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Keywords:

  • ADHD;
  • attention;
  • behavior;
  • dopamine;
  • hyperactivity;
  • impulsivity;
  • mouse;
  • receptor;
  • thyroid;
  • transgenic

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Attention deficit hyperactivity disorder (ADHD) is the most commonly diagnosed childhood psychiatric disorder. We have found that a transgenic mouse bearing a human mutant thyroid receptor (TRβ1) expresses all of the defining symptoms of ADHD—inattention, hyperactivity, and impulsivity—as well as a ‘paradoxical’ response to methylphenidate (MPH). As with ADHD, the behavioral phenotypes expressed by the TRβ transgenic mice are dynamic and sensitive to changes in environmental conditions, stress, and reinforcement. TRβ transgenic mice are euthyroid except for a brief period during postnatal development, but the behavioral phenotypes, elevated dopamine turnover, and paradoxical response to MPH persist into adulthood. Thus, like the vast majority of children with ADHD, the TRβ transgenic mice exhibit the symptoms of ADHD in the complete absence of thyroid abnormalities. This suggests that even transient perturbations in developmental thyroid homeostasis can have long-lasting behavioral and cognitive consequences, including producing the full spectrum of symptoms of ADHD.

Abnormal thyroid hormone levels during gestation can have profound effects on brain development and cognition (Bernal 2002; Thompson & Potter 2000) Neurodevelopmental processes such as cellular differentiation, neurite outgrowth, synaptogenesis, and myelination all depend on proper thyroid homeostasis (Konig & Moura Neto 2002). Normal development of the monoaminergic and cholinergic neurotransmitter systems are also thyroid dependent, and dysfunctions in these transmitter systems have been linked to attention deficits and hyperactivity (Evans et al. 1999; Jin et al. 2001; Oades & Muller 1997; Rastogi & Singhal 1976). It has long been known that cretinism, mental retardation, and other debilitating conditions can result from abnormal thyroid synthesis, action, or metabolism during development. However, the consequences of transient, subclinical thyroid abnormalities are just beginning to be appreciated (Klein et al. 2001; Pop et al. 1995; 2003; Smit et al. 2000 Surks et al. 2004).

In the pituitary gland, the β thyroid receptors (TRβ) are responsible for downregulating thyroid stimulating hormone (TSH), an action that results in decreased production of thyroid hormones thyroxine (T4) and triiodothyronine (T3). A number of mutations in the thyroid receptor β (Thrb) gene have been identified, most of which are in the ligand-binding domain of the TRβ receptor and act to impair the ability of the receptors to bind T3 (Jameson 1994; Yen 2003; Yen et al. 2003). With functional receptors, the mutant receptors form homo- and heterodimers that lack the ability to act at genomic response elements. The result of this dominant negative action is a failure to downregulate TSH. This condition, known as resistance to thyroid hormone (RTH), is a heritable syndrome that typically results in elevated thyroid hormones, normal or elevated TSH, short stature, hearing loss, and tachycardia (Beck-Peccoz et al. 1992; Weiss & Refetoff 2000). In addition, 70% of children with RTH syndrome have attention deficit hyperactivity disorder (ADHD) compared with an incidence of 3–5% in the population as a whole (Burd et al. 2003; National Institutes of Health 1998). Although RTH syndrome is rare, the strikingly high number of RTH patients that exhibit the full spectrum of ADHD symptoms suggests that common mechanisms downstream of the TRβ receptor may be responsible for manifestation of the behavioral phenotypes in both disorders.

Several mouse lines with mutations in the Thrb gene have been created. Knockout mice lacking the Thrb gene exhibit elevated TSH and free and total T3 and T4 but normal behavior (Forrest et al. 1996). Transgenic mice bearing the human PV mutant Thrb gene have been developed (Wong et al. 1997). The PV mutant TRβ1 was derived from a patient (PV) with a mutation in the ligand-binding domain of the Thrb gene, and RTH syndrome characterized by short stature, low body weight, and ADHD (Mixson et al. 1992; Parrilla et al. 1991; Wong et al. 1997). The mice were created using a β-actin promoter to induce ubiquitous expression of the mutant receptor. Consistent with a thyroid resistance phenotype, thyroid hormone levels in the TRβ(β-actin) transgenic mice are increased by approximately 50%, and TSH levels are inappropriately normal. Behaviorally, male TRβ (β-actin) transgenic mice are hyperactive relative to wild-type controls but have normal attention and are not impulsive (McDonald et al. 1998).

In order to better understand the role of thyroid resistance in mediating the behavioral features of ADHD, we tested a transgenic mouse that harbors the PV mutant Thrb gene (Zhu et al. 1999). In this mouse, mutant receptor expression is limited to the pituitary using the mouse glycoprotein hormone α-subunit (αGSU) promoter. The ratio of functional, endogenous receptors to non-functional, mutant receptors in the TRβ(αGSU) transgenic mice is 1:1 (Zhu et al. 1999). Thus, transgenic and wild-type control mice are identical except for 50% of the TRβ1 receptors in the pituitary. However, despite the robust expression of mutant transgene, TRβ(αGSU) transgenic mice exhibit normal thyroid levels as adults (Zhu et al. 1999). The present experiments were designed to test the hypothesis that transient thyroid resistance during development can result in persistent behavioral and neurochemical abnormalities in adulthood, in the TRβ(αGSU) transgenic mice.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Subjects

Transgenic mice bearing the human PV mutant TR-β gene were created using the αGSU promoter, as previously described (Zhu et al. 1999). This promoter limits expression of the transgene to the pituitary. Multiple lines were created and the two lines characterized showed identical phenotypes (Zhu et al. 1999). Mice were backcrossed 12 generations or more to a C57BL6/NIH inbred background and housed by gender in tub cages in groups of 3–5. The vivarium was accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and was maintained on a 14/10-h light/dark cycle with lights on at 0600 h. Except for locomotor activity experiments and hormone assays, which were performed in juvenile mice at the ages specified, all behavioral experiments were conducted in adult mice. Food and water were freely available at all times in their home cages except during operant testing. All experiments described herein were approved by the Institutional Animal Care and Use Committee of Vanderbilt University.

Apparatus

Exploratory locomotor activity was measured in eight identical activity monitors measuring 27 × 27 cm (MED Associates, Georgia, VT). Each apparatus contained 16 photocells in each horizontal direction and 16 photocells elevated 4.0 cm to measure rearing. Beam breaks were recorded and analyzed automatically by a Windows-based microcomputer and MED Associates software. Reaction time, progressive ratio, and delay of gratification experiments were conducted in 12 identical operant chambers, each housed within a ventilated sound-attenuating cubicle (MED Associates). Each operant chamber was outfitted with three nose-poke holes that could be illuminated from the inside, and which had an infrared beam spanning the front for the detection of the nose-poke response. Two nose-poke response holes were on the front wall, equidistant left and right of the receptacle for the delivery of the liquid reinforcer. The third nose-poke hole was located in the center of the opposite wall. A house light above the third nose-poke hole provided a source of low-level illumination. The liquid reinforcer was stored in a reservoir outside the operant chamber but within the sound-attenuating cubicle. A mechanical dipper arm was used to deliver 0.1-ml portions of the liquid reinforcer to the inside of the operant chamber. Operant contingencies were controlled, and data collected, by a Windows-based computer running MED-PC software (MED Associates).

Drug preparation and administration

Methylphenidate (MPH) HCl was dissolved in physiological (0.9%) saline at a concentration of 40 mg salt/kg of body weight and injected intraperitoneally at a volume of 10 ml/kg of body weight. Control mice were injected with an equal volume of saline. The dose of MPH was determined after a series of pilot tests using a small number of mice. Because mice have a high metabolic rate, higher doses are required with mice to achieve the same effects that are evident at lower doses in species such as rats, monkeys, and humans. The 40-mg/kg dose is within the range of doses typically used with mice (Hawken et al. 2004; Itzhak & Martin 2002; McFadyen et al. 2002; Rhodes & Garland 2003; Trinh et al. 2003).

Behavioral methods

Locomotor activity

All behavioral experiments were conducted during the first 6 h of the light cycle, except where noted. Mice were transported to the behavioral testing suite at least 1 h before the start of the behavioral testing sessions. Mice were placed in the monitors for 60 min and allowed to explore freely. Separate groups of mice were tested in 60-min. sessions at 33, 56, or 90 days of age. Repeated locomotor activity sessions were conducted twice daily, in the early light and dark cycles, for three consecutive days followed by two daily sessions a week after the first session.

Sustained attention (simple reaction time)

Sustained attention was assessed using modifications of previously published procedures (McGaughy & Sarter 1995; McGaughy et al. 1996, 1997; McDonald et al. 1998; McGaughy & Sarter 1999). Mice were put on a food-restricted diet of 4-h free access to food per day. Water remained freely available for the course of the experiments. After an initial period of weight loss, mice will gain weight under this food restriction regimen but will continue to be motivated to work for a food reinforcer during the behavioral session. Mice were trained to emit a nose-poke response to receive a reinforcer consisting of the liquid dietary supplement Ensure® (Abbott Laboratories, Columbus, OH) diluted either 1:1 (50%) or 9:1 (10%) with water. Ensure® is a nutritionally complete, sweet vanilla-flavored liquid supplemented with vitamins and other nutrients. The proportions of energy (Kcal) from macronutrients in Ensure® are as follows: carbohydrates 63.9%, fats 22%, and proteins 14.1%. Complete nutritional information can be found at www. abbott.com. Mice were trained gradually so that in the final contingencies the nose-poke holes were illuminated for 3 seconds per trial, and mice were required to respond during this 3-second period in order to earn a reinforcer (4 seconds access to 0.1 ml of Ensure® solution). Responses that resulted in the delivery of reinforcement were termed ‘hits’, and failures to respond within the 3-second period were termed ‘misses’. Each trial began with illumination of the houselight and simultaneous initiation of the ‘precue’ period, a signal to the mouse that the cue was forthcoming after an unpredictable period of time. The duration of the precue period was randomly selected on each trial and ranged from 9 to 21 seconds. (mean = 15 seconds). The intertrial interval (ITI) was fixed at 10 seconds, and responses during this period carried no scheduled consequences. Any response during the precue period in either the nose-poke hole or the food hopper caused the precue period to reset to its initial value for that trial. In this way, mice learned to refrain from responding during the precue period, and anticipatory responses in all mice were reduced to near-zero levels. A chart depicting the flow of events in a reaction-time session is included in Fig. 1. After reaction-time sessions with variable reinforcer magnitudes, performance of both groups of mice was re-stabilized on Ensure® diluted 3:1 (25%) with water, at which time three sessions were conducted in which only water was delivered for a correct response. Although this is not technically an extinction schedule, water holds little or no value as a reinforcer because the mice were not deprived of water. This was confirmed by the inability of the water outcome to maintain high levels of performance.

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Figure 1. Flow charts depicting the order of events in the three operant tasks. The logical order of events for the reaction time, delay of gratification, and progressive ratio tasks. Detailed descriptions of the tasks are found in the results section.

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Progressive ratio

The progressive ratio task is a commonly-used test of motivation in many species, including humans (Hayward et al. 2002; Mayorga et al. 2000; Paule et al. 1988; Rodefer & Carroll 1996; Rusted et al. 1998; Rocha et al. 2002). Mice are trained to emit a nose-poke response to earn reinforcement of 4 seconds access to either 50 or 10% Ensure® solution. Each reinforcer requires two more responses than the previous reinforcer, i.e. the first reinforcer requires two responses, the second four, the third six, and so on. The session continues until the mouse does not respond for 5 min. The number of responses required for the last reinforcer of the session is termed the ‘break point’ and is a measure of the mouse's motivation to work for the food reinforcer. A chart depicting the flow of events in a progressive-ratio session is included in Fig. 1.

Delay of gratification

The adjusting amount version of the delay of gratification (DoG) task was modified from those published by de Wit and Richards (de Wit et al. 2002; Richards et al. 1999a; Richards et al. 1999b; Farrar et al. 2003). Food-restricted mice were trained to respond in the left and right nose-poke holes to receive reinforcement of a 25% Ensure® solution, delivered by raising a dipper from the outside to the inside of the chamber. We reduced the concentration of Ensure® in this experiment for practical reasons. The higher concentration (50%) was sticky and difficult to clean thoroughly after the daily session. The 25% concentration was thinner but still maintained a high level of responding. Contingencies were gradually changed, so in the end mice were required to make a choice between immediate reinforcement for a response in the right nose-poke hole and delayed reinforcement for a response in the left nose-poke hole. The magnitude of the reinforcer was manipulated by changing the duration that the dipper was available to the mouse, starting from the moment that the mouse broke the infrared beam spanning the entry to the dipper well. The duration of the delayed reinforcer remained constant at 10 seconds. The duration of the immediate reinforcer started at 4 seconds on the first trial of each session and increased or decreased by 15% with each trial. In this way, the median dipper duration for all trials after trial 30 can be used as an indication of the mouse's indifference point, i.e. the point at which the mouse does not show a greater preference for one reinforcer over the other. A shorter dipper duration is indicative of preference for the immediate reinforcer and is considered a measure of impulsive behavior (Mischel & Gilligan 1964; Rapport et al. 1986; Schweitzer & Sulzer-Azaroff 1988; Mischel et al. 1989; Sonuga-Barke et al. 1992; Richards et al. 1999a, 1999b; Tripp & Alsop 2001; de Wit et al. 2002; Farrar et al. 2003; Sonuga-Barke et al. 2003). A chart depicting the flow of events in a DoG session is included in Fig. 1.

Catecholamine assays

Experimentally naïve, 3-month-old mice were killed after a brief isoflurane anesthesia. Brains were rapidly removed and sliced coronally into 1- or 2-mm thick sections using a brain matrix on wet ice. A 2-mm diameter punch was taken from the right striatum in a section extending from 0 to 1 mm rostral to bregma. A 1-mm diameter, punch was taken from the nucleus accumbens in a section extending from 1 to 2 mm rostral to bregma. The anterior cingulate and surrounding cortex were removed from the same section. The hypothalamus was removed from the section containing 0–2 mm caudal to bregma. All samples were immediately frozen on dry ice and stored at −80 °C until homogenization. Each frozen tissue sample was homogenized by ultrasonication in 250 µl of 0.1 m TCA, which contains 10−2 m sodium acetate, 10−4 m EDTA, and 10.5% methanol (pH 3.8). Samples were spun in a microcentrifuge at 13 000 g for 8 min. The supernatant was removed and stored at −80 °C (Cransac et al. 1996). The pellet was stored at −80 °C until used for protein analysis. The supernatant was thawed and spun for 20 min. Samples of the supernatant were analyzed for biogenic monoamines by a specific HPLC assay utilizing an Antec Decade (oxidation: 0.7) electrochemical detector. Twenty-microliter samples of the supernatant were injected using a Water 717 + autosampler onto a Waters Nova-Pak C18 HPLC column. Biogenic amines were eluted with a mobile phase consisting of 89.5% 0.1 m TCA. Solvent was delivered at 0.7 ml/min using a Waters 515 HPLC pump. Using this HPLC solvent, the following biogenic amines elute in the following order: norepinephrine (NE), 3-methoxy-4-hydroxyphenylglycol (MHPG), epinephrine, DOPAC, dopamine (DA), 5-HIAA, HVA, 5-HT, and 3-MT (Lindsey et al. 1998). HPLC control and data acquisition were managed by Millennium 32 software.

Hormone assays

Blood was drawn from mice at 33 or 56 days of age after a brief (approximately 20 seconds) isoflurane anesthesia. Triiodothyronine (T3), thyroxine (T4), and TSH assays were conducted using radioimmunoassay as previously described (Zhu et al. 1999).

Statistical analyses

Single-session locomotor activity data were analyzed using a 2-factor repeated measures analysis of variance (RMANOVA), with genotype as a between-subjects factor and 5-min period as the repeated measure. Multi-session locomotor activity data were analyzed using a hierarchical RMANOVA, with genotype and gender as between-subjects factors. Session was nested within day and within phase of the light cycle. Data from male and female mice were pooled when there was no genotype–gender interaction; for locomotor activity tests and thyroid hormone assays, gender data were analyzed separately.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

TRβ transgenics exhibit situational hyperactivity and a paradoxical response to MPH

Exploratory locomotor activity was assessed in individual groups of mice of various ages. There were no significant genotype differences in locomotor activity in initial activity sessions at any age (all F's < 0.5; all P 's > 0.480; data not shown). However, after repeated sessions during both light and dark phases of the daily light/dark cycle, young adult male TRβ transgenic mice became hyperactive in the dark phase compared with wild-type mice. There was a significant genotype × day × 5-min period interaction (F33,627 = 1.85, P = 0.0030), indicating that the more male transgenics were exposed to the open field, the less they habituated within the session (Fig. 2). There were no differences in activity among female mice in single or repeated sessions (all F 's < 1.5; all P 's > 2.5; data not shown).

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Figure 2. Situational hyperactivity in male TRβ transgenics. Mice were exposed to the open field on 4 days for 1 h in the light cycle and 1 h in the dark cycle. Black bars signify dark-cycle sessions. TRβ transgenic mice were no different than wild-type controls after initial exposure to the novel environment. After repeated exposures, genotype differences emerged among the male mice during the dark phase of the light/dark cycle. By the fourth dark-phase session, male TRβ transgenics were significantly more active overall and habituated less within the session than wild-type controls. There were no genotype differences among female mice on any parameter of locomotor activity.

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Experimentally naïve 56-day-old mice were injected intraperitoneally with 40 mg/kg MPH or saline immediately before being placed in open-field activity monitors. At this age, the activity of uninjected transgenic mice was no different than that of wild-type controls (Fig. 2). After saline injection, however, male TRβ transgenic mice were significantly hyperactive compared with wild-type controls, suggesting a differential response to injection stress in the male transgenic mice (F1,10 = 45.53, P < 0.0001). Methylphenidate significantly increased normal response to this stimulant drug (Fig. 3; F1,7 = 20.98, P = 0.0025). In contrast, male TRβ transgenics started and finished the session more active, but were less active under MPH than under saline for most of the 1-h session (Fig. 3A; drug × time F11,143 = 4.17, P < 0.0001). Female transgenic mice were not hyperactive under saline (F1,10 = 0.01, P = 0.938) but had an attenuated response to methylphenidate compared with controls (Fig. 3B).

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Figure 3. Altered response to methylphenidate in male TRβ transgenics. Locomotor activity in naïve 56-day-old mice immediately after intraperitoneal injection of saline or 40 mg/kg methylphenidate (MPH). (a) Male TRβ transgenics were significantly more active than wild-type mice under saline. Activity of male wild-type mice increased after injection of MPH, consistent with this drug's action as a psychostimulant. In contrast, the activity of the TRβ transgenic mice was lower under MPH than under saline for most of the 1-h session. This paradoxical response to psychostimulants is a hallmark of ADHD. (b) Female TRβ transgenic mice were not hyperactive under saline. However, they exhibited an attenuated response to MPH similar to that observed in the male transgenics.

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TRβ transgenic mice are inattentive and impulsive

Sustained attention was measured using an operant reaction-time task. Mice were trained to emit a nose-poke response as quickly as possible after illumination of a cue light, to receive a sweet liquid reinforcer (the dietary supplement Ensure®) diluted either 1:1 (50%) or 9:1 (10%) with water. Responses that resulted in the delivery of reinforcement were termed ‘hits’, and failures to respond within the 3 seconds cue period were termed ‘misses’. Humans and animals with attentional impairments have fewer hits and slower reaction times on this type of task (Sunohara et al. 1999; Borger & van der Meere 2000). Figure 4 illustrates that when the reinforcer magnitude was large (50%), there were no genotype differences in reaction times or percentage of hits. However, when the reinforcer concentration was reduced to 10%, TRβ transgenic mice had significantly slower reaction times than wild-type controls, before returning to baseline (Fig. 4A; F1,28 = 5.25; P = 0.0297; genotype × session F5,140 = 6.83; P < 0.00001). The change in hit rate showed a similar pattern, although the difference was not statistically significant (Fig. 4B; F1,28 = 1.54; P = 0.225; genotype × session F5,140 = 2.20; P = 0.0574). This decline in performance under conditions of degraded reinforcement suggests that the reaction-time deficits may result from differential motivation and not an inability to attend per se. This effect dissipated after several sessions with the lower concentration, suggesting that the deficits were not a function of the absolute value of the reinforcer but rather the result of the change to a lower reinforcer magnitude.

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Figure 4. Attention deficits in TRβ transgenics are sensitive to reinforcement magnitude. Mice were trained to respond quickly to a briefly-presented stimulus in order to receive a reinforcer of diluted Ensure®, a sweet liquid dietary supplement. (a, b) Using a concentrated reinforcer (50% Ensure® solution), there were no genotype differences on the reaction-time task. Under low reinforcement (10% Ensure®) conditions, TRβ transgenics had slower reaction times (a) and reduced hit rates (b) compared to wild-type controls. After several sessions with 10% Ensure®, performance of both groups returned to baseline levels.

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Motivation for the liquid reinforcer was assessed using the progressive ratio task. Mice were trained to make multiple responses in a nose-poke response hole for a reinforcer. The required number of responses increased by two for each subsequent reinforcer until the subject did not respond for 5 min, at which time the session ended. The number of responses required for the last reinforcer of the session is referred to as the break point and is a commonly used measure of motivation. At the higher reinforcer magnitude (50%), the TRβ transgenic mice had higher break points than wild-type littermates, suggesting increased motivation for the reinforcer (Fig. 5; F1,22 = 4.90; P = 0.0375). When the reinforcer magnitude was reduced (10%), the break points for both groups decreased to similar values (F1,22 = 1.15; P = 0.295). These data suggest two possible explanations for the performance of the TRβ transgenic mice on the reaction-time task. The increased motivation of the TRβ transgenic mice for the higher magnitude reinforcer may have masked their attentional deficit on the reaction-time task. When reinforced with the lower magnitude reinforcer, the attentional deficit in the TRβ transgenic mice was revealed. Alternatively, the TRβ transgenics may be more sensitive to decreases in reinforcer magnitude. The TRβ transgenic mice exhibited a larger decrease in break point when the reinforcer magnitude was reduced and a larger increase in reaction times on the reaction-time task when the reinforcer magnitude was reduced.

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Figure 5. Altered motivation in the TRβ transgenic mice. In the progressive ratio test of motivation, the number of responses required to earn a reinforcer increased by two on each trial until the mouse stopped responding for 5 min. The number of responses required for the last reinforcer of the session is termed the ‘break point’ and is a measure of motivation. When 50% Ensure® was used as a reinforcer, TRβ transgenics were more motivated to work for reinforcement than wild-type controls. When the reinforcer concentration was reduced to 10%, break points were similar for both groups.

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Impulsivity is an inability to inhibit a response or responding without adequate assessment of the context or consequences (Hinson et al. 2003). Response to extinction is commonly used to assess impulsive behavior (Douglas & Parry 1983; Sagvolden et al. 1993b; Iaboni et al. 1997; Wigal et al. 1998; Johansen et al. 2002). To measure this, we fully degraded the reinforcer in the operant reaction-time task, i.e. administered water as a consequence for a ‘hit’. All other environmental events were identical to previous reaction-time sessions. By the third day under these extinction conditions, the percentage of hits decreased to approximately 50% from approximately 98%, and reaction time slowed to approximately 1.5 seconds from approximately 0.85 seconds, in both transgenic and wild-type mice (Fig. 6a, b; hits F1,28 = 1.55, P = 0.223; reaction time F1,28 = 2.92, P = 0.099). However, on the hit trials, transgenic mice were more likely to approach the food well after a response (Fig. 6c; F1,28 = 13.23 P = 0.0011). This type of behavior suggests that the TRβ transgenic mice have an impaired ability to inhibit the food-well approach response. The slowed reaction times and decreased proportion of hits (Figs 6a, b) suggest that both groups of mice understood the contingencies equally, i.e. that no reinforcer was forthcoming. Nevertheless, the TRβ transgenic mice continued to approach the food well while the wild-type mice were better able to inhibit this response.

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Figure 6. TRβ transgenics are more resistant to extinction. Impulse control, measured by the persistence of dipper approaches in the presence of a water-only reinforcer on the reaction-time test. Reinforcer magnitude was reduced to 0% Ensure®, i.e. only water was given for a correct response. Because mice had free access to water throughout the study, water held little or no value as a reinforcer. (a) By the third session with a water outcome, reaction times slowed in both groups of mice. (b) Similarly, hit rate was reduced in both groups of mice by the third session. (c) After a hit, the likelihood of entering the food well decreased with successive trials with a water outcome, in both groups. However, the TRβ transgenics persisted in approaching the food well more than wild-type mice, indicative of increased impulsive behavior.

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An additional test of impulsive behavior was conducted using the DoG procedure. Mice were given a choice between a small reinforcer delivered immediately or a large reinforcer delivered after a delay (dipper delay). The delay to the large reinforcer remained constant within a session but increased from 0 to 32 seconds on consecutive daily sessions each week. Reinforcer magnitude was controlled by manipulating the duration of access to Ensure® (dipper duration). The duration of the delayed reinforcer was 10 seconds and remained constant throughout the experiment. The duration of the immediate reinforcer was titrated up or down 15% on each trial, depending on the outcome of the previous trial, but was typically less than 1 seconds. The duration of the adjusting dipper (immediate reinforcer) is the primary dependent measure and is an index of impulsive behavior. When the delay to the large reinforcer was 8 seconds or shorter, mice of both genotypes were able to wait for the large reinforcer (Fig. 7a–c). At the 32-second dipper delay, both groups had a strong preference for the small but immediate reinforcer (Fig. 7e). There were no genotype differences at these dipper delays (all F's < 0.9; all P's > 0.365). However, at the 16-second dipper delay, TRβ transgenics were significantly more likely than wild-type controls to choose the immediate reinforcer (Fig. 7d; F1,13 = 5.9, P = 0.0301; genotype × trial F1,18 = 6.0, P = 0.0149). The median dipper duration of the immediate reinforcer calculated from all trials after the 30th trial is called the indifference point. Because the adjusting dipper duration typically reaches an asymptote by the 30th trial, the indifference point represents the duration at which mice have no strong preference for either reinforcer. As shown in Fig. 7f, the TRβ transgenic mice had a lower indifference point than wild-type control mice when the dipper delay was 16 seconds (F1,13 = 7.26, P = 0.0184).

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Figure 7. TRβ transgenics have poor impulse control on the delay of gratification procedure. Mice were given a choice between a small, immediate reinforcer (adjusting dipper duration) or a large, delayed reinforcer (10-second dipper duration). (a–e) Trial-by-trial data are presented only for the number of trials that all mice in both groups completed, for any given session. Dipper delay remained constant within a session, at 0, 4, 8, 16, or 32 seconds. All sessions began with an adjusting dipper duration of 4 seconds. The adjusting dipper duration increased or decreased by 15% on each trial but was typically less than 1 second. Shorter dipper durations result from a preference for the immediate reinforcer and are indicative of impulsive behavior. At dipper delays of 8 seconds or shorter (a–c), mice of both genotypes were able to wait out the delay for the larger reinforcer, and at the 32-second delay, (e) neither group was willing to wait for the larger reinforcer. (d) However, at the 16-second dipper delay, TRβ transgenics showed a distinct preference for the immediate reinforcer. (f) The log of the median adjusting dipper duration was calculated for all trials after trial 30, a point at which response patterns are relatively stable. At the 16-second dipper delay, TRβ transgenics were more likely to choose the immediate reinforcer compared with wild-type controls. (g) Percent initial choice of the immediate reinforcer. At dipper delays of 16 seconds and longer, TRβ transgenics initiated 15–25% more trials by choosing the immediate reinforcer than wild-type mice. (h) At any time after choosing the delayed reinforcer to initiate the trial, mice were able to respond in the other nose-poke hole to receive the immediate reinforcer and end the trial. This behavior was termed a defection. TRβ transgenic mice defected more than wild-type control mice at the 16-second delay. (i) Total proportion of trials on which mice chose the immediate reinforcer, either as the initial choice or as a defection. TRβ transgenics were nearly twice as likely as wild-type mice to choose the immediate reinforcer at the 16-second delay.

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In addition to this measure of impulsivity, we assessed the tendency of mice to switch from the delayed to the immediate alternative during the DoG procedure. At any time during the delay, mice were free to choose the immediate reinforcer after having chosen the delayed reinforcer to start the trial. This behavior was termed a ‘defection’ and resulted in the immediate delivery of the smaller reinforcer. In addition to initially choosing the immediate reinforcer more often than wild-type mice (Fig. 7g), TRβ transgenics were more likely to defect to the immediate reinforcer (Fig. 7h) when the dipper delay was 16 seconds (initial choice F1,13 = 3.75, P = 0.0749; defections F1,13 = 3.29, P = 0.0929). Although neither of these measures was statistically significant, a composite measure of both behaviors shows that the TRβ transgenics were nearly twice as likely as wild-type mice to choose or defect to the immediate reinforcer, when the dipper delay was 16 seconds (Fig. 7i; F1,13 = 5.37, P = 0.0375). This inability to tolerate the delay is indicative of impulsive behavior (Rapport et al. 1986; Schweitzer & Sulzer-Azaroff 1988; Tripp & Alsop 2001; Sonuga-Barke et al. 2003).

The behavioral tasks in the present experiment were designed to assess specific behaviors. However, as with any complex behavioral task, there is much more involved than the specific behavior or cognitive process being assessed. For example, performance in the reaction-time task may be affected by differences in locomotor activity, visual abilities, and memory for the rules of the task. All of the operant tasks include a number of measures designed to detect differences in ‘adjunct’ behaviors during the experimental session. For example, we measured ITI response rates, responding in inactive nose-poke holes, entries into the food well both after a correct response and at inappropriate times, latency to enter the food well after a correct response, response rate and postreinforcement pause during progressive ratio, and resets induced by inappropriate responding during the precue period in the reaction-time task. Inappropriate responding may suggest a misunderstanding of the rules of the task, hyperactivity, or an inability to inhibit responding. Failure to approach the food well after a correct response may suggest a lack of motivation, misunderstanding the rules of the task, or a sensorimotor deficit. Slow response times, both to experimental stimuli and to the food well after a correct response, may indicate a sensorimotor deficit, lack of motivation, or general slowing. With the exception of the probability of entering the food well after a correct response with a water outcome (Fig. 6), the only genotype difference on any of the control measures was a genotype × session interaction on ITI responding (F5,140 = 3.6, P = 0.0046). After the change from 50 to 10% Ensure, ITI response rates increased in TRβ transgenic mice but decreased in wild-type controls. The significant interaction was the result of group differences on the first session on 10% Ensure only, and response rates in all mice remained low. During this session, the majority of the mice of both genotypes had zero ITI responses per min. Three wild-type mice and two TRβ transgenics had 0.15 responses per min., three transgenics had 0.3 responses per min., and one transgenic had 0.6 responses per min. By the following session and on all other sessions, ITI response rates were not different between genotypes. Because there were so few genotype differences in control measures, we can be confident that group differences in adjunct performance variables were not responsible for the observed differences in attention, motivation, and impulsive behavior.

Male TRβ transgenics exhibit a transient thyroid resistance during postnatal development

As adults, TRβ transgenics have normal T3, T4, and TSH levels (Zhu et al. 1999). Because the behavioral phenotypes demonstrated by the TRβ transgenics must ultimately be due to non-functioning TRβ1 receptors in the pituitary, we expected to detect thyroid abnormalities at 3–6 weeks postnatally. During this time, the thyroid system is the most dynamic and thyroid levels are highest in mice. At 33 days, the male TRβ transgenic mice had significantly elevated TSH, compared with wild-type controls (Fig. 8a; F1,7 = 42.42, P = 0.0003). T3 levels were higher in male transgenics compared with wild-type controls, although this difference was not statistically significant (Fig. 8b; F1,7 = 5.08, P = 0.059). T4 levels were not different between genotypes (Fig. 8c; F1,7 = 2.28, P = 0.175). This hormone profile is consistent with the known action of mutations in the ligand-binding domain of TRβ1 and with the phenotype of thyroid hormone resistance. In the TRβ transgenics, half of the TRβ in the pituitary are functional. Thus, under most conditions they are able to downregulate TSH sufficiently so as to maintain normal thyroid levels. However, during the postnatal thyroid surge the system is challenged to such an extent that the transgenic mice are not able to downregulate TSH efficiently, resulting in elevated thyroid hormones and an even greater increase in TSH. There were no genotype differences in hormone levels among female mice at 33 days of age (all F's < 2.7; all P's > 0.110; data not shown). By 56 days of age and into adulthood (Zhu et al. 1999), the thyroid hormone and TSH concentrations of both male and female transgenic mice were no different from those of wild-type control mice (all F's < 3.95; all P's > 0.080; data not shown).

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Figure 8. Transient thyroid resistance in male TRβ transgenic mice. Levels of T3, T4, and TSH were measured at postnatal day 33. (a–c) Male TRβ transgenic mice exhibit a classic thyroid resistance phenotype, i.e. increased T3 (a) and T4 (b) in the presence of elevated TSH (c). Ordinarily, TSH will be reduced in the presence of elevated circulating thyroid hormones. Female transgenics do not show this phenotype at 33 days and were not different than wild-type control mice.

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TRβ transgenics have elevated striatal DA turnover

Adult mice were killed, and brain areas were rapidly dissected and frozen in liquid nitrogen. Striatal and prefrontal levels of DA and NE were assessed using high-pressure liquid chromatography (HPLC). Levels of their respective metabolites, homovanillic acid (HVA) and MHPG, were also measured. Except for a gender difference in prefrontal NE regardless of genotype, there were no genotype or gender differences in any of the neurotransmitters or their metabolites in either brain area (all F's < 3.2, all p's > 0.097). However, DA turnover in the striatum, as measured by HVA/DA ratio, was significantly elevated in male and female TRβ transgenic mice, compared with wild-type controls (Fig. 9; F1,15 = 11.97, P = 0.0042). Elevated striatal DA turnover has also been observed in other models of ADHD and is suggestive of DA transporter dysfunction (Jones et al. 1998; Zhuang et al. 2001).

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Figure 9. TRβ transgenic mice have elevated dopamine (DA) turnover. (a, b) There was no genotype difference in either DA or homovanillic acid (HVA). (c) In contrast, the TRβ transgenics had significantly elevated dopamine turnover, as indicated by the ratio of HVA to DA.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Although the thyroid resistance phenotype in the TRβ transgenics was detected only at 33 days of age, behavioral abnormalities were evident in euthyroid, 56-day-old mice, and persisted into adulthood. Thus all of the demonstrated behavioral phenotypes in the TRβ transgenic mice occurred in the presence of normal levels of T3, T4, and TSH. This suggests that the transient thyroid resistance observed in 33-day-old transgenics resulted in permanent changes in the brain. Thyroid hormone directly regulates the development of several brain systems associated with attention, locomotor activity, motivation, and impulsive behavior. For example, genes that code for proteins involved in myelination and the development of cholinergic, dopaminergic, and noradrenergic neurotransmitter systems are all regulated by thyroid hormone. The attenuated response to methylphenidate and increase in DA turnover in the TRβ transgenic mice suggests that these permanent brain changes involve the catecholaminergic system. This is consistent with data showing that rats made transiently hyperthyroid as pups, but not as adults, are hyperactive and exhibit elevated DA turnover (Rastogi & Singhal 1976). A number of catecholaminergic receptors and transporters have been implicated in hyperactivity and attentional deficits, including the DA transporter, DA D4 receptor, and the α2 adrenergic receptor (Giros et al. 1996; Clifford et al. 1998; Arnsten 2000). Methylphenidate inhibits the DA and NE transporters, resulting in increased neurotransmitter concentrations in catecholaminergic synapses. Considerable evidence suggests that DA turnover rates are associated with DA transporter function. The DA transporter knockout and knockdown mice both exhibit hyperactivity, reduced DA uptake, and elevated DA turnover (Jones et al. 1998; Zhuang et al. 2001). Other perturbations that reduce DA uptake also increase turnover, such as aging, fatigue, or exposure to environmental toxins such as the ubiquitous polychlorinated biphenyls (PCBs) or the organophosphate insecticide chlorpyrifos (Karen et al. 2001; Haycock et al. 2003; Mizokawa et al. 2003; Richardson & Miller 2004).

Interestingly, locomotor hyperactivity in male transgenic mice was only evident after repeated sessions or after a saline injection in an initial activity session. The fact that multiple sessions were required before genotype differences emerged suggests that male transgenic mice were only hyperactive in a familiar environment. The asymptotic locomotor activity of the male transgenics increased with repeated dark-cycle sessions, suggesting that the hyperactivity was more than a simple failure to habituate. Importantly, the hyperactivity exhibited by the male TRβ transgenics was an increase in normal murine activity in a familiar environment, with none of the repeated circling, head-bobbing, or stereotypic behaviors characteristic of many animal models of hyperactive syndromes (Giros et al. 1996; Wilson 2000). Naïve male transgenics were only hyperactive after a saline injection (Fig. 3a). It is difficult to know to what extent this putative stress-induced hyperactivity interacted with the attenuated response to methylphenidate. If the male transgenics had not been hyperactive relative to wild-type controls under saline, the methylphenidate response would have been in the same direction as that of the wild-type mice, albeit a smaller response similar to what was observed in the female mice. Alternately, if injection stress was the source of behavioral change it is plausible to assume that both saline and methylphenidate groups were affected. Whether injection stress is manifest similarly under saline and methylphenidate is not known. Current investigations examining home-cage running-wheel activity with methylphenidate administered through the drinking water should address some of these concerns. This situational hyperactivity in the TRβ transgenic mice is consistent with expression of attention deficits and hyperactivity in humans and in animal models, which are exhibited primarily in familiar environments and are exacerbated under stressful conditions (American Psychiatric Association 1994; National Institutes of Health 1998; Drolet et al. 2002; Carboni et al. 2003).

Transgenic mice performed as well as wild-type mice under the high reinforcement conditions (50% Ensure) in the reaction-time task. When the concentration of the reinforcer was reduced to 10%, performance of the male transgenic mice deteriorated significantly more than that of wild-type controls. This is consistent with the idea that ADHD-related phenotypes in humans and non-humans are particularly sensitive to changes in reinforcement (American Psychiatric Association 1994; Douglas & Parry 1983,1994; Sagvolden et al. 1993a, 1998). Although this may be conceived of as a motivational deficit (Haenlein & Caul 1987; Slusarek et al. 2001), our progressive ratio data suggest that there was no genotype difference in motivation at the lower reinforcer magnitude. In fact, the transgenic mice had higher progressive ratio break points when working for 50% Ensure, suggesting that attentional deficits at the higher reinforcer concentration may have been masked by increased motivation to work for the food reinforcer. When Ensure was replaced with water as a reinforcer, there were no differences in hit rate or reaction time between the two genotypes. Although this may seem inconsistent with the change from 50 to 10% Ensure, in fact there is a fundamental difference in that it is a change from reinforcement to no reinforcement, rather than from strong-to-weak reinforcement. The mice were not water deprived, and the rapid drop in hit rate to 20% in both groups after three sessions is evidence that water held little or no value as a reinforcer.

The persistence of TRβ transgenic mice to approach the food well with only a water outcome is characteristic of resistance to extinction (Sagvolden et al. 1998), which may be mediated by the presence of conditioned reinforcers in the operant-testing environment. Johansen and Sagvolden (2004) showed the importance of conditioned reinforcers using the spontaneously hypertensive rat (SHR) model of ADHD. Using a multiple-component reinforcement schedule, performance of water-restricted SHR rats for water reinforcement was similar to that of controls. However, under the extinction component of the schedule, SHR rats were significantly more likely to enter the water receptacle in the presence of conditioned reinforcer (a light that previously signaled reinforcement). In the absence of conditioned reinforcers, SHR mice were no more likely than controls to enter the water receptacle. In the present experiment, there were a number of conditioned reinforcers operating on each hit trial, the most salient of which was extinguishing of the cue light at the moment of each correct response. Each correct response was also signaled by a click, which was used during training to facilitate learning and maintained throughout the experiment. Immediately following these events, the dipper was raised, which was accompanied by the quiet but distinct sound of the motor driving the dipper arm. All of these events occurred on every hit trial, regardless of reinforcer concentration, and probably served as conditioned reinforcers. Resistance to extinction can be induced by lesions of the dorsal noradrenergic bundle (DNB) and reversed by inhibitors of the NE transporter (Mason & Fibiger 1979; Mason & Iversen 1979; Pisa et al. 1988). An extensive set of experiments conducted by Mason & Iversen (1977, 1978, 1979; Mason 1979) determined that resistance to extinction induced by DNB lesions could not be explained by conditioned reinforcement, locomotor activity, perseveration, internal inhibition, frustrative non-reward, or motivation. Instead, they found that this DNB extinction effect was the result of impaired selective attention in the failure to ignore relevant stimuli in the experimental situation. In contrast, Steketee et al. (1989) found that that the DNB extinction effect resulted from differential response to novelty and not attentional impairments. Although further investigation is required to determine the behavioral and neurochemical processes involved in the persistence of TRβ transgenics to enter the food well in the absence of reinforcement, it is consistent with deficits observed in the SHR model and with a disruption in the noradrenergic system.

Although the incidence is greater among children with ADHD than in children without ADHD, detected thyroid abnormalities, including RTH, are rare in children with ADHD (Weiss et al. 1993). Like the TRβ transgenic mice, most children with ADHD are hyperactive, impulsive, and inattentive in the presence of normal thyroid hormone profiles. In the TRβ transgenics, these symptoms arise from a brief period of thyroid resistance during brain development, but symptoms persist much longer. In contrast, the initiating cause of the vast majority of ADHD cases is unknown. A recent study suggests that subclinical maternal thyroid abnormalities may be involved in a greater number of cases of ADHD than previously thought—even among children with normal thyroid levels at the time the symptoms are expressed (Haddow et al. 1999). Estimates of the prevalence of subclinical thyroid abnormalities range as high as 23% (Canaris et al. 2000; Glinoer & Delange 2000; Hollowell et al. 2002; Klein et al. 1991; Ladenson et al. 2000; Volzke et al. 2003). In addition, exposure to a number of environmental toxins has been implicated in ADHD (Berger et al. 2001; Hauser et al. 1998; Rice 2000). Many of the most common environmental toxins act directly on the thyroid system (Gauger et al. 2004), and in some cases may induce thyroid hormone resistance (Kuriyama et al. 2003). In many if not most cases, subclinical maternal or neonatal thyroid dysfunction or perinatal exposure to environmental toxins goes undetected or unmeasured. Thus, it is not known the extent to which these factors contribute to the prevalence of ADHD in the general population. Our data are consistent with the notion that transient, undetected, or subclinical thyroid disruption during gestation or postnatal brain development may contribute to some cases of ADHD in which juvenile thyroid levels are completely normal.

Interestingly, the thyroid resistance phenotype was not observed in female transgenics, despite their expressing largely similar behavioral phenotypes as male mice. The fact that female transgenics exhibited an attenuated response to methylphenidate and increased DA turnover suggests that the changes in the catecholamine system are also similar in male and female transgenics. At this point, we do not know the cause of the behavioral, pharmacological, or neurochemical phenotypes in the female transgenics, but as with male transgenics, it probably involves a transient thyroid resistance. There are a number of possible reasons for this gender difference. There is evidence in rats that the postnatal TSH surge begins later in females than in males (Simpkins et al. 1976). This temporal difference may be attributable to the fact that female rats have a higher density of pituitary TRβ than male rats (Donda et al. 1987) and thus may be better able to maintain physiological TSH levels. There is sexual dimorphism in both the number of substance P (SP)-containing cells in the anterior pituitary (AP) as well as the number of those cells that colocalize with TSH (Brown et al. 1991). The function of the colocalization is not known, but it is thought that SP modulates TSH release. SP levels in the AP are regulated both by thyroid hormones and estrogen (Brown et al. 1991). Estrogen receptors are also found in the AP and may compete with thyroid receptors for coactivators and DNA response elements (Pfaff et al. 1994). Estrogen increases thyroidal 131I uptake in mice, starting at 30 days of age (Cidlowski et al. 1975), and modulates thyroid growth in a gender-specific manner from 30 to 45 days of age (Banu et al. 2001). Other changes, such as gender differences in hippocampal granule cell number, are directly mediated by thyroid hormone during development (Madeira et al. 1988). Any or all of these mechanisms and more may contribute to the observed gender difference in the thyroid resistance phenotype in the TRβ transgenics at 33 days of age.

The TRβ transgenic mice exhibit all of the core symptoms and several adjunct features of ADHD, including a paradoxical response to methylphenidate. It is notable that the behavioral phenotypes expressed by the TRβ transgenic mice are dynamic and sensitive to changes in environmental conditions, stress, and reinforcement. Such differential reactivity has also been observed in children with ADHD, is altered by methylphenidate, and may be responsible for the observed gender differences (Iaboni et al. 1997; Johansen et al. 2002). This suggests that the mechanisms involved in ADHD converge at some point with mechanisms downstream of inactive TRβ1 receptors in the transgenic mice. The increased DA turnover in the transgenic mice supports this notion. Thyroid hormones regulate complex cascades of gene expression during development, including many of the genes implicated in ADHD. Exactly which genes are perturbed to produce the constellation of ADHD symptoms is not yet known. Thus, the TRβ transgenic mouse is a powerful tool that may be used to discover basic mechanisms and novel therapeutics involved in a large number of cases of ADHD of differing etiologies.

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  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
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Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Technical assistance was provided by Michelle Jacobs and Lindsay Wood. Support was provided by NINDS (1R21NS043581–01A1), NICHD (5P30HD015052-23), and the Nicholas Hobbs Society. Thyroid Assays were conducted by Wanda Snead of the Hormone Assay Core in the Mouse Metabolic Phenotyping Center, Vanderbilt Diabetes Center. Catecholamine assays were conducted by Ray Johnson in the Neurochemistry Core, Vanderbilt Center for Molecular Neuroscience.