In the MeCP2Bird model (18), male and female mice appeared normal till the third postnatal week, afterward both sexes developed gross abnormalities (with sex-dimorphic time course) consisting of a stiff, uncoordinated gait and reduced spontaneous movements. Most animals subsequently developed hind-limb clasping and irregular breathing. Uneven wearing of the teeth and misalignment of the jaws was also recurrent. Testes of MeCP2-null mice were always internal. A distinct feature of the phenotype was varying body weight, which was dependent on the genetic background (C57Bl/6 gave rise to underweight animals, whereas, after crossing to a 129 strain, F1 animals became heavier than siblings since they were 8 weeks of age). In MeCP2Bird hemizygous males, variable progression of pathology occurred between the fourth and seventh postnatal week leading to rapid weight loss and death (around eighth postnatal week) (see Table 8.1). By contrast, females initially showed no symptoms and raised normal litters, and only after the twelfth postnatal week, acquired inertia and hind-limb clasping phenotypes. More pervasive unambiguous symptoms (e.g. irregular breathing) are present only in one half of the heterozygous females by nine months.
Also in the MeCP2Jae model, onset of symptoms has been extensively described (25). As early as 4 weeks of age mutant male mice could be occasionally identified by an altered gait. A significant loss of body weight, body tremors, and shaking paws were evident by 5 weeks of age, whereas piloerection and periods of laboured breathing were noted as early as 6 weeks of age. Heterozygous mutant females, although their body weight was slightly reduced when compared with wild type (WT) females by 5 weeks of age, seemed normal for the first four months and began to show symptoms such as reduced activity and ataxic gait at a later stage. In association with the appearance of symptoms, females were also reported to gain weight. Piloerection, hind-limb clasping, and heavy breathing took six months or longer to develop.
Mice with a truncated mutation (MeCP2-308) similarly recapitulate many RTT features (19). Mutant male mice exhibited no apparent abnormalities until around 6 weeks of age, when tremors were detectable while suspending mice by the tail. At 4 months of age, tremors were apparent by visual observation alone. After 5 months of age, forty percent of animals developed kyphosis; after 8 months of age, the fur of mutant mice was noticeably more oily and dishevelled than that of wild types. Spontaneous behavioral myoclonic jerks and seizures were observed in several mutant mice. Body weight was within the normal range, and mice were fertile. Truncated MeCP2-308 male mice were anecdotally reported to rapidly and repeatedly move their forelimbs, often bringing them together and sometimes holding them together for several seconds when they were undisturbed in their home-cage or suspended by the tail. Moreover, as early at as 6 weeks of age, mutant male mice are reported to develop periocular inflammation and bleeding usually accompanied by bacterial infections. In a mixed background (129SvEv × C57Bl/6) (26), ataxia and breathing abnormalities appear (27). Most MeCP2-308 male mice survived at least 1 year of age, whereas heterozygous female mice displayed milder and variable features starting from 1 year of age (see Table 8.1).
Although their characterization is quite far from complete, also the other RTT models have been reported to recapitulate RTT symptoms. In the forebrain knockout, for instance, no obvious initial phenotypic differences were noted between the hemizygous mice and sex-matched controls. At 16 weeks of age, however, mutant mice became heavier than WT and started showing hind-limb clasping. In addition, weight gain went on increasing as they grew older (28).
In another MeCP2-null model, the MeCP2Tam, depending on the background, hemizygous male mice have been reported to survive up to 20 weeks of age. Unusual gait, hind-limb clasping, dishevelled fur, labored breathing, tremors, and seizures were observed in this mouse model. At about 5 weeks of age, hemizygous male mice start failing to thrive. No differences between the heterozygous and WT females were evident until 13 weeks of age (21).
A longer lifespan has been observed in the truncated MeCP2-168 model. Hemizygous male mice have been reported to survive until about 12 weeks of age and to show, by 7 weeks of age, significant hind-limb atrophy and clasping, hypoactivity, and breathing irregularities (29). Interestingly, forelimb stereotypes have been reported for this mouse model as well as for the MeCP2-308 (22). Heterozygous females showed significant symptoms (i.e. hind-limb clasping and breathing irregularities) by approximately 6 months and survived more than 1 year.
Hypothalamus knockout mice have been reported to start showing symptoms at about 7 weeks of age, when both stereotypies and kyphosis appeared (23). However, no reduced lifespan has been observed in this RTT mouse model. Interestingly, loss of MeCP2 in Sim1-expressing neurons resulted in mice that recapitulated the abnormal physiological stress response that is seen when MeCP2 is absent in the entire brain. Quite surprisingly, this MeCP2 conditional knockout mice were reported to be aggressive, hyperphagic, and obese (23).
As a whole, age at the onset of symptoms varied largely in those models, with the earliest onset in MeCP2Bird male mice (3 weeks), and the latest in truncated MeCP2-308 hemizygous mice. The same profile is evident in the lifespan of the MeCP2Bird mice living till the 8thpostnatal week. Table 8.1graphically shows the progression of the disease in the different mouse models so far available. Interestingly, motor stereotypes have been reported in all the models, but they are referred to involve forelimbs only in the two models carrying truncated mutations of MeCP2 gene.
3 Behavioral Phenotyping of the Presymptomatic Phase:The Earlier the Better
Particularly in models of neurodevelopmental disorders, it seems critical to conduct behavioral phenotyping during the developmental period in order to document precisely the onset of symptoms, examine transient signs, and provide a basis for the timing of early intervention. Moreover, in transgenic and knockout mice, developmental analysis can shed light on gene effects not accessible when studying adulthood alone. Behavioral testing during ontogeny can help our understanding of how a genetic manipulation affects the central nervous system function in ontogeny and it can represent an appropriate strategy to identify possible compensatory and/or unexpected effects (30).
These considerations become particularly relevant when taking into account RTT. Both RTT patients (1) and MeCP2 mutant mice (17–19) (see Table 8.1) have been reported to show an apparently normal period before clinical symptoms start to develop, and this feature of the disease is at the moment one of the diagnostic criteria for RTT (DSM-IV for the fourth, and current edition). Increasing evidences from both clinical and animal studies, however, support the presence of earlier defects (i.e., during that developmental phase previously regarded as asymptomatic) (31–33).
Studies of family home videos, recorded before the disorder was clearly manifested (31, 32), would confirm that girls with RTT, during the first months of life, are not so asymptomatic as was thought. Motor deficits during the first 6 months of life (e.g. abnormal general movements and finger movements) (34) as well as alterations in communicative behaviors during the first 2 years of life (e.g. limited gestural communication) (35) have been reported. Developmental delays and preregression abnormalities correlate in the RTT girls with the severity of symptoms shown later on during development (36). Importantly, tongue protrusion and/or asymmetric eye opening during the first 6 weeks post term age in combination with abnormal fidgety movements at 3-4 months have been recently reported to be early specific signs of RTT (37).
In RTT mouse models, early molecular and cellular impairments have been described. Specifically, an imbalance between inhibitory and excitatory synaptic transmission in the ventrolateral medulla (i.e. a strong depression in GABAergic synaptic transmission) was reported in the MeCP2Bird model starting as early as postnatal day (PND) 7 (38). MeCP2Bird mice at 2 weeks of age were also reported to display an increased glutamatergic synapse number (39). Overexpression of genes that are induced during the stress response by glucocorticoids was highlighted in early symptomatic as well as in presymptomatic knockout male mice (40). In addition, Viemari and colleagues (41) suggested that central respiratory deficits in knockout mice may arise early during development. Although obvious breathing abnormalities (i.e. irregular breathing rhythm) were not fully displayed before 1 month of age, in vitro recordings in medullary slices obtained from MeCP2Bird mice at PND 14-21 revealed an increased variability in the duration of respiratory cycle that was also associated with a significant reduction of norepinephrine (NE) content. The rhythm produced by such isolated respiratory network was stabilised by exogenous NE application, confirming this hypothesis.
Anatomical as well as functional modifications have also been reported during the presymptomatic phase in the RTT mouse models, which provide important hints on neurobehavioral dysfunctions that precede the noticeable behavioral changes. In particular, volumetric changes in certain brain areas have been reported to be already evident during the presymptomatic phase in both MeCP2Bird and MeCP2Jae mice (25, 42). Both the models presented an overall reduction in brain size, but a significant reduced volume has been reported for the motor cortex and the corpus callosum in the MeCP2Bird mice of 5-8 weeks of age (42) and for the amigdala, the hippocampus and the striatum in MeCP2Jae mice at postnatal day 35 (25). A longitudinal study (from birth to postnatal day 42) on the concentrations of major neurotransmitters in the brain of MeCP2Bird mice, reported smaller concentrations of biogenic amine in the brain of mutant mice when compared with wild type (WT). Interestingly, this difference became larger with increasing age. Brain concentration of GABA was also reported to be altered, but the difference between mutant and WT mice did not show clear age dependency (43). Moreover, the modifications observed in symptomatic MeCP2Jae mice in BDNF expression and cortical synaptic plasticity were confirmed in presymptomatic mice (44, 45). Different results have been obtained regarding the hippocampal synaptic plasticity, which resulted to be impaired in symptomatic mutant mice (MeCP2Bird and MeCP2Jae), but showed no significant changes during the presymptomatic phase (46). More recently, a comparative study between MeCP2Bird and MeCP2Jae mice has been published, which describes morphological changes in the early neuronal morphology (47). Interestingly, brain region-specific changes were clearly defined during the presymptomatic phase, but not necessarily they overlapped between the two models.
These results suggest that some subtle alterations are already evident in both RTT patients (quite before the preregression period) and mouse models, a better investigation and characterization of the presymptomatic phase in RTT mouse models could therefore be extremely worthwhile. As a matter of fact, detailed developmental analysis in such models, exploiting the already mature methodology of behavioral and physiological phenotyping (30), can provide an early window of opportunities on which potential therapeutic strategies could be tested. Starting from the early phases of development would make a potential rescue more likely. Moreover, the identification of precocious altered patterns is crucial for the identification of the early behavioral and molecular markers to be used as early diagnostic tools. So far, however, besides the abnormal ultrasound vocalization evidenced during the first postnatal week in MeCP2Jae mice (33) (see below, Sect. 8.3.1), few data are available concerning the behavioral development of RTT mouse models.
3.1 Neonatal Phase
To date, behavioral phenotyping of RTT mouse pups has been conducted only in MeCP2Bird and MeCP2Jae models (33, 48) (see Table 8.2, Neonatal phenotype). Yet, a fine-grain behavioral phenotyping of truncated MeCP2-308 pups is currently in progress (49).
Table 8.2
Presymptomatic phase in RTT mouse models
Neonatal phase | Postweaning phase (from weaning to the onset of symptoms) | |||
|---|---|---|---|---|
Motor abilities | Communicative/emotional | Motor abilities | Communicative/emotional | |
MeCP2Bird (Onset of symptoms: 3 weeks) | M/F: delay in postural and negative geotaxis reflex. F: surface righting reflex disturbed (48) | Not investigated | In this RTT mouse model this phase overlaps with the early symptomatic (see Table 8.1) | |
MeCP2Jae (Onset of symptoms: 5 weeks) | M/F: delay in cliff aversion test, hindlimb and placing; F: level-screen test; M: hindlimb placing (33) | M/F: ↑ USVs during the first postnatal week (33) | Not investigated | Not investigated |
MeCP2Tam (Onset of symptoms: 5 weeks) | Not investigated | Not investigated | Not investigated | Not investigated |
Forebrain knockout (Onset of symptoms: 12 weeks) | Not investigated | Not investigated | Not investigated | Not investigated |
Hypothalamus knockout (Onset of symptoms: 7 weeks) | Not investigated | Not investigated | Not investigated | Not investigated |
MeCP2-308 (Onset of symptoms: 6 weeks) | M: Locomotor coordination deficits, delayed acquisition of spontaneous movements (49) | M: ↓USVs during the first 10 days of postnatal life (49) | Not investigated | Not investigated |
For MeCP2Bird pups, the analysis conducted by means of a scale adapted to very young rodents capable of verifying the onset of maturation of a series of single reflexes and motor skills (49–51) from PND 4 to 21, revealed a delay in the acquisition of the postural and negative geotaxis reflexes in mutant pups of both sexes. In females only, a significant reduction of body weight (in line with adult data) and impaired righting reflex were found when compared with WT littermates (48).
For MeCP2Jae pups, a similar analysis revealed that overall somatic growth did not differ between the mutant mice and sex-matched WT controls. Righting, fore-limb grasping, fore-limb placing, vertical-screen test and geotaxis did not differ between the wild type and sex-matched mutants. However, a mutation-associated transient delay was noted, when performing the cliff aversion test, the hind-limb grasping and placing, and the level-screen reflex, with slight sex-different profiles.
Interestingly, on PND 5 mutant males were characterised by a more than threefold increased emission in ultrasonic vocalizations (USVs) and a different pattern of calls throughout the first postnatal week when compared to WT controls. Females also showed an increase in ultrasonic vocalizations during the whole first week with a peak on PND 7 (33). These kinds of vocalizations are emitted by pups when separated from the mother. They are considered a precocious and reliable index of pups communicative/social behaviour, also thought to provide a window on emotional /affective condition early in development (52).
In MeCP2-308 mice, during the neonatal phase, male pups were followed for the expression of spontaneous movements and emotional communicative behaviour – namely, ultrasonic vocalizations (49). Subtle anomalies in spontaneous general movements, namely increased motor arousal accompanied by a general picture of impaired communicative capacity emerged in MeCP2-308 pups.
3.2 Postweaning Phase
Onset of symptoms in RTT mouse models has been described to be extremely variable depending on the kind of MeCP2 mutation (see Table 8.1). As a consequence, the presymptomatic phase can overlap with the neonatal one, as in the case of the MeCP2Bird model (18), or it can last longer (e.g. up to 6 weeks of age in the truncated MeCP2-308 (19)).
Yet, except from the three studies mentioned above (33, 48, 49), that actually found some early impairments in neonatal mutant mice (for details see “Neonatal phenotype” and Table 8.2), other analyses have not been carried out so far to evaluate the presence of early behavioral deficits detectable before the onset of obvious symptoms in RTT mouse models (see Table 8.2).
Paucity of developmental data is likely due to the fact that to carry out such kind of studies early during development, many experimental variables need to be considered in the experimental design. These include the necessity to investigate and score blind mouse pups of an unknown genotype. Also, the behavioral assessment is based on a limited behavioral repertoire characteristic of pups’ ontogenetic niche (50, 53). Further, unavoidable procedures by the experimenter such as repeated handling or maternal separation, have to be minimised to reduce stressful events for pups and dam (52).
4 Symptomatic Phase in RTT Mouse Models
Tables 8.3and 8.4graphically summarise the behavioral alterations so far reported in different RTT mouse models during the early and the clearly symptomatic phases, respectively.
Table 8.3
Early symptomatic phase in RTT mouse models
Motor performance | Sensory abilities | Anxiety-like behaviors | Cognitive abilities | Social behaviors | |
|---|---|---|---|---|---|
MeCP2Bird (3-5 weeks) | M/F : ↓ latency fall off the rotarod (48) | M/F: some failed to respond to sound (18) | M/F: not found (48) | Not investigated | Not investigated |
MeCP2Jae (5-8 weeks) | M/F : ↓spontaneous activity; ↓ambulatory movements; ↓motor coordination; ↓grip strength; ↓swim performance (25) | M/F: not found (25) | M/F: ↑freezing in fear conditioning and zero maze; ↑time in open arms of zero maze and elevated plus maze; ↓activity in open field; ↑thigmotaxis in water maze; ↑double transitions on zero maze (25) | M: ↓freezing in the cued fear conditioning; ↓time with the displaced object in an open field. F: ↓time with the displaced and substituted objects in an open field (25) | Not investigated |
MeCP2Tam (5-8 weeks) | Not investigated | Not investigated | Not investigated | Not investigated | Not investigated |
Forebrain KO (12-16 weeks) | M: ↓motor coordination (28) | Not investigated | Not investigated | Not investigated | Not investigated |
MeCP2-308 (6-16 weeks) | M: not found | M: not found |
Table 8.4
Clearly symptomatic phase in RTT mouse models
Motor performance | Sensory abilities | Anxiety-like behaviors | Cognitive abilities | Social behaviors | |
|---|---|---|---|---|---|
MeCP2Bird (From 5 weeks) | M/F: ↓ activity in the open field (18) | Not investigated | Not investigated | Not investigated | Not investigated |
MeCP2Jae (From 8 weeks) | Not investigated | Not investigated | Not investigated | Not investigated | Not investigated |
MeCP2Tam (From 8 weeks) | M: ↓ activity on freewheels over 7 days; ↓motor coordination (21) | Not investigated | M: ↓ time in open arms of an elevated plus maze (21) | M: ↓ freezing in the cued and contextual fear conditioning (21) | Not investigated |
Forebrain KO (From 16 weeks) | M: ↓ locomotor activity; ↓motor coordination (26) | M: hearing or pain sensitivity alterations not found (28) | M: ↓ time in open arms of an elevated plus maze; ↓ activity in open field (28) | M: ↓freezing in cued fear conditioning (28) | M: ↓ social interactions; ↓ social recognition (28) |
MeCP2-308 (From 16 weeks) | M: ↓latency to fall in a rotarod modified version; ↓time to fall in a dowel test; ↓time hanging in a wire suspension test; ↓ score in a vertical pole test; ↓ total distance travelled, ↓ time spent moving, abnormal exploratory response in the open field (19) | Not investigated | M: ↑anxiety in the open field, the elevated plus maze and the light/dark box (57) |
4.1 Breathing Dysfunction
Although respiratory impairments have been anecdotally reported for all the mouse models, a more accurate analysis of breathing patterns has been performed in the MeCP2Bird and the CNS knockout models only (41, 54).
Using a pulmonary plethysmograph, an instrument for measuring changes in volume within lungs, a progressive worsening of breathing disturbances has been highlighted in MeCP2Bird mice starting from their appearance at 1 month of age (41). In particular, alternating periods of fast and slow respiratory frequencies as well as apneas of variable durations were reported in the knockout male mice at 6 weeks of age. One week later, mice worsened and showed a significant reduction in mean breathing frequency, accompanied by very frequent long lasting apneas.
More recently, to evaluate the effects of the organ distribution of MeCP2 deficiency, breathing patterns of heterozygous females from both knockout and CNS conditional knockout models have been investigated (54). When studied under hypoxic conditions at about 5 months of age, both the models showed hyperventilation, but only knockout females showed a subsequent respiratory depression followed by a prolonged apnea. An ubiquitous deficiency of MeCP2 seems therefore to be necessary to observe this breathing pattern.
Interestingly, the depression observed to follow hyperventilation in knockout females was not longer highlighted if 4% carbon dioxide was added to the hypoxic gas mixture. These results, together with the observation that tidal volume and lung volume in knockout mice were larger than in controls, would suggest, according to the authors, that the peculiar response of knockout females to hypoxia would be due to hypocapnia, a diminished concentration of carbon dioxide in the blood (54).
In addition to the in vivostudies, an analysis of the breathing phenotype of MeCP2Bird mice has been performed using the perfused working hearth-brainstem preparation (55). This technique has allowed the authors to identify a dysfunction of the central and vagal postinspiratory activity in this RTT mouse model, which could potentially explain most of the respiratory abnormalities that affect the RTT patients.
4.2 Motor Abnormalities
In MeCP2Bird mice, only a reduced latency to fall off the rotarod (a paradigm mostly used for evaluating motor skills) at 5 weeks of age in both sexes, indicative of a motor coordination deficit has been highlighted (48).
More information is available for MeCP2Jae mice: they start to exhibit severe motor deficits when compared with WT, by 5 weeks of age. One week later, during the dark cycle, mutant mice of both sexes were less active and made less ambulatory movements than their sex-matched WT controls. Abnormalities in grip strength, reduced motor coordination assessed by rotarod testing, and severely impaired swim performance were also reported (25).
In MeCP2-308, mice motor abnormalities are definitively more subtle. When assessed for motor deficits at 8-10 weeks of age, using a variety of tests, mutant male mice did not show any significant impairments in exploratory behaviors, locomotion or coordination, apart from shorter latencies to drop in the wire-suspension test (19, 27). After 3 months of age, however, motor deficits were evident in dowel test, vertical pole test and in a modified version of the rotarod (rod covered with duct to prevent the grips) (19). Lower locomotor activity in an open field has also been reported. Although similar speed and amount of rearings have been observed, both total distance traveled and time spent moving were progressively reduced during the subsequent three intervals of 10 min (19).
Mild abnormalities in circadian motor activity were also detected in the MeCP2-308 mice, namely a decrease of activity in the dark phase associated with a light-phase hyperactivity (27). Mutant male mice also show a significant impairment in nesting behavior in the home cage that according to the authors, could correspond to the apraxia of hand use seen in Rett patients, since in the same animals motor deficits were excluded (27). As for the forebrain knockout mice, the rotarod test, when performed at both 12 and 16 weeks of age, revealed an impairment of those mutant mice in motor coordination (28).
Although, as mentioned above, all the RTT mouse models have been reported to show limb stereotypies, no attempt to fully, and formally, characterise these interesting motor deficits has been carried out so far.
4.3 Sensory Deficits
Sensory abilities appear basically spared in the main RTT mouse models. In MeCP2Bird mice, no significant genotype-based differences were reported (18). Also in MeCP2Jae mice, no sensory deficits were detected in visual acuity, shock reactivity and auditory acuity (25), and no hearing or pain sensitivity alterations were found in the forebrain knockout model (28).
In truncated MeCP2-308 mice, the performance in the food buried test (matching control levels) allowed to fully exclude olfactory deficits in male subjects (27); threshold current intensity values required to elicit stereotyped behavioral responses also did not differ between the mutant and WT male mice (56).
4.4 Increased Expression of Anxiety-like Behaviours and Stress Response
Emotional profile did not appear altered in 4-week-old (both mutant males or females) MeCP2Bird mice tested in both open field and in the elevated plus maze tests (48). By contrast, anxiety-like profiles in MeCP2Jae mice appear altered at 8 weeks of age, but the effect of the genotype was rather unclear (25): although mutants spent more time than controls in the open arms of both the zero maze and the elevated plus maze, they also spent a greater amount of time freezing on zero maze.
At 4-6 months of age, increased anxiety-like behaviors were also evident in forebrain knockout mice when tested in the elevated plus maze as well as in the open field (28). The same tests have highlighted an impairment also in MeCP2-308 mice (19). Moreover, McGill and colleagues (57) indicated that increased anxiety-like behaviours were accompanied by an enhanced physiologic response to stress in MeCP2-308 male mice at 4 months of age. The authors detected a decrease in time spent in (1) centre of the arena of an open field; (2) open arms of an elevated plus maze and (3) lit side of a light/dark box, all these effects not being attributable to hypoactivity or motor impairment. Recently, an increased anxiety-like behavior has been reported to be already evident in MeCP2-308 mice as early as 2 months of age (49)
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