as it was developed in the social psychological literature. This observation suggests that common principles exist that cause both humans and animals to discover causal relations in their environment. Such a speculative observation probably requires a number of prior explanations. Attribution theory is the study of the manner in which certain events are explained in terms of their potential causes. Born from the field of social psychology -a historic coincidence in Kelley's view (1967) -the main topic of attribution research was people's causal analysis of the behaviour of others and one's self. In other words, on the basis of which rules do I infer the "why" behind my own or other people's actions? But in essence, a much broader question is at stake in attribution theory: how does one make causal inferences between all sorts of events? The question even arises whether a clear distinction ought to be made between a causal interpretation of events and a causal interpretation of actions. The distinction between "cause" and "reason" is key here, and it was also the focus of a recent discussion (Buss, 1978;Harvey & Tucker, 1979;Kruglanski, 1979;Buss, 1979). Only causal interpretations of events will be discussed below. To put it in more trivial terms, when a rat is administered a shock by the experimenter, it might ask itself: What is this shock due to (asks after the cause)? It does not ask: why did this experimenter give me a shock (asks after the reason)? Both are "why" questions, but they are logically different from each other. Second, a distinction should be made between the attribution process and the content of the attributions (Nisbett & Wilson, 1977;Kruglanski, 1979). At the level of contents, it is obvious that any animal-human comparison would be a tenuous one. But this is also true for a comparison between mutual humans, if only because of cultural differences (Kruglanski, 1979). As regards attribution as process, it is probably possible to arrive at more general statements about the heuristics that apply to both humans and animals. A third introductory remark concerns the status of the concept of attribution. Attribution is intended as a "mediating" concept that can either be assigned a reality value or an "as if" nature. This is true for most "cognitive" concepts that were designed to mediate between input and output (consider, for instance, the concept of "expectancy"). In our view, there is a trend towards increasing emphasis on the "as if" nature of attributions in social psychology. The most common descriptions -somewhat schematically -present this sequence as follows: 1. something occurs (S) -2. the organism asks itself "why" -3. following deliberation, it arrives or does not arrive at a judgement -4. it acts in a manner that is consistent with this (R). If assigned a reality value, it is possible to render the typically non-observable links 2. and 3. observable in humans by simply inquiring after them. It would not be exaggerated to state that this is the focus of most attribution research. And any study of attributions in animals is of course impossible in this respect. But it has been asked more and more whether the "links" do not acquire a different status precisely because they are made observable. Let us again briefly go back to Seligman's anecdote about the béarnaise sauce. If Seligman is asked: "Why did you become sick?", he will answer: "Because I had a flu attack." In other words, does a "conscious" reflection on the occurrence of an event not respond to different rules than the total original experience of this occurrence? Is this not where the truth lies of Pascal's statement that "le coeur a ces raisons que la raison ne connaît pas"? In a rather extensive article, Nisbett and Wilson (1977) defended the proposition that these cognitive mediating processes circumvent every form of introspection. To support their argument, they cited a number of statements by cognitive psychologists, including Neisser and Mandler, that we would like to cite here. For instance, Neisser writes that "the constructive processes (of encoding perceptual sensations) themselves never appear in consciousness, their products do" (Neisser, 1967, p. 301). And Mandler considers that "there are many systems that cannot be brought into consciousness, and probably most systems that analyze the environment in the first place have that characteristic. In most of these cases, only the products of cognitive and mental activities are available to consciousness" (Mandler, 1975, p. 245). Although Nisbett and Wilson's proposition is debatable (Smith & Miller, 1978), Langer (1978) does not hesitate to go one step further: she simply denies the mediating role of conscious cognitions in most of our day-today actions: "Much psychological research relies on a theoretical model that depicts the individual as one who is cognitively aware most of the time, and who consciously, constantly, and systematically applies "rules" to incoming information about the environment in order to formulate interpretations and courses of actions. Attribution theorists rely on this model in attempting to uncover the sources of regularities in human behaviour. But if in fact it can be demonstrated that much complex human behaviour can and does occur without these assumed cognitive assessments, then we must question both pervasiveness of attribution making as a cognitive process and the assumptions made by most social psychologists" (Langer, 1978, p. 35). We find a similar plea to look for very simple heuristics to explain the notion of attribution in Kahneman and Tversky (1973), Pryor and Kriss (1977) and especially Taylor and Fiske (1978) who concluded that most attribution processes "seem to occur automatically and substantially without awareness, and as such, they differ qualitatively from the intentional, conscious, controlled kind of search which we like to think characterises all our behaviour" (Taylor & Fiske, 1978, p. 283).
These introductory explanations probably create more room for the proposition that there is something common about the way that humans and animals infer causal relations. We rely on a recent overview article by Kelley and Michela (1980) on attribution theory to buttress this claim in a more direct way. They first offer the general scheme that is implicitly contained in the study field of attribution (Figure 2).
As indicated above, any type of direct research into (2) is evidently impossible with animals. One has to limit oneself to manipulations of (1) and inferring what happens in (2) from a change in (3). But as also noted above, this limitation probably also applies to studies of attribution in humans. On the antecedent side (1) then, there is a clear similarity between the factors influencing the nature of attributions and conditioning. Let us illustrate this using the principles that Kelley and Michela distilled from attribution literature. These principles hold that certain aspects of the information that the organism is confronted with lead to attributions. Almost every one of these is a principle that we already mentioned in our discussion of the factors that influence conditioning.
• Covariance: The ANOVA model. This principle was primarily emphasised by Kelley himself (Kelley, 1967(Kelley, , 1973. "The effect is attributed to that condition which is present when the effect is present and which is absent when the effect is absent" (Kelley, Figure 2: Schematic model of attribution research (after Kelley & Michela, 1980). 1967. This covariance principle is of course heavily analogous, if not identical, to the role of contingency in classical conditioning. This raises a twofold observation: first, there is no reason to suppose, as Kelley does, that the influence of this covariance principle must revert to a model of the human as a "naïve" scientist who thinks according to an ANOVA model (he probably only does so in the context of attribution experiments!). As noted above, the influence of contingency does not necessarily imply that the organism has any notion of contingency. In addition, there is the dilemma of moving from a correlation judgement to a causal judgement. A causal relation after all implies a correlation, but the reverse does not hold. Is a causal judgement only possible when one implicitly also has knowledge of the mechanisms that connect cause and effect? Or are other conditions necessary in addition to perfect correlation for two events to be perceived in a cause-effect relation? This dilemma brings us to the question posed by Michotte (1954): Is causality a phenomenal experience or a "post hoc" reflection? It is interesting to note in this regard that Testa (1974) relied on Michotte's findings to explain flavour aversion. • Saliency: "The notion here is that an effect is attributed to the cause that is most salient in the perceptual field at the time the effect is observed." (Kelley & Michela, 1980, p. 466). This "saliency" is again a factor that plays a role in conditioning (see below). • Similarity and Contiguity: The principle of contiguity does not require much explanation to be related to conditioning. Rescorla and Furrow (1977) convincingly demonstrated the role of "similarity", which has always been seen as an associative principle, within a conditioning paradigm. • Primacy: "The general notion here is that a person scans and interprets a sequence of information until he attains an attribution from it and then disregard later information or assimilates it to his earlier impression" (Kelley & Michela, 1980, p. 467). Conditioning literature analogies also exist for this. It for instance takes a long time for the animal to recognise a "random" relationship such as when a tone and a shock are "randomly" presented but this random series begun with a contingency between both events. The reverse is also true: a contingency is also learned with difficulty in the case of a random start and subsequent contingency (Alloy & Seligman, 1979).
Conditioning literature parallels also exist for the interaction between the nature of the information on the one hand and the existing "beliefs" or causal models and the motivational component (Figure 2) on the other. The "blocking" phenomenon can be considered a causal model that interferes with the learning of other causal relations: both humans and animals do not look for every possible cause but instead suffice themselves with one sufficient cause. In addition, the motivational component has always been central to conditioning. To explain this using anthropomorphic terms, the animal only asks itself a why-question when something important occurs. We here touch on a point that made us relate the notion of attribution to findings on conditioning. We prefer the term attribution over the term association to denote what happens during conditioning. Not only because "association" is a historically heavily charged concept, but because the term does not permit a distinction between the propositions "event X reminds me of event Y" and "I ascribe event X to event Y". To again illustrate this using Seligman's example: when he becomes nauseous, he can perfectly remember the full dinner event, but only one relation, one "attribution" is made with the béarnaise sauce. Remembrance is a necessary but not a sufficient condition to establish a causal relation between two events. Winograd (1971) described this as follows: "Let us imagine that I emerge from my house in the morning and find a flat tire on my car. It occurs to me immediately that around nine o'clock the previous evening, while driving home, I heard a disturbingly loud noise as I drove over something in the road. Now, 12 hours later, I "associate" the flat tire with the impact. This is not an association in the usual S-R contiguity sense; rather, I have related two events which were separated by a long period of time. I can do this only if I have a record of the earlier event, or memory. In fact, I have many memories of previous events, and that is the problem. The question to be dealt with is one of trace selection or contact, of how I have related these two particular events" (Winograd, 1971, p. 272-273). This, precisely, is the dilemma posed by the Garcia effect, looked at from a different perspective. It is why use of the term "attribution" rather than the term "association" becomes even more imperative when we keep the phenomenon of flavour aversion in mind. As Revusky and Garcia write, "Probably, the rat can really associate these events, but will not attribute the production of shock to the flavored water. In other words, a rat can learn that consumption of flavored water precedes shock, but will not readily learn that consumption of flavoured water produces shock" (Revusky & Garcia, 1970, p. 41). A bit further, both authors write: "This paper would probably be more precise if, whenever the term "association" is used, "attribution" were to be substituted" (p. 43). Does, after this discussion, it still seems absurd that an animal responds "as if" it were making an attribution?

Conclusion
Of course, a change in terminology is only a pseudo-solution to a dilemma that has been key since Pavlov's dog: How is a relation between two events learned? This contribution offers only limited insight into this question, and it is quite fortunate that the effective learning of such relations does not depend on its explanation. But the search for such an explanation becomes imperative the moment it is established that the learning of relations fails. Because this probably constitutes a true breeding ground for human and animal suffering: the inability to explain an important event.
Finally, this contribution might foster the impression that contemporary conditioning psychology tries to anthropomorphise the rat too much, when in the past humans were seen too much as rats. But a rat is a rat and a human a human. Nevertheless, it does not seem very fruitful to me to hermetically seal off both study domains. Whereas Estes notes that "the thought arises that the processes and mechanisms of human cognition represent specializations and elaborations of processes and mechanisms which can advantageously be studied in animals that learn as well as in machines that think" (Estes, 1975, p. 6), this contribution was written from the conviction that Estes' first alternative continues to be valuable. For as long as a computer does not salivate upon seeing a chunk of meat, "Pavlov's dog" continues to be a fascinating phenomenon.

Note
1 The use of X-rays in this experiment probably calls for some explanation about the prior history of the Garcia effect. Commissioned by the ministry of defence, Garcia and his collaborators completed a series of investigations into the influence of ionising radiation on animal behaviour during the fifties. Their most important finding was that such radiation -even when administered in small doses -had an aversive nature to the animal, and the behavioural component that this could most clearly be inferred from was the strongly reduced drink and food intake. It proved much more difficult, however, to use these radiation effects to teach spatial avoidance behaviour (Garcia, Kimeldorf & Hunt, 1961).

Competing Interests
The author has no competing interests to declare.