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Chapter 46: Behavioral Phenotypes of Neurodevelopmental Disorders 627 DEFINITION AND CHARACTERIZATION disorder of purine metabolism clearly leads to the overpro- duction of uric acid and renal stones generic 100 mg viagra capsules visa, but the pathway to The study of behavioral phenotypes emphasizes the discov- the movement disorder and self-injury is not direct and may ery purchase genuine viagra capsules line, among individuals with known chromosomal viagra capsules 100mg discount, genetic, be mediated through effects on the arborization of dopa- or neurodevelopmental disorders, of those mental and be- mine neurons (19). Moreover, there are variants of LND havioral features causally related to the underlying condi- with different degrees of enzyme deficit, ranging up to 20%, tion. Examples are the characteristic self-mutilation of fin- that have clinical effects. When present, the behavior suggests the syn- disorders, have increased probability of occurring and do drome. As Nyhan suggested, these are 'syndromes of behav- not occur in all cases; they are not be fully expressed in ior' (14). Still, despite their behavioral presentations, not all affected individuals; (b) the genetic background of the all individuals with the disorder show the classic behavioral individual may affect the phenotypic expression; (c) the pos- features, but the probability is greater that they will. The sibility exists that environmental factors may modify expres- essential issue is that the behavior suggests the diagnosis. Harris proposed that behavioral phenotypes der; and (e) variability occurs in mouse models in which are stereotypic patterns of behavior that are reliably identi- there may be species-specific factors so mutant mouse fied in groups of individuals with known neurodevelopmen- models do not replicate the behavioral features. One must tal disorders and are 'not learned' (15,16). They may be consider the genetic background, strain differences, and the the consequence of neurodevelopmental abnormalities that differences in the rodent physiology. This approach to definition is deficient mouse has a uricase enzyme that breaks down uric a phenonic approach that takes as its starting point observa- acid. Therefore, it is not a model for the hyperuricemic tions of the behavior itself rather than beginning with a metabolic disorder, but it still may be a useful model to discrete and genetically identifiable condition, such as study dopamine deficiency in the brain. Using the phenomic approach, the behav- the disorder may be modeled in transgenic mice or in other ioral phenotype of Rett syndrome (17), with its characteris- species. The canine model of narcolepsy (20) genetic origin was recognized. Moreover, the phenomic ap- is an interesting example of an approach to a human clinical proach does not discount acquired disorders, such as fetal disorder. Mutations for canine, autosomal recessive, narco- alcohol syndrome, as having behavioral phenotypes. The lepsy were identified by linkage analysis in canine back- impact of alcohol on cellular signaling is now well known, crosses, and homology was demonstrated between human with its consequences of cell death, abnormal midline brain chromosome 6 and canine chromosome 12. Canine narco- development, behavioral problems, and learning disabilities lepsy is caused by a disruption of a G-protein–coupled re- (16,23). However, human narcolepsy is not as- following definition that includes the characteristic types sociated with frequent hypocretin gene mutation (20). This does not mean that the behavior is present in all instances but that the probability of its occurrence is PSYCHOPATHOLOGY AND BEHAVIORAL increased. In the future, more may be learned about brain PHENOTYPES mechanisms by comparing persons with behavioral involve- ment with others who have the same syndrome but without Numerous neurogenetic disorders are associated with non- the behavioral features. Although some investigators have sought to limit the These include attention problems, hyperactivity, impulsiv- study of behavioral phenotypes to known genetic disorders ity, self-injury, aggression, autistic-like behavior, and pre- (11), knowledge of the genetic disorder is only the first step. Such presentations indicate vulnerabil- Links from gene to behavior are complicated in that one ity of the developing brain and perturbation of brain systems gene may lead to the encoding of many, perhaps ten or resulting in these clinical conditions. However, because more, different proteins; the number of genes and type of these behaviors occur across many syndromes, they lack mutation determine complexity. For example, in LND, the specificity and do not qualify as specific behavioral pheno- 628 Neuropsychopharmacology: The Fifth Generation of Progress types. Still, these behavioral features should be included in are highlighted, findings on origin are discussed, and poten- the description of the disorders. For example, the relation- tial neurochemical and neuroanatomic abnormalities are re- ship between aggression and antisocial behavior has been viewed. Behavioral and pharmacologic therapies have had suggested in monoamine oxidase A (MAOA) deficiency. Neuroanatomic studies, brain imaging studies, single large Dutch kindred (21). The affected males differed and continuing investigations of neurotransmitter systems, from unaffected males in that they tested in the borderline endocrine rhythms, and sleep studies may provide informa- range of mental retardation and demonstrated increased im- tion that will be helpful in the future in treatment. Yet a specific psychiatric diagnosis was not made in four affected males who were examined Lesch–Nyhan Disease by psychiatrists. Because MAOA deficiency leads to in- LND is a rare (1:380,000) sex-linked recessive disease creased 5-hydroxytryptamine (5-HT) levels, the aggressive caused by an inborn error of purine nucleotide metabolism. HPRT, which is involved in the purine salvage (purine base suggested that even if a possible association between MAOA recycling) pathway (25). LND is of psychosocial and psychiatric tors noted that genes are essentially simple and code for importance because of the lifelong suffering experienced by proteins, whereas behavior is complex; thus, a direct causal the involved child and his family, the uniqueness of the relationship between a single gene and a specific behavior behavioral phenotype, and the resources needed for lifelong is highly unlikely. In MAOA deficiency, complexity is patient supervision. Moreover, an understanding of the neu- shown by the variability in the behavioral phenotype and robiological basis of this disease may contribute to a better by the highly complex consequences of MAOA deficiency understanding of brain mechanisms involved in self-inju- on neurotransmitter function. Thus, the full pathway from rious and compulsive behaviors. Still, a great deal may be learned by considering such path- Genetic and Metabolic Aspects ways in neurogenetic syndromes. The HPRT-encoding gene is located on the X chromosome in the q26-q27 region and is made up of nine exons and eight introns totaling 57 kilobases (kb). The HPRT gene is PREVALENCE transcribed to produce a mRNA of 1. More than 270 With increasing attention to neurogenetic disorders, the mutations throughout the coding regions have been identi- number of identifiable behavioral phenotypes is increasing. Techniques that provide information on the Careful observations of behavior are necessary when consid- three-dimensional structure of the HPRT protein make it ering intervention for neurogenetic disorders. Although possible to correlate structure and function of the enzyme standardized rating scales and personality profiles have been (26). Besides The gene involved in LND is on the X chromosome, so behavioral phenotypes, isolated special abilities that occur the disorder occurs almost entirely in males; occurrence in in genetically based syndromes require assessment. The metabolic abnormality is the include special abilities in calculation and in music (24). This enzyme is normally present in each posed modular organization of the central nervous system. Its absence prevents the normal metabolism of hypoxanthine and results in excessive uric acid production BEHAVIORAL PHENOTYPES OF SPECIFIC and manifestations of gout without specific drug treatment NEURODEVELOPMENTAL DISORDERS (i. The full disease requires the virtual ab- sence of the enzyme. Other syndromes with partial HPRT The sections that follow discuss four syndromes in which deficiency are associated with gout without the neurologic behavioral phenotypes have been identified:LND, PWS/ and behavioral symptoms. Page and Nyhan reported that AS, fragile X syndrome, and WMS. Characteristic behaviors HPRT levels are related to the extent of motor symptoms, Chapter 46: Behavioral Phenotypes of Neurodevelopmental Disorders 629 the presence or absence of self-injury, and possibly the level findings was documented on quantitated neurologic exami- of cognitive function (27). The study of variant cases with motor symptoms but with no self-injurious be- Self-injurious behavior usually is expressed as self-biting; havior suggests that reductions in dopamine receptor den- however, other patterns of self-injurious behavior may sity are not a sufficient explanation of the self-injury.

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Decreased HVA in CSF can be expected after a compound in advance buy 100 mg viagra capsules amex. It is doubtful that any substantial mixed Types A and B MAO inhibition in the brain (52) decisions concerning doses of compounds affecting either and might be relevant to assessing DA uptake inhibition purchase 100 mg viagra capsules otc. DA uptake inhibition order viagra capsules 100mg online, did not decrease HVA in the CSF (40,44). ACTH/Cortisol Prolactin Stimulation of the hypothalamic–pituitary–adrenal axis as reflected by increases of ACTH and/or cortisol has also been Prolactin has the potential to be used as a marker for drugs used as a marker of drug action in the CNS. This approach affecting these systems, because either DA or 5HT can affect has been most extensively applied to evaluation of putative this circulating hormone. In the simplest and most wide- 5HT agonists, sometimes coupled with pretreatment with spread instance, it has been used as an index of D2 antago- whatever antagonists were available (e. Unlike typical neuroleptics, atypical neuroleptics pro- dolol) (63). Such studies can generate evidence of apparent duce no elevations of prolactin at therapeutic dosages (59, selectivity in postsynaptic receptor responses to indirect and 60). By extension, absence of prolactin elevation after anti- direct agonists. ACTH release induced by 5-hydroxy-L- psychotics could be considered a surrogate of low D2 recep- tryptophan (5HTP) is argued to occur through indirect acti- tor occupancy in the striatum and, hence, imply low to vation of 5HT2 receptors because it is antagonized by ri- absent motor side effects (61,62). Pindolol does, however, antagonize ACTH re- to increase extracellular 5HT in the brain including sponses to a variety of agents classified as partial to full fenfluramine, clomipramine, l-tryptophan, and 5- 5HT1A agonists (64,79,80). Again, given the complexities hydroxytrytophan; and (b) those that stimulate various of the ACTH/cortical response and the imperfect selectivity types of 5HT receptors, including meta-chlorophenylpiper- of the pharmacologic agents, quantitative conclusions as to azine (mCPP) and some, but not all, putatively selective degree of specific receptor activation or antagonism are not 5HT1A agonists (63–66). Growth Hormone Temperature decreases are consistently observed follow- ing 5HT1A agonists (63,83), and hence can serve as a surro- There was a period in which plasma growth hormone (GH) gate marker of 5HT1A agonist effects in the CNS. Evaluat- was used as a surrogate for increased noradrenergic transmis- ing the ability of a novel compound to antagonize the sion in human brain (presumably at the level of the hypo- hypothermia produced by a 5HT1A agonist may be the easi- thalamus) after, for instance, the 2 agonist, clonidine, or est way to see if it antagonizes 5HT1A receptors in the an NE uptake inhibitor (71,72). More recently, stimulation of plasma GH has been con- sidered evidence of activation of both 5HT1A and 5HT1B/ LIMITATIONS OF CURRENT SURROGATE 1Dreceptors (see Table 34. Sumatriptan MARKERS increases GH, apparently through activation of the 5HT1B/ 1D receptors (73,74), with the most recent evidence using As described, there is no validated linkbetween the concen- the more brain penetrant, zolmitriptan, implicating 5HT1D tration of drug in blood (or even CSF) and a specific bio- postsynaptic receptors (75). It has been suggested that stim- chemical effect in human brain (not to mention a specific ulation of the 1B/1D receptors inhibits somatostatin release brain region). Thus, even in the case of SSRIs, the most Chapter 34: Proof of Concept 463 widely prescribed class of drugs in neuropsychopharmacol- By contrast, positron emission tomography (PET), an- ogy, we do not know how closely platelet 5HT depletion other procedure to estimate rCBF, requires an onsite cyclo- reflects 5HT uptake inhibition in brain. Although known tron, so it is not available in many areas. Recent third-party therapeutic doses of SSRIs invariably have been shown to camera reimbursement of some PET procedures (primarily deplete platelet 5HT, the converse is not clear; that is, any [18F]fluorodeoxyglucose (FDG)) are making PET cameras dose that is effective in platelets will be therapeutic. Matters more available, but cyclotrons are still relatively scarce. The are even less certain when it comes to using available surro- two most widely used radioisotopes used in PET are 11C gates of NE uptake inhibition to establish the dose of a with a half-life of 20 minutes and 18F with a half-life of drug. And, as already noted, primary dosing decisions are 110 minutes. Additionally, 15O, with a half-life of 2 minutes not made on the basis of whether compounds affect prolac- is primarily employed in brain perfusion studies. The most tin, growth hormone, or ACTH/cortisol responses. The problem is that even in vantage is lackof specificity. Furthermore, in vivo ani- ies demonstrate increases in rCBF in the limbic system and mal studies raise questions about species differences; there- decreases in the basal ganglia as manifestations of craving fore, how does one select the dose for clinical studies when for cocaine following exposure to videotapes suggesting co- testing a new compound that is well tolerated with a wide caine use (91). How does one know that the target in question has been blocked ESTIMATION OF DOPAMINE RECEPTOR or stimulated so as be sure that one is testing the hypothesis OCCUPANCY BY ANTIPSYCHOTIC DRUGS that such an effect produces therapeutic benefit? The answer is, one does not know with the surrogates (discussed in the It has been hypothesized that positive symptoms of schiz- preceding). This brings us to a discussion of the promise ophrenia, such as hallucinations and delusions, result from of direct measures of drug effect in the brains of living increased stimulation of postsynaptic dopamine (DA) re- humans. This DA hypothesis of schizophrenia is substantiated by the observation that positive symptoms of schizophrenia abate when DA receptor blocking drugs, IMAGING STUDIES such as neuroleptics, occupy the postsynaptic DA receptors. In 1988, Farde and colleagues demonstrated the concept Measures of Substrate Metabolism that effective neuroleptic dosages for schizophrenia corre- Regional cerebral blood flow (rCBF) is a well-established spond to 80% to 90% occupancy of DA type-2 receptors surrogate marker widely used for both clinical diagnostic (D2Rs) by the drug (95). Thus, occupancy of 80% to 90% procedures and new drug development. Furthermore, clinically 123 99m equivalent doses of neuroleptics are estimated by comparing with radioligands labeled with Ior Tc; for example, [123I]iodoamphetamine, [99mTc]ethylenediylbis-L-cystein proportions of receptors occupied by various psychothera- diethylester (ECD), and [99mTc]hexamethylpropylenea- peutic agents (93,94). Several examples of this approach are mine oxime (HMPAO). Although repeated assessments given in the following. Furthermore, Tc is relatively available in practically every hospital with a radiology or The occupancy of D2Rs by neuroleptics has been extensively nuclear medicine department worldwide; therefore, SPECT evaluated by PET and SPECT. Historically, this began with procedures can be performed widely on a clinical basis. On the other tients with schizophrenia utilizing [11C]raclopride (95,96), hand, doses of greater than 30 mg/day olanzapine are associ- spiperone derivatives, including [11C]N-methyl-spiperone ated with greater than 80% D2R occupancy as well as dyski- (NMSP) (97), [18F], or 76Br (98). This research has estab- nesias and prolactin elevation. Olanzapine exhibits 59% to lished that a therapeutic response corresponds to occupancy 63% D2Rs occupancy after single 10-mg dosing. Further- of 65% to 90% of the D2Rs by the drug; however, occu- more, olanzapine shows a greater (68% to 84%) occupancy pancy of greater than 90% of the D2Rs is not associated of D2Rs than clozapine (20% to 67%) after 10 to 20 mg with a greater therapeutic effect. Therefore, occupancy of daily dosing in patients (60). These characteristics suggest 65% to 90% of D2R2 is correlated with a therapeutic dose that olanzapine differs from clozapine. Additionally, quetia- of typical neuroleptics as well as other clinical manifestations pine is characterized by abatement of psychotic symptoms of pharmacologic efficacy. For example, patients with acute in association with a transient increase in D R occupancy 2 extrapyramidal side effects were found to have higher DA (62). Wolkin and colleagues (1989) found a comparable occupancy of haloperidol in responders versus nonrespond- Receptor Occupancy as a Surrogate ers indicating that treatment nonresponse is not a function Marker of Clinical Efficacy? Kapur and colleagues recently confirmed that the D2 One of the most important questions about D2R and occupancy is an important mediator of beneficial and ad- 5HT2A receptor occupancy is the prediction of doses yield- verse effects (101) in a study of first episode schizophrenia ing clinical efficacy. D2R occupancy predicted the clinical and haloperidol. Although the patients showed a wide range improvement of 22 first episode schizophrenic patients ran- of D2R occupancy (38% to 87%), the degree of D2R occu- domly assigned to 1 or 2.

An RatesCalculated from 15NMRSand13C alternative strategy is to use isotopic precursors that exclu- MRS Results sively introduce label into the glia purchase viagra capsules pills in toronto. Analysis of the flow of isotope from the glia into the neuronal glutamate pool yields To obtain an independent measurement of Vgln and Vana purchase genuine viagra capsules line, 15 15 the rate of total neuronal/glial glutamate trafficking purchase viagra capsules no prescription. Com- N MRS was used to measure the rate of N-labeled am- 13 parison with the rate calculated using [1- C] glucose gives monia incorporation into the N5 position of glutamine and the fraction of neuronal/glutamate trafficking due to the the unresolved resonance of N2 glutamate plus glutamine glutamate/glutamine cycle (27,36). A mathematical analysis based on the model was used The initial use with MRS of the strategy of glial selective to derive Vgln from the MRS measurement of the time 15 15 precursors to calculate the fraction of glutamate trafficking course of [5- N]glutamine and [2- N] glutamate gluta- due to the glutamate/glutamine cycle measurement was by mine. The labeling in the first hour was almost exclusively Shen et al. Under hyperammo- using N and N labeled ammonia (62,69). The low ini- 15 nemic conditions the rate of N ammonia incorporation tial rate of anaplerosis allows the rates determined from the 15 into the N5 and N2 position of glutamine is the same in N NMRstudy to be compared with the rates measured 13 the glutamate/ -ketoglutarate cycle because only the an- by C NMRunder normal physiologic conditions. The aplerotic pathway of glutamine synthesis is present. To distinguish these models, the endpoint 15N enrichment of the N2 positions Validation of the 13C MRS Measurement of glutamate and glutamine were calculated relative to the of the Glutamate/Glutamine Cycle, and glutamine N5 position for each model using the N5 gluta- Assessment of Alternate Models of mine labeling curve as an input and compared with experi- mental values. Several alternative models to the glutamate/glutamine cycle An additional test of the glutamate/glutamine cycle (Fig 25. In one alternative model model was recently performed using 2-13C] glucose as an the 13C labeling of glutamine represents an internal glial isotopic precursor (27). Label from [2-13C] glucose enters glutamate/glutamine cycle as opposed to trafficking be- the inner positions of glutamate and glutamine only tween the neuron and glia. Label enters C4-glutamine from through pyruvate incorporation into the TCA cycle by py- [1-13C] glucose in this model through exchange in the glial ruvate carboxylase, which is localized to the glia (27,74). In this pathway glu- tamate taken up by the glial cell is transaminated into - ketoglutarate and enters the TCA cycle. Reactions in the TCA cycle convert -ketoglutarate to oxaloacetate, which is then converted to pyruvate by the action of malic enzyme. The pyruvate formed from glutamate is oxidized in the TCA cycle through the action of pyruvate dehydrogenase. Calculated 15N2/15N5 fractional enrichment ratios mate lost to the brain by this pathway is then replaced by of glutamine and glutamate for three models of glial glutamine synthesis. Three models of neuronal glutamate completion were anaplerosis through pyruvate carboxylase. Evidence of this compared with experimental results in which the time course of pathway is derived primarily from isolated cell cultures. It [5-15N] glutamine and [2-15N] glutamine and glutamate were 15 has been proposed that the fraction of glutamate going measured by N nuclear magnetic resonance (NMR) in the cortex of a rat infused with 15N-labeled ammonia at 7 T (36). The mea- through this pathway increases with brain electrical activity sured ratio at the end of the infusion is in excellent agreement (64). If instead the MRS measurement of the glutamate/glutamine cycle is the cycle was internal to the astrocyte the N2/N5 glutamine rela- to cause the fraction of glutamine synthesis of net an- tive 15N enrichment would be two times higher than measured aplerosis to be overestimated and Vcycle to be consequently and no labeling would have been observed in N2 glutamate (model b). If glutamate neurotransmitter repletion took place underestimated, because the labeling of the internal posi- through the astrocytes providing the neurons with -ketogluta- tions of glutamine from the two pathways from [1-13C] rate (model c, which is diagrammed in Fig 25. The unambiguous in vivo anaplerotic and total glutamine synthesis would be similar and 13 the N5/N2 ratio of glutamine would be close to 1. A similarlabeling strategyhas recently flow from glutamate to pyruvate to be measured (10,63). Suggestive evi- dence of this pathway is the finding in several studies that the rate of anaplerosis under normal ammonia conditions tamine (27). Subsequently, neuronal/glial cycling moves the calculated from labeling of glutamine by 13C labeled glucose label to the neuron where it labels the large glutamate pool. In contrast the rate An alternate possibility is that rather than glutamate oxida- of labeling of glutamine from a [1-13C] glucose precursor tion this extra labeling reflects cycling between oxaloacetate is a measure of the glutamate/glutamine cycle. In vivo and and pyruvate to generate reduced nicotinamide adenine di- in vitro 13C MRS at 7 T was recently used to measure nucleotide phosphate (NADPH) reducing equivalents in the labeling time course of glutamate and glutamine in the the glia, a pathway that has been shown to be highly active cerebral cortex of rats under hyperammonemic and nor- in the liver (75). The rate calculated for the Validation of the 13C MRS Glutamate/ neuronal/glial glutamate cycle was similar, with both labels Glutamine Cycle Measurement by indicating that the glutamate/glutamine cycle is the major Correlation with Brain Electrical Activity pathway of neuronal/glial glutamate trafficking accounting for between 80% and 100% of total glutamate trafficking. A If the 13C labeling measured in glutamine by 13CMRSis similar conclusion was recently reported for human cerebral due to the glutamate/glutamine cycle, then the calculated cortex using [2-13C] acetate as a precursor (38), which selec- rate of this pathway should correlate with brain electrical tively introduces label into glutamate and glutamine activity. Neuronal glutamate release is known to increase through glial pyruvate dehydrogenase. To test this prediction, 13C MRS was used to measure the rates of neuronal glucose oxidation and the glutamate/ glutamine cycle in the rat cerebral cortex at three levels of cortical electrical activity: isoelectric EEG induced by high- dose pentobarbital anesthesia, and at two milder levels of anesthesia (26). During isoelectric conditions, under which minimal glutamate release takes place, almost no glutamine synthesis was measured, consistent with the conclusion that the 13C MRS measurement of glutamine synthesis primarily reflects the glutamate/glutamine cycle. Above isoelectricity, the rates of the glutamate/glutamine cycle and neuronal glucose oxidation both increased with higher electrical activ- ity. The relationship measured in this study between the rate of the glutamate/glutamine cycle and neuronal glucose oxidation is described below (see Determination of the In Vivo Coupling Between the Rate of the Glutamate/Gluta- FIGURE 25. In vivo 13C NMR time course of the human occipi- mine Neurotransmitter Cycle and Neuronal Glucose Oxi- tal/parietal lobe: the time course from one subject of the concen- trations of [4-13C] glutamate and [4-13C]glutamine during a dation). At time 0 on the plot an intravenous infusion of [1-13C] glucose was started. The model is shown to provide an 13C MRS Measurements of the Rate of 13 excellent fit to the data. The rise of [4- C] glutamine is clearly seen to lag the labeling of [4-13C] glutamate, consistent with neu- the Glutamate/Glutamine Cycle in Human ronal glutamate being the main precursor for glutamine synthesis Cerebral Cortex via the glutamate/GABA/glutamine cycle. Determination In 1994 we first demonstrated that in vivo C NMRmay of the rate of the glutamate-glutamine cycle in the human brain be used to measure the rate of glutamine labeling (12,18) by in vivo 13C NMR. Proc Natl Acad Sci USA 1999;96:8235–8240, from [1-13C] glucose in human occipital/parietal cortex. However, the rate of the glutamate/glutamine cycle was not uniquely determined in the initial experiments due to the (mean SD, n 6). In agreement with studies in rat inability to distinguish the glutamate/glutamine cycle from cortex, the glutamate/glutamine cycle is a major metabolic other sources of glutamine labeling. To determine whether flux in the resting human brain with a rate approximately there is a similar high rate of the glutamate/glutamine cycle 80% of the rate of total glucose oxidation. A high rate A time course from the study of Shen and co-workers of the glutamate/glutamine cycle was measured using a two- (29) showing the rapid labeling of C4-glutamine and C4- compartment model, similar to the model used by Shen glutamate from [1-13C] glucose in a single subject is shown and co-workers (29). A best fit of the metabolic model is plotted tion provided by the higher field strength at 4 T allowed through the data. A lag is clearly shown in the labeling of the additional positions of the C2 and C3 resonances of C4-glutamine relative to C4-glutamate, which is consistent aspartate, glutamate, and glutamine to be incorporated into with the large neuronal glutamate pool being the main pre- the modeling.