Anthony Sclafani: Consummate scientist

Accepted Manuscript Anthony Sclafani: Consummate scientist Joseph R. Vasselli, Gerard P. Smith PII:

S0195-6663(16)30959-X

DOI:

10.1016/j.appet.2016.12.024

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APPET 3269

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Appetite

Received Date: 20 October 2016 Accepted Date: 18 December 2016

Please cite this article as: Vasselli J.R. & Smith G.P., Anthony Sclafani: Consummate scientist, Appetite (2017), doi: 10.1016/j.appet.2016.12.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Anthony Sclafani: Consummate Scientist

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Joseph R Vassellia and Gerard P Smithb

New York Obesity-Nutrition Research Center, Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY, USA

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Department of Psychiatry, Weill Cornell Medicine, Payne Whitney Westchester, New York-Presbyterian Hospital, 21 Bloomingdale Road, White Plains, NY

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Address for Correspondence:

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Joseph R. Vasselli, Ph.D. Columbia University Obesity-Nutrition Research Center Russ Berrie Medical Pavilion 1150 St. Nicholas Avenue – Suite 121 New York, NY 10032-3702 Phone: 212 342-6978 Fax: 212 851-5579 Cell: 203 640-4078 E-mail: [email protected]

Keywords: hyperphagia, VMH syndrome, diet-induced obesity, palatability, carbohydrate appetite, taste preference

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Abstract In this article we review the scientific contributions of Anthony Sclafani, with specific

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emphasis on his early work on the neural substrate of the ventromedial hypothalamic

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(VMH) hyperphagia-obesity syndrome, and on the development of diet-induced obesity

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(DIO). Over a period of 20 years Sclafani systematically investigated the neuroanatomical

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basis of the VMH hyperphagia-obesity syndrome, and ultimately identified a longitudinal

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oxytocin-containing neural tract contributing to its expression. This tract has since been

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implicated in mediating the effects of at least two gastrointestinal satiety factors. Sclafani

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was one of the first investigators to demonstrate DIO in rats as a result of exposure to

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multiple palatable food items (the “supermarket diet”), and concluded that diet palatability

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was the primary factor responsible for DIO. Sclafani went on to investigate the potency of

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specific carbohydrate and fat stimuli for inducing hyperphagia, and in so doing discovered

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that post-ingestive nutrient effects contribute to the elevated intake of palatable food

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items. To further investigate this effect, he devised an intragastric infusion system which

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allowed the introduction of nutrients into the gut paired with the oral intake of flavored

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solutions, an apparatus her termed the “electronic esophagus”. Sclafani coined the term

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“appetition” to describe the effect of intestinal nutrient sensing on post-ingestive appetite

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stimulation. Sclafani’s productivity in the research areas he chose to investigate has

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been nothing short of extraordinary, and his studies are characterized by inventive

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hypothesizing and meticulous experimental design. His results and conclusions, to our

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knowledge, have never been contradicted.

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Abstract Word Count: 245

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1. Introduction If those of us who know Anthony (Tony) Sclafani were asked to provide a term describing his qualities as an investigator, the term most often used would undoubtedly

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be some version of “consummate scientist”. This is the character of the man we have

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come to know through his prolific contributions to the study of feeding behavior and its

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neurological and physiological bases. His work spans more than four decades of

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sustained research, and includes studies of hypothalamic hyperphagia, diet palatability,

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dietary obesity, intestinal bypass, carbohydrate appetite, and more recently flavor-nutrient

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conditioning and the role of intestinal nutrient receptors in ingestive behavior.

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Remarkably, Sclafani has made landmark discoveries and original contributions to our

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knowledge and techniques in all of these areas. His studies are characterized by

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inventive hypothesizing and meticulous experimental design, and his productivity is

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nothing short of extraordinary.

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Sclafani first became interested in the study of feeding as a result of a seminar he

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took with Sebastian (“Pete”) Grossman as a beginning graduate student at the University

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of Chicago in 1966. “In my first year at Chicago I took a seminar course with Grossman

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in which each student had to review a brain area. I chose the hypothalamus, and among

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the topics I reviewed (temperature control, aggression, thirst, hunger, etc.) I became most

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interested in hunger and feeding. Therefore, when I joined Pete’s lab in the second year,

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that was my area of research.” (Sclafani, personal communication to JRV, July 13, 2016).

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This was indeed a fortunate development for the study of feeding behavior and its

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biological bases. The young Sclafani entered the area of feeding behavior when the

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reigning notion regarding the neurological basis of appetite and body weight regulation

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ACCEPTED MANUSCRIPT 4 was the Dual Center hypothesis, The hypothesis was based on landmark hypothalamic

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lesion studies by Hetherington & Ranson (1940) and Anand and Brobeck (1951), and

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formalized in the now classic motivational review by Eliot Stellar (Stellar, 1954).

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According to the hypothesis, the lateral hypothalamus (LH), a “hunger center”, is inhibited

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by signals from the ventromedial hypothalamus (VMH), a “satiety center”. Support for this

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notion came from studies demonstrating that electrical stimulation of these areas results

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in feeding or satiety, while selective destruction of them leads to aphagia or hyperphagia,

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respectively. Hyperphagia and obesity resulting from medial hypothalamic damage was

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termed the VMH syndrome, and the belief at the time was that the ventromedial nucleus

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(VMN) was the actual locus of feeding-inhibitory impulses. The Dual Center model led to

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hundreds of research papers many of which, but not all, seemingly supported this

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hypothesis. At the time Sclafani began his work on the VMH syndrome, however, the

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hypothesis was already being called into question (Mogenson, 1974). We will review the

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evolution of his studies, and the ingenious development of his research strategy in

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addressing this issue, in the next section.

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When one examines the entirety of Sclafani’s research career, one of the most reliable themes which emerges is how issues arising in one area of investigation led him

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productively into the next. This trend started with his study of the VMH syndrome itself,

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which as we know is characterized by a dependence on diet palatability. Following

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extensive studies of finickiness and willingness to work for food in normal and

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hypothalamic hyperphagic rats, Sclafani introduced the dual lipostat model of hunger and

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appetite, which integrated seemingly disparate results reported by numerous

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investigators in this area (Sclafani & Kluge, 1974). This model brought into focus the

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ACCEPTED MANUSCRIPT 5 effects of palatable diets in stimulating body weight gain and altering the body weight

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“setpoint” in an upward direction. A direct result of this hypothesis was his pursuit of the

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effects of palatable diets on the development of obesity not only in hypothalamic obese

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but also in normal animals (Sclafani & Berner, 1976), which led ultimately to his landmark

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studies of dietary obesity. True to form, Sclafani systematically examined the role of age,

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sex, exercise, diet type, and diet selection on the development of dietary obesity in

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normal rats, thus expanding his research scope well beyond that of hypothalamic effects.

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These contributions will also be reviewed in detail below.

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2.1 Challenging the Dual Center Hypothesis

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student, was designed to directly test the Dual Center hypothesis by making long bilateral

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parasagittal transections between the medial and lateral hypothalamus without significant

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damage to either area. To make the transections, the investigators used a retractable

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wire “knife” first introduced in this article (Sclafani & Grossman, 1969). The transections

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resulted in significantly increased food intake and body weight over a 15-day post-

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surgical period, supporting the notion that inhibitory fibers arising medially, when severed,

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release feeding excitatory fibers in the lateral hypothalamus, resulting in sustained

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hyperphagia. It is interesting to note that in this paper, Sclafani and Grossman reported

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that one knife cut animal also had significant bilateral damage in the posterior

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hypothalamus (presumably an effect of the surgical procedure), and displayed

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hyperphagia and weight gain far in excess of the rest of the knife cut animals. In an early

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clue which presaged Sclafani’s later thinking, the authors cited the possibility that the

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VMH “may have direct caudal output to the midbrain through the posterior hypothalamus”

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Sclafani’s first published study with Grossman, conducted while he was a graduate

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(Sclafani & Grossman, 1969, p. 536), a possibility originally suggested by Hetherington

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and Ranson (1942).

100 Sclafani next undertook his dissertation work in Grossman’s laboratory, which

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involved a systematic series of experiments utilizing bilateral VMH electrolytic lesions,

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bilateral long and short parasagittal knife cuts, and bilateral coronal transections either

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anterior or posterior to the VMH. An improved version of the knife cut device, labeled the

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“encephalotome”, was introduced in the article reporting this work (Sclafani, 1971).

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Sclafani’s primary findings indicated that long parasagittal knife cuts, extending into the

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anterior hypothalamic area (AHA), produced a VMH syndrome very similar to that

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produced by VMH lesions in terms of degree of hyperphagia and body weight gain, and

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in numerous behavioral tests including finickiness, food motivation, irritability, latency to

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eat, and wood gnawing. In contrast, shorter parasagittal knife cuts limited to the region

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between the ventromedial nucleus and lateral hypothalamus failed to generate a VMH-

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like feeding and weight gain syndrome. Since both the long and shorter knife cuts should

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in theory have severed inhibitory fibers arising medially and coursing to the lateral

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hypothalamus, this latter observation was not consistent with the Dual Center hypothesis.

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The coronal knife cuts also had no effect, probably because they were too medially

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placed to transect hyperphagia-related fibers (Sclafani, 1971).

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These results prompted Sclafani to suggest a possible “rostrolateral orientation of

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VMH feeding-related fibers” (Sclafani, 1971, p. 90), a possibility he would pursue avidly in

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future studies. In fairness, others had also demonstrated VMH-like syndromes following

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long transections between the medial and lateral hypothalamus (Albert & Storlien, 1969;

ACCEPTED MANUSCRIPT 7 Gold, 1970; Grossman & Grossman, 1971), with Gold questioning the existence of satiety

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fibers originating in the VMH, since his effective cuts were anterior and lateral to the

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VMH. This was a very active time in the pursuit of the neuroanatomy of feeding behavior,

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and a lively and productive competition was developing between numerous laboratories,

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including those mentioned above.

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Sclafani, now working in his own laboratory at Brooklyn College, published three

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additional reports that raised serious doubts concerning the validity of the Dual Center

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hypothesis. In the first of these (Sclafani & Maul, 1974), the investigators reasoned that

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effective parasagittal knife cuts between the medial and lateral hypothalamus,

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presumably severing inhibitory fibers coursing to lateral hypothalamus, should prevent

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electrical stimulation of the VMN from inhibiting feeding in the rats, as it did prior to

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surgery. This was not the case, however, again prompting Sclafani to consider the

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possibility that rostral or caudal connections from the VMH may mediate its inhibitory

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feeding effects.

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In a second report (Sclafani, Berner & Maul, 1973), Sclafani and his co-

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investigators demonstrated that parasagittal hypothalamic knife cuts differing in their

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lateral plane from the midline differentially affected expression of the VMH syndrome,

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with cuts at lateral 1.0 mm (just lateral to the VMN) being far more effective in provoking

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hyperphagia and weight gain that those at lateral 1.5 mm (closer to the boarder of the

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lateral hypothalamus). In a straightforward interpretation of the Dual Center hypothesis,

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this result should not have been the case. In an inventive bit of original theorizing,

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Sclafani reasoned in the third paper (Sclafani, Berner & Maul 1975) that these results

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ACCEPTED MANUSCRIPT 8 could still be consistent with the Dual Center hypothesis if some feeding excitatory fibers

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were located in the perifornical region (between the medial and lateral hypothalamus),

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while the remainder were located in the lateral hypothalamus (see Fig. 1). Knife cuts at

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lateral 1.0 mm should then sever all of the medial fibers inhibiting both sets of feeding-

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excitatory neurons, while knife cuts at lateral 1.5 mm would sever feeding-inhibitory

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axons on lateral hypothalamic feeding excitatory cells, but in addition sever the feeding-

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excitatory axons in the perifornical area entering the lateral hypothalamus. Thus

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parasagittal knife cuts made at lateral 1.5 mm could result in the less pronounced

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expression of hyperphagia, a result previously observed by the investigators.

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Sclafani further reasoned that if this were the case, then a lateral 1.0 mm cut made following an initial lateral 1.5 mm cut should make no difference in the relatively weak

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expression of hyperphagia seen in 1.5 mm cut rats. However, as demonstrated in this

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study (Sclafani et al., 1975), this turned out not to be the case, i.e., a lateral 1.0 mm cut

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made following a lateral 1.5 mm cut resulted in full hyperphagia and excess body weight

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gain. In a major departure from existing thinking, Sclafani concluded that “the results of

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this study…challenge the basic assumption that the VMH inhibits the LH feeding system;

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and this necessitates a reevaluation of hypothalamic feeding circuitry” (Sclafani et al.,

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1975, p. 215). It should be noted that Sclafani generated these results in response to his

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own theorizing and systematic testing, a strategy he used repeatedly in his work.

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2.2 Identification of a Longitudinal Feeding Inhibitory Pathway By this point, Sclafani was convinced that the hyperphagia and obesity produced by hypothalamic damage was due to interruption of a longitudinal feeding inhibitory

ACCEPTED MANUSCRIPT 9 pathway. Acknowledging earlier reports demonstrating that overeating and obesity could

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be produced by coronal knife cuts either anterior or posterior to the hypothalamus (Albert,

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Storlien, Albert & Mah, 1971; Grossman, 1971; Palka, Leibelt & Critchlow, 1971), Sclafani

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returned to the use of coronal cuts as an essential strategy in defining the course of a

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longitudinal tract passing through and beyond the hypothalamus. In designing his next

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study (Sclafani & Berner, 1977), Sclafani also made use of the strategy of asymmetric

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knife cuts, as demonstrated earlier by Gold and co-workers (Gold, Quackenbush &

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Kapatos, 1972; Gold, Jones, Sawchenko & Kapatos, 1977). Sclafani was able to show in

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this study (Sclafani & Berner, 1977) that unilateral parasagittal hypothalamic knife cuts

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combined with contralateral posterior hypothalamic or midbrain tegmental coronal cuts

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resulted in hyperphagia and rapid body weight gain. In a direct comparison of the effects

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of bilateral parasagittal vs. bilateral coronal posterior hypothalamic knife cuts, both

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manipulations resulted in highly significant hyperphagia-weight gain effects, although the

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magnitude of the effects was greater in rats bearing parasagittal knife cuts. Addressing

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this difference, Sclafani speculated that the parasagittal cuts may have severed more

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feeding inhibitory fibers than the coronal cuts at their respective anatomical locations.

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Finally, in results similar to those of the earlier Sclafani et al. (1975) report, bilateral

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coronal posterior hypothalamic cuts, followed 20 days later by bilateral parasagittal knife

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cuts at lateral 1.5 mm, resulted in significantly greater hyperphagia and obesity than

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obtained with the 1.5 mm lateral cuts alone.

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In the Discussion section of this paper Sclafani presented a long and carefully

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reasoned analysis of the probable course of the feeding-inhibitory tract, utilizing his

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results and the results of others to chart the probable course of the tract through the

ACCEPTED MANUSCRIPT 10 medial hypothalamic area (Figure 2). Here he states that “..the feeding inhibitory fibers

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responsible for the hyperphagia syndrome course in the perifornical region of the medial

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forebrain bundle (MFB), and turn medially just rostral to the ventromedial nucleus”

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Sclafani & Berner, 1977, p. 1014). After considering possible candidates for the identity of

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this tract, including ascending noradrenergic and serotonergic fiber systems, he is careful

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to state that “..it is possible that it is the destruction of a descending fiber system that is

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responsible for hypothalamic hyperphagia.” (Sclafani & Berner, 1977, p. 1016), citing the

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existence a descending pathway from the AHA to the midbrain recently identified by

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Conrad and Pfaff (1975). This was in marked contrast to Gold’s hypothesis that

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ascending noradrenergic and serotonergic pathways terminating in the paraventricular

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nucleus (PVN), when transected, result in the overeating-obesity syndrome (Gold, 1973;

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Kapatos & Gold, 1973).

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In an early attempt to trace the anatomical course of the longitudinal feeding-

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inhibitory tract, Sclafani and colleagues studied axonal fiber degeneration using the Fink-

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Heimer stain following hyperphagia-inducing parasagittal hypothalamic knife cuts in the

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medial perifornical region of the hypothalamus (Mufson, Sclafani & Aravich, 1980).

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Axonal damage radiated both anteriorally and posteriorally from the knife cut locus,

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starting in the AHA, passing through the perifornical area of the hypothalamus along the

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course of the medial forebrain bundle (MFB), and ultimately terminating in the midbrain

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tegmental area. Medially and dorsally, axonal degeneration was seen in the AHA and

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PVN. Sclafani’s own results, and the results of others (Swanson & Kuypers, 1980;

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Swanson, Sawchenko, Wiegand & Price, 1980) now focused his attention specifically on

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the PVN. As noted above, Gold and his co-workers had proposed that the PVN itself

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could be the terminus for ascending longitudinal feeding inhibitory fibers (Gold et al.,

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1977). Moreover Leibowitz, Hammer & Chang (1981) had recently demonstrated that

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lesions restricted to the PVN induced overeating and obesity in the rat.

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221 In their next study Aravich & Sclafani (1983) addressed the issue of whether PVN and VMH damage produced similar syndromes of hyperphagia and obesity. If true, this

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would support the notion of a feeding inhibitory tract originating in the PVN which, when

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interrupted at either locus, would result in the hypothalamic hyperphagia syndrome. In a

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direct comparison of the effects of VMH knife cuts and PVN lesions, the authors found

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that the feeding, body weight, and behavioral effects of the two types of damage were

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remarkably similar, although parasagittal VMH knife cuts resulted in greater hyperphagia

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and body weight gain. The authors concluded that “..the neuronal substrate responsible

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for the hypothalamic hyperphagia syndrome is not confined to the paraventricular

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nucleus, but is more diffusely organized in the medial and anterior hypothalamus”

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(Aravich & Sclafani, 1983, p. 980). However, based on the fact that most effective

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damage to the PVN was localized to the caudal parvocellular area of the nucleus, which

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is rich in oxytocin-expressing cell bodies, the authors hypothesized that a descending

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oxytocin-containing pathway from the PVN, when severed, is most likely responsible for

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the hyperphagic effects specifically of PVN damage (Sclafani & Aravich, 1983).

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In an ambitious set of studies designed to define the caudal path of the

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longitudinal feeding-inhibitory tract, and identify its neurotransmitter substrate, Sclafani

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and his graduate student Annette Kirchgessner utilized asymmetrical knife cuts

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consisting of combinations of parasagittal, coronal, and oblique transections

ACCEPTED MANUSCRIPT 12 (Kirchgessner & Sclafani, 1988). The investigators found that unilateral parasagittal knife

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cuts in the hypothalamus combined with contralateral coronal knife cuts in either the

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ventrolateral pons or ventrolateral medulla resulted in significant hyperphagia and weight

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gain. This was also the case for unilateral parasagittal knife cuts combined with

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contralateral oblique knife cuts directly under the dorsal vagal complex. Importantly, the

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combination of bilateral parasagittal hypothalamic knife cuts with a unilateral coronal

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ventrolateral pontine cut produced no greater feeding effect than the parasagittal

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hypothalamic knife cuts alone. This indicates that the asymmetrical knife cuts were

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effective because they transected the same feeding-inhibitory fibers on both sides of the

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brain. It remained true, however, that the asymmetrical knife cut conditions did not

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generate as great a degree of hyperphagia and weight gain as bilateral parasagittal

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hypothalamic knife cuts, suggesting that the asymmetrical cuts did not sever all of the

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feeding-inhibitory fibers involved in the VMN hyperphagia syndrome. The authors

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concluded that the caudal course of the PVN-feeding inhibitory pathway is through

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posterior hypothalamus along the MFB, then passing through the ventrolateral pons and

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medulla (probably in the hindbrain reticular formation), with a sharp dorsomedial turn

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beyond this point towards the dorsal vagal complex. Although not demonstrated in this

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study, the authors hypothesized that many of the feeding-inhibitory fibers terminate in the

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nucleus of the solitary tract (NST) and dorsal motor nucleus of the vagus nerve (DMX),

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components of the dorsal vagal complex which modulate orosensory, viscerosensory,

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and autonomic information in relation to feeding.

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In their final study, Sclafani and Kirchgessner utilized combinations of unilateral and bilateral hypothalamic and ventral pontine knife cuts, and immunocytochemical

ACCEPTED MANUSCRIPT 13 analysis, to map the entire course of the PVN-hindbrain feeding-inhibitory tract, and

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identify the neurotransmitter mediating its effects (Kirchgessner, Sclafani & Nilaver,

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1988). The investigators first used horseradish peroxidase (HRP) applied to the site of a

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unilateral coronal ventrolateral pontine reticular knife cut to identify HRP-labeled neurons

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rostral to the cut (labeling results from the retrograde transport of HRP by cut axons to

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the parent cell nucleus). Anteriorally, HRP-labeled neurons were found in the PVN,

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particularly in the oxytocin-rich caudal parvocellular region, and in other anterior and

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forebrain regions not involved in feeding, but none were found in the VMN. Posteriorally,

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HRP-labeled neurons were found in the NST, although this labeling could be attributed to

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the retrograde transport of HRP to ascending afferent neurons from the NST (an effect

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consistent with later results, as detailed below).

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To investigate whether oxytocin is the mediating signal in the PVN-hindbrain

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feeding inhibitory tract, and identify its caudal terminus, the investigators utilized an

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immunoperoxidase oxytocin staining technique, and traced the effects of bilateral and

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unilateral cuts at the hypothalamic and midbrain pontine levels. With this procedure,

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transection of oxytocin-containing axons results in an accumulation of the neuropeptide in

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stumps proximal to oxytocin-expressing cell bodies, and severe depletion of the

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neuropeptide in stumps distal to the cut, including at the terminal synapse. Following the

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knife cuts made in this study, a heavy concentration of oxytocin immunoreactive (OT-IR)

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activity was found in transected axons and cell bodies in both the magnocellular and

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parvocellular divisions of the PVN. In contrast, marked depletion of OT-IR was seen in

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axon fibers along the entire course of the PVN-hindbrain tract, including in the posterior

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hypothalamus and midbrain and up to, but not including, NST cell bodies (Kirchgessner

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et al., 1988).

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hyperphagia-inducing knife cuts transect a longitudinally coursing oxytocin-containing

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tract from the PVN to the NST/DMX complex in the hindbrain, and interruption of this tract

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is a major contributor to the VMH hyperphagia-obesity syndrome (see Fig. 3). Thus

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almost 20 years after starting his investigation of the neural substrate underlying the VMH

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hyperphagia-obesity syndrome, Sclafani and his co-workers successfully identified a

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major pathway involved in its expression. The authors were careful to conclude, however,

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that “The fact that PVN lesions are less hyperphagia-promoting than are MH knife cuts

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indicates that the PVN-hindbrain pathway is only part of the anatomical circuit involved in

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the MH hyperphagia syndrome” (Kirchgessner & Sclafani, 1988, p. 527). They suggest

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that the VMH hyperphagia-obesity syndrome may result from two or more independent

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neural systems that separately promote these effects, one PVN-associated, and the other

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consisting of fiber projections from the VMN which mediate insulin release, and result in

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hyperinsulinemia when severed (Kirchgessner & Sclafani, 1988).

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2.3 Current Status of the PVN-Hindbrain Pathway

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One major bit of unfinished business pointed out by Sclafani and colleagues in the

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discussion of his 1988 histology study was the issue of the physiological role of the PVN-

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hindbrain pathway in feeding (Kirchgessner et al., 1988). Although a role in mediating

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satiety can be assumed for this tract, based on the effects of its transection, no direct

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evidence for its normal function existed at the time. This issue has been specifically

ACCEPTED MANUSCRIPT 15 addressed in a recent series of studies by several investigators. In the first of these

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reports, oleoylethanolamide (OEA), an endogenous small-intestinal lipid amide that

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signals feeding inhibition via the vagus nerve, was shown to selectively activate neurons

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in the PVN and NTS (Rodriguez de Fonseca, Navarro, Gómez, Escuredo, Nava et al.,

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2001). Further work identified the central release of oxytocin in the PVN in response to

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peripheral OEA administration as the mediator of this satiety effect, and demonstrated

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that blockade of central oxytocin receptors by the intracerebroventricular infusion of an

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oxytocin antagonist prevented the anorectic effects of OEA (Gaetani, Fu, Cassano,

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Dipasquale, Romano et al., 2010). In further support of an PVN-NTS oxytocin-mediated

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satiety pathway Grill and colleagues (Ong, Alhadeff & Grill, 2015) showed that

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microinjection of oxytocin into the fourth ventricle or medial NTS of rats dose-dependently

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inhibits food intake. Finally, in a study investigating the nature of ascending stimulation

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from the NST which activates PVN oxytocin neurons Romano, Potes, Tempesta,

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Cassano, Cuomo et al. (2015) demonstrated that chemical lesion of A2 noradrenergic

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cell bodies in the NST, which provide direct excitatory input to the PVN, prevents OEA-

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induced satiety in rats. Expression of oxytocin in the PVN was virtually eliminated in rats

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receiving the NST A2 cell body lesions. These results demonstrated that an ascending

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noradrenergic NST-PVN pathway mediates the activation of PVN-oxytocin satiety

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signaling. Thus, experimental interruption of either the ascending noradrenergic or

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descending oxytocinergic PVN-NST pathway can induce a satiety deficit.

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Additional support for the notion of a satiety function for PVN-NTS interconnections is

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found in work by Rinaman and colleagues (Rinaman, Hoffman, Dohanics, Le, Stricker &

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Verbalis, 1995), who demonstrated that the gut peptide cholecystokinin (CCK) activates

ACCEPTED MANUSCRIPT 16 primarily noradrenergic neurons in the NTS and ventrolateral medulla that project to

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oxytocin-expressing regions of the PVN, presumably mediating CCK satiety effects. That

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CCK activates oxytocin-expressing neurons in the PVN had earlier been demonstrated

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by Verbalis (Verbalis, Stricker, Robinson & Hoffman, 1991). Thus at least two intestinal

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satiety factors may share use of the PVN-hindbrain pathway. Finally, recent studies in

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mice have demonstrated that both oxytocin and oxytocin receptor knock-out lead to adult-

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onset obesity, albeit without the expression of hyperphagia (Camerino, 2009; Takayanigi,

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Kasahara, Onaka, Takahashi, Kawada & Nishimori, 2008). However, a study in which

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oxytocin expression was inhibited or antagonized specifically in the hypothalamus of mice

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demonstrated both increased food intake and body weight gain (Zhang, Bai, Zhang,

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Dean, Wu, Li, Guariglia, Meng & Cai, 2011). Indeed, these and other studies have

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provided a rationale for the potential use of oxytocin inhibitors as a treatment for obesity

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(Blevins & Baskin, 2015).

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3.1 Sclafani and Diet-Induced Obesity

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productive, Sclafani expanded it in the early 1970s to investigate diet-induced obesity

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(DIO). When asked about this, he replied, “In my early studies of hypothalamic obesity I

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was very interested in dietary finickiness and included palatable (high fat and sweetened

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condensed milk) and unpalatable (powdered chow, quinine chow, cellulose) diets in my

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experiments. In 1971 or 1972 I met Stanley Schachter and his then student, Judy Rodin,

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at Columbia. They were writing about the similarities of obese humans and VMH rats

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which further sparked my interest in comparing hypothalamic obese and dietary obese

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animals. Schachter, as you may recall, was one of the founders of the Appetitive

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Seminar and it was my contacts with him that got me invited to the inaugural meeting of

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the Seminar” (A. Sclafani, personal communication to GPS, April 27, 2016).

364 At first, Sclafani was unsuccessful in producing DIO: “In our hypothalamic studies

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we fed rats the then standard high-fat diet (33% Crisco, 67% powdered chow) that

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induced enhanced weight gains in the VMH rats but not much weight gain in the controls

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(Sclafani, Springer & Kluge, 1976). As I recall we then tried a higher fat diet (~60%) like

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that used in the Peckman, Entenmen & Carroll (1962) study cited in our 1976 cafeteria

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study, but it also produced relatively little weight gain. That prompted us to go all out with

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supermarket foods. I had previously used sweetened condensed milk diets and Fruit

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Loops in prior studies so suspected that these types of foods would be more effective

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than Crisco-chow diets” (A. Sclafani, personal communication to GPS, April 27, 2016).

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The supermarket, cafeteria diet consisted of a high-fat diet (33% Crisco fat, 67% Purina powder), sweetened condensed milk (Magnolia brand mixed with water, 1:1) and

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a variety of other palatable supermarket foods including chocolate chip cookies, salami,

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cheese, banana, marshmallows, milk chocolate, and peanut butter. At least 7 different

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foods were available at any one time and the menu was changed frequently except that

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Purina pellets, high-fat diet, and milk were always available (Sclafani & Springer, 1976).

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This palatable diet worked (Figure 4).

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When the manuscript reporting these results was submitted to Physiology &

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Behavior in July, 1974, however, the reviewers were not convinced. They suggested that

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the development of obesity under the conditions of restricted, solitary space and a very

ACCEPTED MANUSCRIPT 18 palatable diet had been shown before (Ingle, 1949) and was commonplace in livestock

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and agricultural businesses (A. Sclafani, personal communication to GPS, April 27,

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2016). Sclafani and Springer responded by repeating their experiment in rats housed in

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isolation but also in group conditions. When female rats housed singly were switched

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from a chow diet to the palatable supermarket diet, they gained weight at a faster rate

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than chow-fed controls, with the difference in weight gain between the two groups

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significant by day 10, and increasing up to day 60 (Figure 5). The authors observed that

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“The 53% weight increase displayed by the experimental group after 60 days on the diet

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is greater than that observed in previous studies using adult rats and high fat diets”

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(Sclafani and Springer, 1976, p. 463).

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Non-systematic observation revealed that the rats ignored the chow and overate the supermarket foods, but intake of the numerous food items could not be measured

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reliably. When the supermarket foods were removed and only chow pellets and water

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were available, the body weights of the obese rats decreased to control values. When

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the supermarket foods were returned to the rats, they again became obese (Figure 5).

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Sclafani and Springer (1976) made a strong inference from this tight correlation:

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“…although the postingestive effects may be of importance to some aspects of the

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syndrome, it is the orosensory properties of the diet that appear to be primarily

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responsible for the development of dietary obesity” (Sclafani & Springer, 1976, p. 463).

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In the same study (Sclafani & Springer, 1976), groups of female rats were

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maintained in “complex environments that consisted of 3 large, wire mesh cages stacked

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on top of each other. The doors of the cages were replaced with a single clear Plexiglas

ACCEPTED MANUSCRIPT 19 door containing wire mesh ramps which connected the 3 levels. The upper two levels of

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the environment had perforated metal floors, while the bottom level contained a metal

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pan filled with wooden shavings. Wooden, metal, and at times, plastic objects were

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located in the upper two levels (Figure 6). Under these enriched environmental

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conditions, rats fed the supermarket diet gained significantly more body weight than rats

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fed Purina chow, but an identical amount of body weight as rats fed the palatable

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supermarket diet and maintained in isolated cages (Figure 7). This demonstrated that the

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opportunity for increased physical activity and sensory and social stimulation experienced

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by the rats in the enriched environment did not influence the degree of DIO stimulated by

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the supermarket diet, an issue raised by the reviewers.

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The supermarket diet did not change the ad lib activity of DIO rats. Access to running wheels reduced the weight gain produced by the supermarket diet, but it did not

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prevent the development of DIO (Figure 7). The gain of body weight of individual rats fed

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the supermarket diet for 60 days, whether maintained in isolation or in the enriched

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environment, was not only larger than rats fed Purina chow, but was also more varied

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(85-200 g for supermarket vs. 10-80 g for chow (Figure 8). The variable susceptibility of

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rats fed the supermarket diet to gain body weight observed by Sclafani & Springer (1976)

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was similar to what had been observed in humans (Garrow,1978) and in rats given

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palatable, high-calorie diets (Schemmel, Mickelson & Tolgay, 1969).

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The accurate measurement of intake of some of the foods in the supermarket diet

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was not possible, so videotape analysis was undertaken. It showed that rats offered the

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supermarket diet “sampled more than one food item during most of their meals.

ACCEPTED MANUSCRIPT 20 434

Furthermore, their number of meals appeared normal and thus, their overeating was

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accomplished by increases in meal size” (Sclafani and Springer, 1976, p. 469).

436 When 24-hour intake of plain milk and quinine-adulterated milk (0.03%) was

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measured in a 3-day intake test, DIO female rats had significantly smaller intakes of

439

unadulterated milk and quinine milk than chow-fed rats (Figure 9, left two panels). The

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larger reduction of intake of quinine-adulterated milk in DIO rats was interpreted as

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finickiness. This was consistent with Maller’s earlier report that DIO rats were finicky to

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cellulose adulteration of their diet (Maller, 1964).

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In the same study (Sclafani and Springer, 1976), DIO rats maintained at 80 or 85

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% of their ad lib body weights bar pressed less for food than control rats (Figure 5, and

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Figure 9, extreme right panel). Thus, DIO rats were apparently less motivated to eat than

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chow-fed rats. The results of a test of the effect of 4 days of food deprivation on wheel

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running were consistent with this interpretation. Chow-fed rats rapidly increased their

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wheel running activity under food deprivation, while the DIO rats maintained their activity

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at baseline levels (Sclafani & Springer, 1976). This effect was replicated in a separate

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study utilizing male DIO rats (Sclafani and Rendel, 1978), in which the DIO group did not

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increase wheel running activity in response to food deprivation until day 9, while control

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rats were more active as early as day 2.

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The longer latency to increase running during food deprivation was interpreted as

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further evidence that DIO rats had reduced motivation to eat. When the latencies to run

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during food deprivation were expressed as percent loss of body weight, however, the

ACCEPTED MANUSCRIPT 21 difference in the latencies disappeared -- both groups increased activity when body

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weights fell to 75-85% of pre-deprivation body weight (Sclafani and Rendel, 1978).

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Rather than reduced motivation to eat, the authors reinterpreted their results to indicate

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that body weight must fall to a critical percentage of predeprivation body weight before

462

activity increases during a fast.

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3.2 A New Animal Model of Human Obesity

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Note that the results with DIO rats were similar to previous results in hypothalamic obese rats: both kinds of rats were finicky to quinine-adulterated diets, bar pressed less

467

for food, and had longer latencies to increase activity when food deprived. Noting that

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the same syndrome had been reported in obese humans, Sclafani concluded, “In view of

469

the fact human obesity is only rarely associated with hypothalamic damage, but is

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associated with the availability of palatable foods and sedentary activity levels, dietary

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obesity may be a more appropriate model for human obesity than is the hypothalamic

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syndrome” (Sclafani and Springer, 1976, p.470).

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Sclafani pursued the characteristics of this new animal model of human obesity. He found that DIO did not appear until after postnatal day 60, the percentage weight gain

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on the cafeteria diet was significantly more in females than in males, and prior experience

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with DIO did not affect its subsequent development (Sclafani and Gorman, 1977).

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Abdominal vagotomy in DIO and hypothalamic obesity was investigated next. Its

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effect depended on the diet: Abdominal vagotomy abolished the hypothalamic syndrome

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in rats given chow, decreased it in rats given 20% sucrose solution, but did not change

ACCEPTED MANUSCRIPT 22 hyperphagia and obesity in rats given cookies, sweet milk, and high-fat ration in addition

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to chow. When the same obesogenic diet was given to brain-intact rats to produce DIO,

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however, abdominal vagotomy decreased the gain of body weight significantly. These

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differential results were the first evidence that the syndromes of hypothalamic and diet-

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induced obesity were not identical (Sclafani, Aravich, and Landman,1981).

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Given the tight correlations between diet and obesity in DIO rats, and his belief

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that diet-induced hyperphagia was the primary cause of DIO, Sclafani began to

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investigate the efficacy of high-carbohydrate and high-fat diets to produce hyperphagia

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and obesity. In male rats, a sucrose solution (32%) produced obesity, but not

492

hyperphagia, while a Polycose solution (hydrolyzed corn starch, 32%) produced obesity

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and hyperphagia (Sclafani and Xenakis, 1984a). When 32% sucrose was given to

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female rats in addition to a composite or self-selected diet, however, it also produced

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hyperphagia and obesity (Ackroff and Sclafani, 1988).

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The form of Polycose was also important. A 32% solution of Polycose produced

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hyperphagia and obesity in female rats, but Polycose had neither effect when it was

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given as a powder or as a powder mixed in the chow diet (Sclafani and Xenakis, 1984b).

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When Polycose was presented as a gel, it was also more potent than when it was

501

presented as a powder (Sclafani, Vigorito & Pfeiffer, 1988).

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To determine if the palatable taste of sucrose or Polycose solutions contributed to

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the hyperphagia, adult female rats were fitted with chronic intragastric catheters and

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given ad lib access to chow and a drinking fluid that was paired with intragastric infusions

ACCEPTED MANUSCRIPT 23 of 32% Polycose (Sclafani, Lucas, and Ackroff, 1996). Drinking Polycose (2%) plus 0.2%

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saccharin under these conditions produced significantly more intake and body weight

508

gain than drinking 2% Polycose with bitter tasting 0.03% sucrose octaacetate. Thus,

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when the oral taste was not preferred, intragastric Polycose produced a much smaller

510

increase of intake and had no effect on bodyweight. The authors concluded that the oral

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palatability of carbohydrate solutions was a major determinant of hyperphagia and

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obesity.

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Complex results also occurred with high-fat diets. Rats fed chow and a separate

515

source of fat consumed more fat and total calories, and gained more body weight when

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the fat source was vegetable shortening or emulsified corn oil than pure corn oil (Lucas,

517

Ackroff, and Sclafani, 1989) .When corn oil or vegetable shortening were presented as

518

gels, however, they increased intake and body weight. The hyperphagia was also

519

greater when chow was the alternative food than when separate sources of carbohydrate

520

and protein were available. Because the orosensory properties of the fat sources did not

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account for the differential hyperphagia, it appeared that long- term fat selection and

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caloric intake were influenced primarily by postingestive reinforcing factors.

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This conclusion was apparently supported when rats self-infused significantly

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more isocaloric high-fat liquid diet (HF) into the stomach than high carbohydrate liquid

526

diet (HC) (Lucas, Ackroff, and Sclafani (1998).

527

infusion of HF diet could also be explained by a reduced satiating effect of HF (Lucas and

528

Sclafani, 1999). The relative contribution of postoral reinforcing and satiating effects of

529

HF intragastric infusions in rats remains to be determined. Recent experiments in mice

However, the increased gastric self-

ACCEPTED MANUSCRIPT 24 demonstrate the importance of the concentration of infused fat in the form of Intralipid for

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the stimulation of intake and the formation of conditioned preferences (Ackroff and

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Sclafani, 2014)*. The threshold concentration of Intralipid for stimulating intake and

533

conditioned preferences was 3.2%. Unfortunately, the threshold for a satiating effect was

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not measured.

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Sclafani’s final two papers on DIO appeared in 2010 and 2012. The first used

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molecular genetic techniques in mice to investigate sugar-induced hyperphagia and

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obesity in 4 strains of mice polymorphic for Tas1r3, the gene that codes for the T1R3

539

sugar taste receptor. The major results were that Tas1r3 genotype did not predict sugar-

540

induced hyperphagia and that susceptibility to sugar-induced obesity varied with strain,

541

and the concentration and type of sugar (Glendining, Breinager, Kyrillou, Lacuna, Rocha

542

& Sclafani, 2010). The last paper used knockout mice that differed in their sensitivity to

543

Polycose and sucrose. The results suggested: (1) sugar solutions must be highly

544

palatable to cause carbohydrate-induced obesity in mice, and (2) palatability could also

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produce increased nutrient utilization because obesity could occur with little or no

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hyperphagia (Glendining, Gilman, Zamer, Margolskee, & Sclafani, 2012).

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Although Sclafani did not discover DIO, his early results in the 1970s convinced

550

many other investigators that DIO was a robust phenomenon accessible to experiment

551

and that it was a better animal model of human obesity than hypothalamic hyperphagia.

552

The experimental demonstration of DIO shattered the prevailing view that food intake in

553

rats under experimental conditions was tightly controlled by the caloric deficit that

ACCEPTED MANUSCRIPT 25 554

developed between meals. This was the basis of Adolph’s famous dictum, “Rats eat for

555

calories” (Adolph, 1947).

556 Sclafani interpreted the importance of diet to mean that hyperphagia, not

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metabolic effects, was the primary effect of an obesogenic diet. The oral effects of

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palatable, carbohydrate diets were adequate stimuli for the syndrome of hyperphagia and

560

obesity. In the case of high-fat diets, however, Sclafani suggested, but did not prove, that

561

postoral stimuli of ingested fat were the adequate stimuli for fat-induced hyperphagia and

562

obesity.

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4.1 Conclusion

Sclafani continued his study of the stimulatory effects of sweet and fat stimuli on appetite. This led to his groundbreaking demonstrations of flavor-nutrient conditioning

567

based on the postingestive effects of nutrients delivered directly to the stomach and

568

proximal small intestine. This work was made possible by his introduction of the

569

“electronic esophagus” for rats and mice, which enabled him to pair the oral intake of

570

flavored solutions with the simultaneous infusion of nutrients directly to the gut (Elizalde &

571

Sclafani, 1990). In a major contribution to the lexicon of the feeding literature, Sclafani

572

coined the term “appetition” to describe the effect of intestinal nutrient sensing on post-

573

ingestive appetite stimulation (Sclafani, 2013). In so doing, he introduced a novel feeding-

574

stimulatory mechanism which promises to be a powerful explanatory concept in our

575

understanding of how palatable foods increase caloric intake and lead to excess body

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weight gain, not only as a function of their oral taste qualities, but also as a result of

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reinforcing effects mediated by small intestinal nutrient sensors.

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ACCEPTED MANUSCRIPT 26 578 579

As noted above, Sclafani’s productivity in the research areas he chose to investigate has been nothing short of extraordinary. Table 1 presents a distribution of

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published articles among major subject areas investigated by Sclafani and his colleagues

582

as of July, 2016. Sclafani’s publications, including research articles and reviews, total

583

over 300 currently. Research areas have been listed roughly in order of appearance in

584

Sclafani’s investigative career. There is inevitably some overlap of areas, but for our

585

purposes the articles are categorized on the basis of their primary research focus. It is

586

remarkable that in over 40 years of experiments on hypothalamic pathways, DIO, and

587

nutrient preferences and conditioning, his experimental results and interpretations were

588

never contradicted.

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Sclafani acknowledges the good fortune of having mentored many dedicated undergraduate, graduate and postdoctoral students in his laboratory who contributed

592

significantly to his research productivity. Not the least of these is his colleague and close

593

friend Karen Ackroff, who joined his laboratory in 1987 as a postdoctoral fellow, and

594

remained for 30 years as his co-investigator and co-director of his laboratory. Over this

595

period, Ackroff and Sclafani jointly authored 76 research articles. The experience of

596

working with Sclafani was described by Ackroff recently: “When I was finishing my

597

dissertation work and trying to write it up, my mentor George Collier provided a wonderful

598

distraction by giving me a stack of manuscripts to read. They were all focused on the

599

maltodextrin Polycose, recounting the various ways Tony Sclafani and his colleagues

600

sought to understand this unusual carbohydrate that animals treated as neither sugar nor

601

starch. Tony had asked George to review them prior to submission for a special issue of

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ACCEPTED MANUSCRIPT 27 Neuroscience and Biobehavioral Reviews, and George in turn asked me. I recall my main

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response as ‘why doesn’t he just come out and say it? There must be a ‘Polycose’

604

receptor! “When I heard that Tony had advertised for a postdoc, I spoke to him at the

605

next Columbia Appetitive Seminar. Perhaps my credentials as a Collier student were

606

good enough for him. And, I knew his Polycose work!”

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“I started in the lab just as Tony and his students were developing the “electronic

609

esophagus”, the system that infused rats intragastrically whenever they licked a drinking

610

spout. This was another mark of Tony’s science – to devise apparatus that supports

611

creative approaches to the physiology underlying the behavior. Tony had begun

612

developing computer controls and interfacing them with the relay rack equipment that had

613

been the standard for behavioral research for so long. With my background from the

614

Collier lab, I became the second computer programmer, modifying the code for new kinds

615

of experiments. The lab was full of post-docs, graduate and undergraduate students, and

616

technicians running studies all day. Tony led by example: he was in the lab early every

617

day, and usually came in on weekends. Although there might be a dozen or more studies

618

in progress, he kept track of them all and discussed them frequently with the

619

experimenters. The most satisfying time in the lab was spent reviewing the data and

620

evaluating possible explanations and next steps, devising plans for the current study and

621

those to follow.”

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“Tony’s leadership of his lab always conveyed his scientific values: focus on the

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problem, devise creative ways to get the answers, and report the information clearly. His

625

hundreds of publications and many invited talks attest to the respect his work has earned.

ACCEPTED MANUSCRIPT 28 626

His lab was an amazing place to conduct great research, and I was fortunate to be able

627

to stay there for my entire postgraduate career” (K. Ackroff, personal communication, to

628

JRV, September 14, 2016).

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629 No description of Sclafani and his contributions would be complete without

mention of his ability to train and motivate his students. Paul Aravich, who earned his

632

doctoral degree with Sclafani in December of 1982, described what it was like being a

633

graduate student of Tony’s: “Tony is one of the most remarkable scientists I have ever

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met. In my dissertation 33 years ago I stated that ‘I have never met, nor do I expect to

635

meet, a more dedicated and cautious scientist.’ Those words are as true now as then. He

636

accepted a kid from the coal fields of Pennsylvania who did not see the ocean until he

637

went to college. As such, he changed my life and the life of my wife, Michele, in more

638

ways than we can count. He taught me how to think critically and to have a broad

639

integrated systems approach. He taught me to be an investigator-educator and gave me

640

significant undergraduate teaching responsibilities along with significant research

641

responsibilities. In the short run balancing these responsibilities was problematic; in the

642

long run balancing them was an invaluable experience.

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“Tony said, ‘I don’t require you to work 7-days a week, but sometimes the

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experiments do.’ As such, no one worked harder than him. Despite that, he regularly

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helped me do knife cut surgeries and brain cannulations. Tony taught me the thrill of the

647

conception of an idea; the thrill of the delivery of an idea; and the sometimes more than 9

648

months of hard labor in between. He taught me that the scientist is the messenger and

649

never the message. When I became a postdoc and a research associate, I appreciated to

ACCEPTED MANUSCRIPT 29 650

an even greater extent the exceptional training he gave me and the amazing access I

651

had to his wisdom and expertise.” (P. Aravich, personal communication to JRV, August 6,

652

2016).

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653 In Sclafani’s long and distinguished academic career, now spanning almost 50 years, there has been no shortage of honors and accolades. In addition to his

656

appointment as Distinguished Professor of Psychology at Brooklyn College in 1994, he

657

was awarded the Tow Professorship at Brooklyn College (1993-1995), an honor

658

bestowed upon “faculty whose talents and accomplishments are of a particularly high

659

order”. In addition to numerous RO1 awards, Sclafani won two consecutive Senior

660

Research Scientist Awards from NIMH for the study of carbohydrate appetite and obesity

661

(1992-2000), and was then honored with an NIH Merit Award over the 10-year period

662

2001–2011. NIH Merit Awards recognize “superior competence and outstanding

663

productivity in pursuit of research in areas of special importance”, and fewer than 5% of

664

NIH investigators receive this honor. Sclafani was one of the first elected presidents of

665

the newly formed North American Association for the Study of Obesity in 1989 (now The

666

Obesity Society). He played a major hand in founding the Society for the Study of

667

Ingestive Behavior, and was elected president of the society in 1992. He cites

668

participation in these groups as having had a major impact on his research career,

669

fostering fruitful collaborations with many distinguished scientists.

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ACCEPTED MANUSCRIPT 30

*Footnote

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There are a number of papers by Sclafani on acquired food preference by oral-

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postingestive nutrient conditioning not discussed here (see review by Sclafani and

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Ackroff, 2012 and chapter by Myers in this Festschrift).

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Acknowledgements

680

The authors would like to thank Dr. Carol Maggio for her assistance in preparation of the

681

manuscript, and her insightful review of the text. We also thank Drs. Karen Ackroff and

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Paul Aravich for their willingness to share their experiences in working with Dr. Sclafani.

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JRV would like to gratefully acknowledge the training and guidance Tony provided during

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his years as a postdoctoral fellow in the Sclafani laboratory at Brooklyn College (1974-

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1976).

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Ackroff, K., &, Sclafani, A. (1988). Sucrose-induced hyperphagia and obesity in rats fed

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a macronutrient self-selection diet. Physiology & Behavior, 44, 181-187.

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Adolph, E.F. (1947). Urges to eat and drink in rats. American Journal of Physiology,

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151, 110-125.

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Albert D.J., & Storlien, L.H. (1969). Hyperphagia in rats with cuts between the

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ventromedial and lateral hypothalamus. Science, 165, 599-600.

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Albert, D.J., Storlien, L.H., Albert, J.G., & Mah, C.J. (1971). Obesity following

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disturbance of the ventromedial hypothalamus: a comparison of lesions, lateral cuts, and

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anterior cuts. Physiology & Behavior, 7, 135-141.

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ACCEPTED MANUSCRIPT 31 Anand, B.K. & Brobeck, J.R. (1951). Hypothalamic controls of food intake in rats and

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cats. Yale Journal of Biological Medicine, 24, 123-139.

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Aravich, P.F., & Sclafani, A. (1983). Paraventricular hypothalamic lesions and medial

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hypothalamic knife cuts produce similar hyperphagia syndromes. Behavioral

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Neuroscience, 97, 970-983.

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Conrad, L.C.A., & Pfaff, D.W. (1975). Axonal projections of medial preoptic and anterior

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hypothalamic neurons. Science, 190, 1112-1114.

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Elizalde, G., & Sclafani, A. (1990). Flavor preferences conditioned by intragastric

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Polycose infusions: A detailed analysis using an electronic esophagus preparation.

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Physiology & Behavior, 47, 63-77.

707

Gaetani, S., Fu, J., Cassano, T., Dipasquale, P., Romano, A., Righetti, L., Cianci, S.,

708

Laconca, L., Giannini, E., Scaccianoce, S., Mairesse, J., Cuomo, V., & Piomelli, D.

709

(2010). The fat-induced satiety factor oleoylethanolamide suppresses feeding through

710

central release of oxytocin. Journal of Neuroscience., 30, 8096-8101.

711

Garrow, J.S. (1978). The regulation of energy expenditure in man. In G.A. Bray (Ed.),

712

Advances in Obesity Research: II. (pp. 200-210). London: Newman.

713

Glendining, J.L., Breinager, L., Kyrillo, E., Kacuna, K., Rocha, R., & Sclafani, A. (2010).

714

Differential effects of sucrose and fructose on dietary obesity in four mouse strains.

715

Physiology & Behavior, 101, 331-343.

716

Glendining, J.L., Gilman, J., Zamer, H., Margolskee, R.F., & Sclafani, A. (2012). The

717

role of T1r3 and Trpm5 in carbohydrate-induced obesity in mice. Physiology & Behavior,

718

107, 50-58.

719

Gold, R.M. (1970). Hypothalamic hyperphagia produced by parasagittal knife cuts.

720

Physiology & Behavior, 5, 23-25.

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ACCEPTED MANUSCRIPT 32 Gold, R. M. (1973). Hypothalamic obesity: the myth of the ventromedial nucleus.

722

Science, 182, 488-490.

723

Gold, R.M., Jones, A.P., & Sawchenko, P.E. (1977). Paraventricular area: critical focus

724

of a longitudinal neurocircuitry mediating food intake. Physiology & Behavior, 18, 1111-

725

1119.

726

Gold, R.M., Quackenbush, P.M., & Kapatos, G. (1972). Obesity following combination of

727

rostrolateral to VMH cut and contralateral mammillary area lesion. Journal of

728

Comparative and Physiological Psychology, 79, 210-218.

729

Grossman, S.P. (1971). Changes in food and water intake associated with an

730

interruption of the anterior or posterior fiber connections of the hypothalamus. Journal of

731

Comparative and Physiological Psychology, 75, 23-31.

732

Grossman, S.P., & Grossman, L. (1971). Food and water intake in rats with parasagittal

733

knife cuts medial or lateral to the lateral hypothalamus. Journal of Comparative and

734

Physiological Psychology, 74, 148-156.

735

Hetherington, A.W. & Ranson, S.W. (1940). Hypothalamic lesions and adiposity in the

736

rat. Anatomical Record, 78, 149-172.

737

Hetherington, A. W., & Ranson, S.W. (1942). The spontaneous activity and food intake

738

of rats with hypothalamic lesions. American Journal of Physiology, 136, 609-817.

739

Ingle, D.J. (1949). A simple means of producing obesity in the rat. Proceedings of the

740

Society for Experimental Biology and Medicine, 72, 604-605.

741

Kapatos, G., & Gold, R.M. (1973). Evidence for ascending noradrenergic mediation of

742

hypothalamic hyperphagia. Pharmacology, Biochemistry &. Behavior, 1, 81-87.

743

Kirchgessner, A.L., & Sclafani, A. (1988). PVN-hindbrain pathway involved in the

744

hypothalamic hyperphagia-obesity syndrome. Physiology & Behavior, 42, 517-528.

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ACCEPTED MANUSCRIPT 33 Kirchgessner, A.L., Sclafani, A., & Nilaver, G. (1988). Histochemical identification of a

746

PVN-hindbrain feeding pathway. Physiology & Behavior, 42, 529-543.

747

Leibowitz, S.F., Hammer, N.J., & Chang, K. (1981). Hypothalamic paraventricular

748

nucleus lesions produce overeating ad obesity in the rat. Physiology & Behavior, 27,

749

1031-1040.

750

Lucas, F., Ackroff, K., & Sclafani, A. (1989). Dietary fat-induced hyperphagia in rats as a

751

function of fat type and physical form. Physiology & Behavior, 45, 937-946.

752

Lucas, F., Ackroff, K., & Sclafani, A. (1998). High-fat diet preference and overeating

753

mediated by postingestive factors in rats. American Journal of Physiology. Regulatory,

754

Integrative and Comparative Physiology, 275: R1511-R1522.

755

Lucas, F., & Sclafani, A. (1999). Differential reinforcing and satiating effects of

756

intragastric fat and carbohydrate infusions in rats. Physiology & Behavior, 66, 381-388.

757

Maller, O. (1964). The effect of hypothalamic and dietary obesity on taste preference in

758

rats. Life Sciences, 3, 1281-1291.

759

Mogenson, G.N. (1974). Changing views of the role of the hypothalamus in the control

760

of ingestive behaviors. In Lederis, K & Cooper, K.E. (Eds.) Recent Studies of

761

Hypothalamic Function (268-293). Basil: Karger.

762

Mufson, E.J., Sclafani, A., & Aravich, P.F. (1980). Fiber degeneration associated with

763

hyperphagia-inducing knife cuts in the hypothalamus. Experimental Neurology, 67, 633-

764

645.

765

Ong, Z.Y., Alhadeff, A.L., & Grill, H.J. (2015). Medial nucleus tractus solitarius oxytocin

766

receptor signaling and food intake control: the role of gastrointestinal satiation signal

767

processing. American Journal of Physiology. Regulatory, Integrative and Comparative

768

Physiology, 308, R800-R806.

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ACCEPTED MANUSCRIPT 34 Palka, Y., Liebelt, R.A., & Critchlow, V. (1971). Obesity and increased growth following

770

partial or complete isolation of ventromedial hypothalamus. Physiology & Behavior, 7,

771

187-194.

772

Paxinos, G., & Watson, C. (1982). The rat brain in stereotaxic coordinates. New York:

773

Academic Press.

774

Peckman, S.C., Entenmen, C., & Carroll, H.W. (1962). The influence of a hypercaloric

775

diet on gross body and adipose tissue composition in the rat. Journal of Nutrition, 77,

776

187-197.

777

Rinaman, L., Hoffman, G.E., Dohanics, J., Le, W.W., Stricker, E.M., & Verbalis, J.G.

778

(1995). Cholecystokinin activates catecholaminergic neurons in the caudal medulla that

779

innervate the paraventricular nucleus of the hypothalamus in rats. Journal of Comparative

780

Neurology, 360, 246-256.

781

Rodriguez de Fonseca, F., Navarro, M., Gómez, R., Escuredo, L., Nava, F., Fu, J.,

782

Murillo-Rodriguez, E., Gluffrida, A., LoVerme, J., Gaetani, S., Kathuria, S., Gall, C., &

783

Piomelli, D. (2001). An anorectic lipid mediator regulated by feeding. Nature, 414, 209-

784

212.

785

Romano, A., Soares Potes, C., Tempesta, B., Cassano, T., Cuomo, V., Lutz, T., &

786

Gaetani, S. (2013). Hindbrain noradrenergic input to the hypothalamic PVN mediates the

787

activation of oxytocinergic neurons induced by the satiety factor oleoylethanolamide.

788

American Journal of Physiology. Endocrinology and Metabolism , 305, E1266-E1273.

789

Schemmel, R., Mickelson, O., & Tolgay, Z. (1969). Dietary obesity in rats: Influences of

790

diet, weight, age, and sex on body composition. American Journal of Physiology, 216,

791

373-379.

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ACCEPTED MANUSCRIPT 35 Sclafani, A. (1971). Neural pathways involved in the ventromedial hypothalamic lesion

793

syndrome in the rat. Journal of Comparative and Physiological Psychology, 77, 70-96.

794

Sclafani, A. (2013). Gut-brain nutrient signaling: appetition vs. satiation. Appetite, 71,

795

454-458.

796

Sclafani, A., & Ackroff, K. (2012). Role of gut nutrient sensing in stimulating appetite and

797

conditioning food preferences. American Journal of Physiology. Regulatory, Integrative

798

and Comparative Physiology, 302, R1119-R1133.

799

Sclafani, A., Aravich, P.F., & Landman, M. (1981). Vagotomy blocks hypothalamic

800

hyperphagia on a chow diet and sucrose solution, but not on a palatable mixed diet.

801

Journal of Comparative and Physiological Psychology, 95, 720-734.

802

Sclafani, A., & Berner, C.N. (1976). Influence of diet palatability on the meal taking

803

behavior of hypothalamic hyperphagic and normal rats. Physiology & Behavior, 16, 355-

804

363.

805

Sclafani, A., & Berner, C.N. (1977). Hyperphagia and obesity produced by parasagittal

806

and coronal hypothalamic knife cuts: further evidence for a longitudinal feeding inhibitory

807

pathway. Journal of Comparative and Physiological Psychology, 91, 1000-1018.

808

Sclafani, A., Berner, C.N., & Maul, G. (1973). Feeding and drinking pathways between

809

medial and lateral hypothalamus in the rat. Journal of Comparative and Physiological.

810

Psychology, 85, 29-51.

811

Sclafani, A., Berner, C.N., & Maul, G. (1975). Multiple knife cuts between the medial and

812

lateral hypothalamus in the rat: a reevaluation of hypothalamic feeding circuitry. Journal

813

of Comparative and Physiological. Psychology, 88, 210-217.

814

Sclafani, A., & Gorman, A.N. (1977). Effects of age, sex, and prior body weight on the

815

development of dietary obesity in adult rats. Physiology & Behavior, 18, 1021-1026.

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ACCEPTED MANUSCRIPT 36 Sclafani, A., & Grossman, S.P. (1969). Hyperphagia produced by knife cuts between the

817

medial and lateral hypothalamus in the rat. Physiology & Behavior., 4, 533-537.

818

Sclafani, A., & Kluge, L. (1974). Food motivation and body weight levels in hypothalamic

819

hyperphagic rats: a dual lipostatic model of hunger and appetite. Journal of Comparative

820

and Physiological Psychology, 86, 28-46.

821

Sclafani, A., Lucas, F., & Ackroff, K. (1996). The importance of taste and palatability in

822

carbohydrate-induced overeating in rats. American Journal of Physiology. Regulatory,

823

Integrative and Comparative Physiology, 271, R1197-R1202.

824

Sclafani, A., & Maul, G. (1974). Does the ventromedial hypothalamus inhibit the lateral

825

hypothalamus? Physiology & Behavior, 12, 157-162.

826

Sclafani, A. & Rendel, A. (1978). Food deprivation-induced activity in dietary obese,

827

dietary lean, and normal-weight rats. Behavioral Biology, 24, 220-228.

828

Sclafani, A., & Springer, D. (1976). Dietary obesity in adult rats: Similarities to

829

hypothalamic and human obesity syndromes. Physiology & Behavior, 17, 461-471.

830

Sclafani, A., Springer, D., & Kluge, L. (1976). Effects of quinine adulterated diets on the

831

food intake and body weight of obese and non-obese hypothalamic hyperphagic rats.

832

Physiology & Behavior, 16, 631-640.

833

Sclafani, A., Vigorito, M, & Pfeiffer, C.L. (1988). Starch-induced overeating and

834

overweight in rats. Influence of starch type and form. Physiology & Behavior, 42, 409-

835

415.

836

Sclafani, A., & Xenakis, S. (1984a). Sucrose and polysaccharide induced obesity in the

837

rat. Physiology & Behavior, 32, 169-174.

838

Sclafani, A., & Xenakis, S. (1984b). Influence of diet form on the hyperphagia-promoting

839

effect of polysaccharide in rats. Life Sciences, 8, 491-508.

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ACCEPTED MANUSCRIPT 37 Stellar, E. (1954). The physiology of motivation. Psychological Reviews, 61, 5-22.

841

Swanson, L.W., & Kuypers, H.G.J.M. (1980). The paraventricular nucleus of the

842

hypothalamus: cytoarchitectonic subdivisions and organization of projections to the

843

pituitary, dorsal vagal complex and spinal cord as demonstrated by retrograde

844

fluorescence double-labeling methods. Journal of Comparative Neurology, 194, 555-570.

845

Swanson, L.W., Sawchenko, P.E., Wiegand S.J., & Price, J.L. (1980). Separate neurons

846

in the paraventricular nucleus project to the median eminence and to the medulla or

847

spinal cord. Brain Research, 197, 207-212.

848

Verbalis, J.G., Stricker, E.M., Robinson, A. G., & Hoffman, G.E. (1991). Cholecystokinin

849

activates c-fos expression in hypothalamic oxytocin and corticotrophin-releasing hormone

850

neurons. Journal of Neuroendocrinology, 3, 205-213.

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856 857 858 859 860 861 862 863

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ACCEPTED MANUSCRIPT 38 864 865 866

Table 1. Anthony Sclafani Research Areas and Number of Publications, 1969-2016

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867 Hypothalamic Pathways

15

Dietary Responsiveness and Finickiness

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Dietary Obesity and It’s Metabolic Effects

28

11

Intestinal Bypass Effects on Feeding and Obesity

10

Carbohydrate Appetite and Sweet Taste Responsivity

51

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Role of the Vagus Nerve and Autonomic Nervous System in the VMH Syndrome

Flavor/Nutrient Preferences and Conditioning

870 871 872 873 874

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Intestinal Nutrient Receptors and Appetition

868

28

119

40

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Figure Legends

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878 Figure 1. Anatomical model proposed by Sclafani et al. (1973) to explain differential

880

feeding effects of 1.0 and 1.5 knife cuts. A knife cut at L 1.0 mm would sever feeding-

881

inhibitory projections to both sets of feeding excitatory neurons, while a knife cut at L 1.5

882

mm would sever feeding inhibitory projections only to LH feeding-excitatory neurons,

883

while also transecting feeding-excitatory projections to the LH. (VMH = ventromedial

884

hypothalamus; LH = Lateral Hypothalamus F = fornix; I = feeding inhibitory neuron E =

885

feeding excitatory neuron). (Adapted with permission from Sclafani et al. 1975, Vol. 88(1),

886

p. 211, Journal of Comparative & Physiological Psychology, American Psychological

887

Assn.).

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Figure 2. Horizontal sections through the hypothalamus illustrating knife cuts that are

890

effective in producing hyperphagia and obesity (Effective Cuts, blue), and those that are

891

not effective (Ineffective cuts, red), based on the findings of Albert, Gold, Grossman,

892

Paxinos, Sclafani, and others. The position of these cuts suggests that the pathway

893

whose destruction is responsible for the hyperphagia syndrome either originates in the

894

preoptic-anterior hypothalamic area (PO-AH) and descends to the mid- or hindbrain, or

895

ascends from the caudal brainstem to terminate in the PO-AH area. (Adapted with

896

permission from Sclafani & Berner, 1977, Vol. 91(5), p. 1014, Journal of Comparative &

897

Physiological Psychology, American Psychological Assn.).

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ACCEPTED MANUSCRIPT 40 Figure 3. Schematic representation of the “feeding” pathway implicated in the MH

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hyperphagia-obesity syndrome. The pathway appears to originate in the PVN and

901

surrounding tissue, and projects laterally and caudally with at least some of the fibers

902

extending to the medulla. The NST and/or DX are likely termination sites for the pathway.

903

Illustrated are a unilateral MH cut and contralateral vM and vP cuts. This horizontal

904

section was adapted from Paxinos and Watson, 1982. (AP = area postrema; DX = dorsal

905

motor nucleus of the vagus; MH = medial hypothalamus; MPO = medial preoptic area;

906

NST = nucleus of the solitary tract; PVN = paraventricular nucleus; vM = ventral medulla;

907

vP = ventral pons; VMN = ventromedial nucleus). (Reprinted with permission from

908

Kirchgessner & Sclafani, 1988, Vol. 42(6), p. 526, Physiology & Behavior, Elsevier Press)

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Figure 4. DIO in a female rat after 3-4 months on the supermarket cafeteria diet

911

(provided by Professor Anthony Sclafani).

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Figure 5. Body weight as a function of days and diet in experimental (EXP) and (CON)

914

groups of female rats (lower left of figure). The EXP group was switched to the cafeteria

915

diet at the interval above the arrowhead. Graph in the upper left shows bar pressing for

916

food reward by the two groups of rats as a function of a fixed ratio schedule. Graph on

917

the right shows decreased body weight gain as a result of pelleted chow diet in EXP and

918

quinine adulteration in CON. The graph in the lower middle illustrates food intake by EXP

919

and CON during the 3-day quinine- adulterated-diet test (Reprinted with permission from

920

Sclafani and Springer, 1976, Vol. 17(3), p. 462, Physiology & Behavior, Elsevier Press).

921

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ACCEPTED MANUSCRIPT 41 922

Figure 6. Enriched group environment in which supermarket cafeteria diet also produced

923

DIO. (Reprinted with permission from Sclafani and Springer, 1976, Vol. 17(3), p. 466,

924

Physiology & Behavior, Elsevier Press).

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925 Figure 7. Mean body weight gains of groups fed supermarket (S) or Purina chow (P)

927

diets and housed in enriched (E), isolated (I), or activity (A) cages. Weight gains based

928

on body weights of rats on (Day 10) just prior to their transfer to the different housing

929

conditions. On Day 0, E-S, IS, and A-S groups were given supermarket diet. On Day 65,

930

indicated by the arrows, I-S and I-P groups were placed in the activity wheels becoming

931

groups IA-S and IA-P, while A-S and A-P groups were returned to isolated cages

932

becoming AI-S and AI-P groups. (Reprinted with permission from Sclafani and Springer,

933

1976, Vol. 17(3), p. 467, Physiology & Behavior, Elsevier Press).

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Figure 8. Frequency histogram of weight gains displayed by 26 experimental (EXP) and

936

26 control (CON) rats after 60 days on the supermarket or Purina chow diets. (Reprinted

937

with permission from Sclafani and Springer, 1976, Vol. 17(3), p. 470, Physiology &

938

Behavior, Elsevier Press).

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Figure 9. Twenty-four hour milk intake (3 left panels) and bar pressing responses

941

(extreme right panel) of dietary obese (filled columns) and control (empty columns)

942

female rats offered plain milk and 0.03% quinine adulterated milk. DIO and chow-fed

943

control rats were reduced to 85% of their ad libitum body weight for the bar-pressing test.

944

(Reprinted with permission from Sclafani and Springer, 1976, Vol. 17(3), p. 464,

945

Physiology & Behavior, Elsevier Press).

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Figure 2

INEFFECTIVE CUTS

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A-6 VM

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Fig. 9