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Keywords:

  • Cerebellum;
  • compensatory eye movements;
  • grip strength;
  • locomotion;
  • motor deficits;
  • motor learning;
  • neurofibromatosis type 1;
  • neurofibromin;
  • Ras

Abstract

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

Neurofibromatosis type 1 (NF1) is an autosomal dominantly inherited disease, characterized by various neurocutaneous symptoms, cognitive impairments and problems in fine and gross motor performance. Although cognitive deficits in NF1 have been attributed to increased release of the inhibitory neurotransmitter γ-amino butyric acid (GABA) in the hippocampus, the origin of the motor deficits is unknown. Cerebellar Purkinje cells, the sole output neurons of the cerebellar cortex, are GABAergic neurons and express neurofibromin at high levels, suggesting an important role for the cerebellum in the observed motor deficits in NF1. To test this, we determined the cerebellar contribution to motor problems in Nf1+/− mice, a validated mouse model for NF1. Using the Rotarod, a non-specific motor performance test, we confirmed that, like NF1 patients, Nf1+/− mice have motor deficits. Next, to evaluate the role of the cerebellum in these deficits, mice were subjected to cerebellum-specific motor performance and learning tests. Nf1+/− mice showed no impairment on the Erasmus ladder, as step time and number of missteps were not different. Furthermore, when compensatory eye movements were tested, no performance deficits were found in the optokinetic reflex and vestibulo-ocular reflex in the dark (VOR) or in the light (VVOR). Finally, Nf1+/− mice successfully completed short- and long-term VOR adaptation paradigms, tests that both depend on cerebellar function. Thus, despite the confirmed presence of motor performance problems in Nf1+/− mice, we found no indication of a cerebellar component. These results, combined with recent clinical data, suggest that cerebellar function is not overtly affected in NF1 patients.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with an incidence of 1:3000 that is caused by heterozygous mutations in the gene encoding the neurofibromin protein on chromosome 17q11.2 (Viskochil et al. 1990; Wallace et al. 1990). NF1 patients exhibit a variety of symptoms, including learning disabilities and cognitive deficits (Krab et al. 2008a). The most prominent part of the cognitive profile of NF1 patients is an impairment in visual–spatial memory (Ozonoff 1999). In analogy, mice that harbor a heterozygous knockout mutation show visual–spatial memory deficits in the Morris Water Maze, a hippocampal learning task (Silva et al. 1997). Studies using Nf1 mutant mice have shown that these hippocampal learning deficits in NF1 are caused by aberrant intracellular signaling. Insufficient neurofibromin-mediated inhibition of the signaling molecule Ras results in increased extracellular signal-regulated kinase – Synapsin I mediated release of the inhibitory neurotransmitter γ-amino butyric acid (GABA) (Costa et al. 2002; Cui et al. 2008; Krab et al. 2008b). Enhanced GABA release then in turn leads to impairments in long-term potentiation (Costa et al. 2002), the putative cellular equivalent of learning and memory (Abbott & Nelson 2000).

Besides the cognitive deficits, problems with fine and gross motor co-ordination as well as decreased muscle strength are commonly observed in NF1 patients (Krab et al. 2008a; Souza et al. 2009). The motor problems often require remedial teaching or physiotherapy and impair the quality of life (Krab et al. 2008a,2009). The mechanisms underlying these motor deficits are unknown, but malfunction of the cerebellum can lead to both motor co-ordination deficits and decreased muscle force (Hilber & Caston 2001), two aspects of the NF1 motor phenotype. Furthermore, NF1 children are impaired in some motor learning paradigms that are sensitive to cerebellar dysfunction, although they performed normal in others (Krab et al. 2011).

From a molecular and cellular point of view, cerebellar dysfunction is also a likely candidate cause for the motor deficits. Purkinje cells are GABAergic cells and the sole output cells of the cerebellar cortex. They are also among the highest neurofibromin-expressing neurons in the brain (Gutmann et al. 1995; Nordlund et al. 1993). Mutations that affect Purkinje cells specifically can cause motor deficits, ranging from problems invisible to the naked eye, as seen in mice with Purkinje cell-specific deletion of PP2B or PKCi function (De Zeeuw et al. 1998; Schonewille et al. 2010), to severe ataxia as seen in Lurcher (Van Alphen et al. 2002) and tottering (Hoebeek et al. 2005). Apart from Purkinje cells, other cerebellar cortical neurons such as Golgi, stellate and basket cells also use GABA as a neurotransmitter, and GABAergic feedforward input of stellate and basket cells has been demonstrated to influence Purkinje cell spiking activity and motor activity (Wulff et al. 2009). However, neurofibromin expression in the cerebellar cortex is limited to Purkinje cells and a subset of Golgi cells (Nordlund et al. 1993). Locomotion can also be hampered by accelerating the kinetics of GABA-filled vesicles in the Purkinje cell to cerebellar nuclear neuron synapse (Fassio et al. 2006). Thus, the high neurofibromin expression and GABAergic nature of Purkinje cells make the cerebellar cortex a promising candidate to be the site of origin for the motor deficits seen in NF1.

Here, we report motor learning deficits in Nf1+/− mice on a non-specific locomotion task, the Rotarod. We found no deficits on the Erasmus ladder, a cerebellum-dependent locomotion paradigm. Short- and long-term vestibulo-ocular reflex (VOR) adaptation was also not affected, arguing against a causative role for the cerebellum in motor deficits observed in Nf1+/− mice.

Materials and methods

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

Mice

Nf1 +/− mice (Jacks et al. 1994) were kindly provided by Alcino Silva and backcrossed into the C57Bl/6 background for more than 30 generations. Male Nf1+/− mutants were crossed once with wild-type 129T2/SvEmsJ mice (Jackson Laboratories) to obtain F1 hybrid mice with 50% C57Bl/6 and 50% 129T2/SvEmsJ contribution. Mice in this particular background were previously shown to have learning and synaptic plasticity deficits (Costa et al. 2002; Y.E. and G.M.W., unpublished data). Experiments were performed on heterozygote Nf1+/− mice and wild-type littermate controls. All mice were between 12 and 26 weeks of age. The animals were housed in group cages during experiments and allowed standard laboratory food ad libitum. They were left on a 12-h light/dark cycle, with light phase between 0700 and 1900 h and all experiments were executed between 0900 and 1700 h. Different experimental groups were used for Rotarod, Erasmus ladder and grip strength. Mice used for eye movement recordings were those first subjected to the Rotarod. All animal experiments were approved by the Dutch Ethical Committee and were in accordance with the Institutional Animal Care and Use Committee guidelines.

Rotarod

Nf1 +/− mice were trained to walk on the accelerating Rotarod (Ugo Basile, Comerio Varese, Italy, 7650). The Rotarod consists of a cylinder with a diameter of 3 cm. The speed of the Rotarod was accelerated from 4 r.p.m. to a maximum speed of 40 r.p.m. after 270 seconds. Maximal walking time was 300 seconds. Latency to fall was recorded on the moment the subject fell down or stopped walking (clinging to the rod) for three consecutive turns. The first day consisted of five trials, with an interval of 45 min. Days 2–5 consisted of two trials with an interval of 45 min.

Grip strength

Grip strength was determined by placing mice with the forepaws on a grid attached to a force gauge (BIOSEB, Chaville, France), and steadily pulling the mice by the tail. Grip strength is defined as the maximum strength produced by the mouse before releasing the grid. Each value is the average of a triplicate test.

Erasmus ladder

The Erasmus ladder is a fully automated test for detecting motor performance and learning deficits, used to analyze cerebellar function in multiple mouse mutants, including ataxic Lurcher mice, and also Cx36−/− mice, which show cerebellar motor learning deficits in the absence of ataxia (Kistler et al. 2002; Van Der Giessen et al. 2008). The Erasmus ladder consists of a horizontal ladder in between two shelter boxes, which are equipped with two pressurized air outlets (Pneumax, 171E2B.T.A.0009, Gosport, UK) to control the moment of departure and speed of the mouse. The ladder has 2 × 37 rungs for the left and right side. All rungs are equipped with pressure sensors (produced at Erasmus MC), which are continuously monitored and which can be used to register and analyze the walking pattern of the mouse instantaneously. Moreover, based upon the prediction of the walking pattern, the rungs can be moved up or down by a high-speed pneumatic slide (Pneumax, 2141.52.00.36.91) with a maximum of 13 mm at any moment in time. The computer system (National Instruments, PXI-1000B, Austin, Texas, USA) that runs the real-time system to record sensor data, adjusts air pressure, predicts future touches, calculates interventions, repositions slides and stores data, operates in a fixed cycle of 2 milliseconds. During the first 4 days, the mice were trained with the even-numbered rungs on the left side and the odd-numbered rungs on the right side in a descended position so as to create an alternated stepping pattern with 30 mm gaps. The mice are trained to walk the ladder for 72 runs per day. We calculated the number of missteps that are sensed by the descended rungs. Conditioning started on day 5 using a 15 kHz tone (Voltcraft, Barking, UK), which gradually increases over 20 milliseconds to 100 dB and which lasts up to 300 milliseconds as the conditioned stimulus, whereas a rising rung, which ascends 12 mm, was used as the unconditioned stimulus. The interstimulus interval was fixed on 285 milliseconds. To keep this time period constant we observed the real-time speed of the mouse and calculated which rung would rise. Mice typically learn that increasing walking speed avoids being hit by the rung, so mice will decrease the time each step takes (step time).

Compensatory eye movements

All mice for eye movement recordings were surgically prepared for experiments under general anesthesia of a mixture of isoflurane (Rhodia Organique Fine Ltd, Bristol, UK) and oxygen. A construct consisting of two nuts was attached to the frontal and parietal bones using Optibond prime and adhesive (Kerr, Bioggio, Switzerland) and Charisma (Heraeus Kulzer, Armonk, NY, USA). After a recovery period of 5 days, the mouse was placed in a restrainer, with its head bolted to a bar. The restrainer was fixed onto the center of the turntable. A cylindrical screen (diameter 63 cm) with a random-dotted pattern (each element 2°) surrounded the turntable (diameter 60 cm). The optokinetic reflex (OKR) and visual-enhanced VOR (VVOR) were evoked by rotating the surrounding screen and turntable, respectively, with an amplitude of 5° at different frequencies (Fig. 3). The surrounding screen and the turntable were driven independently by AC servo-motors (Harmonic Drive AG, Eindhoven, The Netherlands). The table and drum position signal were measured by potentiometers, filtered, digitized (CED Ltd, Cambridge, UK) and stored on a computer. An infrared camera was fixed to the turntable in order to monitor the eye of the mouse. The eye movements were recorded at 240 Hz using the eye-tracking device ISCAN (Iscan Inc., Woburn, MA, USA). Video calibrations and subsequent eye movement computations were performed as described previously (Stahl et al. 2000). Short-term learning was tested by 5 × 10 min of in-phase drum and table stimulation at 0.6 Hz with 5°, resulting in a decrease of VOR gain. Subsequently, mice were subjected to 5 × 10 min of 5° table stimulus with the drum rotating in-phase at 7.5° (day 2) or 10° (day 3–4), to test long-term VOR phase adaptation. Before, after and in between 10-min training periods VOR was measured and between training sessions mice were kept in the dark to avoid extinction.

image

Figure 3. Baseline compensatory eye movements. In addition to locomotion, we examined performance and learning of compensatory eye movements in Nf1+/− mice. Using visual and vestibular stimulation we evoked the OKR and VOR. A gain of 1 equals an amplitude of the eye movement that corresponds perfectly with that of the stimulus. When the eye movement follows the stimulus on the same moment that the stimulus is presented, the phase of the eye movement is zero. (a, b) Over the range of frequencies tested, OKR and VOR in Nf1+/− mice (n = 8) did not differ from that of control mice (n = 8), neither in gain nor in phase. (c) In everyday life, the two complementary flexes are used simultaneously. We tested this visual-enhanced VOR (VVOR) by rotating the mice in the light, providing both visual and vestibular stimulation. The VVOR is also not affected in Nf1+/− mice as both gain and phase did not differ from controls.

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Statistical analysis

In all performance and training paradigms, we analyzed the data using repeated measures analysis of variance, with genotype as the between-subjects factor and trial or day as the within-subjects factor. Grip strength data and consolidation of VOR-gain decrease were tested with a two-sample t test.

Results

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

Nf1+/− mice have a mild non-specific motor performance deficit

To quantify their general level of motor performance, Nf1+/− mice were trained to walk on the accelerating Rotarod. Over the sessions of the first day, wild-type and Nf1+/− mice both significantly improved their Rotarod performance (Fig. 1a, trials: F1,30 = 572.4, P < 0.001), but the difference between the groups did not reach significance (Fig. 1a genotype: F1,30 = 3.0, P = 0.096). However, in a 5-day training paradigm, Nf1+/− mice did demonstrate a deficit compared with wild-type littermate controls (Fig. 1b, genotype: F1,30 = 4.5, P = 0.043). This result is analogous to the reported motor deficits in children with NF1 and to that observed in mice with a deletion of exon23a of the NF1 gene (Costa et al. 2001). Additionally, we found a slightly decreased grip strength in Nf1+/− mice (n = 17; 1.21 ± 0.02 N) compared with wild-type littermate controls (n = 17; 1.29 ± 0.03 N) (t32 = 2.1, P = 0.044, two-tailed Student's t test). This result is consistent with a recent finding of impaired hanging wire test performance (Robinson et al. 2010). Moreover, this finding is consistent with decreased muscular tone observed in NF1 patients (Souza et al. 2009). Taken together, the Nf1+/− mouse model recapitulates NF1-associated motor deficits and decreased muscle strength and allows us to investigate whether cerebellar dysfunction could contribute to these deficits.

image

Figure 1. Rotarod performance of Nf1 +/ mice. Short-term and long-term motor learning was tested in Nf1+/− mice using the accelerating Rotarod. (a) Nf1+/− mice (n = 17) showed a significant (P < 0.001) increase in latency to fall during the first day. This increase was, however, not significantly different from their control littermates (n = 15) (P = 0.09). (b) Five consecutive days of Rotarod training revealed a mild, but significant deficit in Nf1+/− mice compared with control mice (P = 0.04).

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Nf1+/− mice successfully complete the Erasmus ladder test

To elucidate the presence of cerebellar components in NF1, we tested Nf1+/− mice on the Erasmus ladder. The Erasmus ladder is a fully automated system to screen both cerebellar motor performance and learning capabilities of mutant mice in a non-invasive manner (Van Der Giessen et al. 2008). Over 4 days, Nf1+/− mice decreased the number of missteps, indicating a smooth walking pattern on the Erasmus ladder (Fig. 2a, days: F1,23 = 198.6, P < 0.001), showing no difference compared with controls (genotype: F1,23 = 1.8, P = 0.19). On day 5, we started a conditioning experiment, in which a tone was paired with a rising rung. Following conditioning, Nf1+/− mice increased their speed when hearing the tone (Fig. 2b, days: F1,23 = 121.1, P < 0.001) comparable to controls (genotype: F1,23 = 1.1, P = 0.30).

image

Figure 2. Erasmus ladder training. To identify the contribution of the cerebellum to the Rotarod deficits, Nf1+/− mice were subjected to the Erasmus ladder motor learning paradigm. (a) During the first 4 days, mice are trained to walk the ladder. The starting number and decrease of number of missteps did not differ between Nf1+/− mice (n = 14) and controls (n = 11). (b) From days 5 to 8, mice were conditioned to accelerate upon a tone (conditioned stimulus). As unconditioned stimulus, a rung would rise. Again Nf1+/− mice learned to increase their speed to avoid the rung, in a manner not significantly different from their control littermates.

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Compensatory eye movements are not affected in Nf1+/− mice

The results on the Erasmus ladder argue against a cerebellar component in the motor deficits of NF1. Although the Erasmus ladder can detect cerebellar dysfunction, locomotion remains an activity in which many different brain regions are involved. Therefore, next we tested motor performance and learning using eye movement measurements, a well-established, reflex-based paradigm to assess cerebellar function (Schonewille et al. 2010; Wulff et al. 2009). Motor performance is normal as Nf1+/− mice have a normal OKR, vestibulo-ocular reflex and visually enhanced vestibulo-ocular reflex (Fig. 3, all P > 0.05 for genotype).

Next, we tested motor learning by subjecting the mice to VOR adaptation training. During the first day of 5 × 10 min training session, Nf1+/− mice decreased their VOR gain significantly (Fig. 4a, trials: F1,14 = 765.4, P < 0.001), but this decrease was not different from that seen in control animals (genotype: F1,14 = 0.2, P = 0.65). Mice were placed in the dark overnight and tested again the next day. Consolidation, the percentage of the change in gain carried forward to the next day, was also not significantly different between Nf1+/− and control mice (Fig. 4b, t14 = 0.05 P = 0.96, two-tailed Student's t-test). Ultimately, mice were trained over the next 3 days to increase the phase of their VOR to 180°, effectively reversing the direction of this compensatory eye movement. Surprisingly, Nf1+/− mice were capable of reversing the phase of their VOR in a manner similar to that seen in control mice (Fig. 4c, trials: F1,14 = 82.8, P < 0.001, genotype: F1,14 = 0.38, P = 0.55).

image

Figure 4. Cerebellar motor learning in Nf1 +/ mice. To complete the analysis of motor learning, we subjected Nf1+/− mice to VOR gain decrease and VOR phase reversal, one of the most sensitive cerebellar learning paradigms. (a) VOR gain decrease training (day 1) consisted of 5 × 10 min of training in the light with both turntable and drum moving in phase at 0.6 Hz with 5° amplitude (see left). Before, in between and after the trials, we measured the VOR. Gain decrease was comparable between Nf1+/− mice (n = 8) and controls (n = 8). (b) Animals were kept in the dark overnight and, using VOR gain values from the next day, the percentage of memory consolidation was calculated. This consolidation was again not different between Nf1+/− and control mice. (c) Finally, as the ultimate test of cerebellar function in Nf1+/− mice, we attempted to reverse the phase of the VOR over multiple training days. To do so, we increased the amplitude of the visual stimulation to 7.5° (on day 2) to 10° (on days 3 and 4), with the turntable still rotating in phase at 5°. This training induced a large shift of the VOR phase in control mice. Markedly though, Nf1+/− mice demonstrated a comparable shift in VOR phase, arguing against a role for the cerebellum in Nf1-associated motor deficits.

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Discussion

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

Motor problems and learning disabilities are the most common complication of NF1. NF1 children are not clearly ataxic, but frequently display clumsiness (Cnossen et al. 1998; Hofman et al. 1994; Levine et al. 2006) and often receive physiotherapy (Krab et al. 2008a). Remarkably, however, studies on the origin of the motor deficits are scarce. One study assessing motor learning in children with NF1 more precisely obtained some mixed results and no clear cerebellar deficit emerged (Krab et al. 2011). To overcome limitations in studies with human subjects, we used mice to evaluate the role of the cerebellum in NF1 in greater detail.

The Nf1+/− mouse model we used here suffers from a heterozygous loss-of-function mutation, comparable to that found in NF1 patients, and has been used as a model for NF1 for more than a decade (Shilyansky et al. 2010b). Nf1+/− mice, like NF1 patients, have learning and memory deficits that are restricted to specific types of learning, e.g. visual–spatial motor learning (Silva et al. 1997). The motor deficits we observed on the Rotarod are in line with, and support the use of, Nf1+/− mice as a mouse model for NF1.

On the basis of the high expression of Neurofibromin in Purkinje cells, and the GABAergic nature of NF1 cognitive problems and Purkinje cell output, a cerebellar component in the motor deficits was hypothesized. Hence, we set out to distinguish the cerebellum-specific motor deficits from the non-specific deficits. Surprisingly, we found no deficits when motor performance and learning were tested using more cerebellum-specific motor tests.

For any disorder, identifying the site of origin of the deficits, as well as the specific cellular and molecular pathways that lead to the deficits, is crucial for a possible cure. Previous experiments have revealed that in the hippocampus GABAergic inhibition is enhanced in Nf1+/− mice. Together with the normally high expression of Neurofibromin in Purkinje cells, this suggest that GABAergic inhibition from Purkinje cells onto vestibular nucleus neurons or deep cerebellar nuclei neurons may well be increased. Enhanced GABA-release kinetics from Purkinje cells onto vestibular nucleus neurons have been correlated to motor deficits before (Fassio et al. 2006). Then why were no cerebellar deficits observed here? Theoretically, it is possible that a cerebellar component is present, but cannot be detected by the tests chosen here. However, this is the first mutant that demonstrated a deficit on the Rotarod, but not on the Erasmus ladder. Conversely, mutants with the opposite phenotype have been found (Frisch et al. 2005; Kistler et al. 2002; Van Der Giessen et al. 2008). This seemingly paradoxical finding might be explained by differences in the nature of the two tasks. The Rotarod directly demands a fitting response of the animal in terms of strength, endurance and motor co-ordination, whereas Erasmus ladder allows the animal more time and space for its response, predominantly requiring good motor co-ordination.

To complement the locomotion-based test, we also subjected mice to the VOR phase reversal paradigm (Gonshor & Jones 1976). This is a well-established, highly cerebellum-dependent task, but more reflexive by nature and thus presumably less dependent on other brain areas. The absence of a phenotype in both tasks argues against a role for the cerebellum in the Nf1+/− mice.

The main question that emerges from these results of course is: what is causing the motor deficits observed in Nf1+/− mice and patients, if not the cerebellum? It is tempting to speculate that Rotarod deficits are related to the decreased grip strength we observed. Decreased grip strength can be attributed to many regions, e.g. the cerebellum (Hilber & Caston 2001), or the peripheral nervous system (Norreel et al. 2001). Children with NF1 have been assessed with a variety of non-specific motor tasks, all of them including requirement of some type of higher brain function. An example is the Beery Visual-Motor Integration task which has been frequently reported to be impaired in NF1. This test indicates problems not only in a variety of brain areas, including the cerebellum, but also the right hemisphere, the primary motor cortex of the dominant hand, subcortical nuclei and/or the corpus callosum (Beery 2004). In addition, hypoactivation of corticostriatal pathways has been observed in NF1 (Shilyanski et al. 2010a). Future studies aimed at finding the source of motor deficits in NF1 patients should be focused on these brain areas.

In summary, although the origin of the NF1-associated motor deficits remains unknown, the data presented here, combined with recent clinical data (Krab et al. 2011), argue against clinically meaningful cerebellar deficits.

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  1. Top of page
  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

The authors like to thank M. Elgersma, M. Aghadavoud, R. de Avila Freire, A. Cupido and M. Vinueza Veloz for their technical support. This work was supported by an Erasmus University Fellowship (MS), the Dutch Organization for Medical Sciences (YE, CIDZ), Life Sciences (MS, CIDZ), Senter Neuro-Bsik (CIDZ), Prinses Beatrix Fonds (CIDZ), the SENSOPAC, C7 and CEREBNET programs of the European Community (CIDZ) and the Dutch Neurofibromatosis Foundation.