The peer teachers' perception of intrinsic motivation and rewards.

Peer-assisted learning (PAL) is an educational method commonly applied in academic teaching. It is characterized by the interplay between peer teachers and learners who are at a similar academic level. Although it has been shown that peer teachers benefit from participating in PAL, little is known about their perception of motivation and rewards. Here we designed a questionnaire and measured the perception of intrinsic motivation and rewards of peer teachers from three different PAL programs. Overall, peer teachers were highly intrinsically motivated. The reward category Supporting Others was appreciated the most, followed by the reward categories Self-Improvement, Feedback, and Financial. The perception of rewards reflected the features of the three PAL programs. For example, the item "learning the teaching matter themselves" was most valued by peer teachers who were enrolled in a PAL program that deployed their peer teachers primarily to convey knowledge. In contrast, "actively shaping the teaching situation" was appreciated most by peer teachers of the PAL program that enables their peer teachers to conceive their teaching sessions independently. These findings go toward recommendations of the implementation and further development of PAL programs. If PAL programs clearly define their features and aims, they could specifically attract (and select) peer teachers and meet their needs as well as expectations, providing opportunities to gain knowledge and teaching experience. Ultimately, these PAL programs could better support the learners.

The role of agrin, Lrp4 and MuSK during dendritic arborization and synaptogenesis in cultured embryonic CNS neurons.

The role of agrin, Lrp4 and MuSK, key organizers of neuromuscular synaptogenesis, in the developing CNS is only poorly understood. We investigated the role of these proteins in cultured mouse embryonic cortical neurons from wildtype and from Lrp4- and MuSK-deficient mice. Neurons from Lrp4-deficient mice had fewer but longer primary dendrites and a decreased density of puncta containing excitatory and inhibitory synapse-associated proteins. Neurons from MuSK-deficient mice had an altered dendritic branching pattern but no change in the density of puncta stained by antibodies against synapse-associated proteins. Transfection of TM-agrin compensated the dendritic branching deficits in Lrp4-deficient but not in MuSK-deficient neurons. TM-agrin transfection increased the density of excitatory synaptic puncta in MuSK-deficient but not in Lrp4-deficient mice and reduced the number of inhibitory synaptic puncta irrespective of MuSK and Lrp4 expression. Addition of purified soluble agrin to microisland cultures of cortical neurons revealed an Lrp4-dependent increase in the size and density of glutamatergic synaptic puncta and in mEPSC but not in mIPSC frequency and amplitude. Thus, agrin induced an Lrp4-independent increase in dendritic branch complexity, an Lrp4-dependent increase of excitatory synaptic puncta and an Lrp4- and MuSK-independent decrease in the density of puncta containing inhibitory synapse-associated proteins. These results establish selective roles for agrin, Lrp4 and MuSK during dendritogenesis and synaptogenesis in cultured CNS neurons.

Impaired muscle spindle function in murine models of muscular dystrophy.

KEY POINTS: Muscular dystrophy patients suffer from progressive degeneration of skeletal muscle fibres, sudden spontaneous falls, balance problems, as well as gait and posture abnormalities. Dystrophin- and dysferlin-deficient mice, models for different types of muscular dystrophy with different aetiology and molecular basis, were characterized to investigate if muscle spindle structure and function are impaired. The number and morphology of muscle spindles were unaltered in both dystrophic mouse lines but muscle spindle resting discharge and their responses to stretch were altered. In dystrophin-deficient muscle spindles, the expression of the paralogue utrophin was substantially upregulated, potentially compensating for the dystrophin deficiency. The results suggest that muscle spindles might contribute to the motor problems observed in patients with muscular dystrophy. ABSTRACT: Muscular dystrophies comprise a heterogeneous group of hereditary diseases characterized by progressive degeneration of extrafusal muscle fibres as well as unstable gait and frequent falls. To investigate if muscle spindle function is impaired, we analysed their number, morphology and function in wildtype mice and in murine model systems for two distinct types of muscular dystrophy with very different disease aetiology, i.e. dystrophin- and dysferlin-deficient mice. The total number and the overall structure of muscle spindles in soleus muscles of both dystrophic mouse mutants appeared unchanged. Immunohistochemical analyses of wildtype muscle spindles revealed a concentration of dystrophin and ß-dystroglycan in intrafusal fibres outside the region of contact with the sensory neuron. While utrophin was absent from the central part of intrafusal fibres of wildtype mice, it was substantially upregulated in dystrophin-deficient mice. Single-unit extracellular recordings of sensory afferents from muscle spindles of the extensor digitorum longus muscle revealed that muscle spindles from both dystrophic mouse strains have an increased resting discharge and a higher action potential firing rate during sinusoidal vibrations, particularly at low frequencies. The response to ramp-and-hold stretches appeared unaltered compared to the respective wildtype mice. We observed no exacerbated functional changes in dystrophin and dysferlin double mutant mice compared to the single mutant animals. These results show alterations in muscle spindle afferent responses in both dystrophic mouse lines, which might cause an increased muscle tone, and might contribute to the unstable gait and frequent falls observed in patients with muscular dystrophy.

Acetylcholine receptors in the equatorial region of intrafusal muscle fibres modulate mouse muscle spindle sensitivity.

KEY POINTS: Acetylcholine receptors are aggregated in the central regions of intrafusal muscle fibres. Single unit muscle spindle afferent responses from isolated mouse extensor digitorum longus muscle were recorded in the absence of fusimotor input to ramp and hold stretches as well as to sinusoidal vibrations in the presence and absence of the acetylcholine receptor blockers d-tubocurarine and α-bungarotoxin. Proprioceptive afferent responses to both types of stretch were enhanced in the presence of either blocker. Blocking acetylcholine uptake and vesicular acetylcholine release by hemicholinium-3 also enhanced stretch-evoked responses. These results represent the first evidence that acetylcholine receptors negatively modulate muscle spindle responses to stretch. The data support the hypothesis that the sensory nerve terminal is able to release vesicles to fine-tune proprioceptive afferent sensitivity. ABSTRACT: Muscle spindles are complex stretch-sensitive mechanoreceptors. They consist of specialized skeletal muscle fibres, called intrafusal fibres, which are innervated in the central (equatorial) region by afferent sensory axons and in both polar regions by efferent γ-motoneurons. Previously it was shown that acetylcholine receptors (AChR) are concentrated in the equatorial region at the contact site between the sensory neuron and the intrafusal muscle fibre. To address the function of these AChRs, single unit sensory afferents were recorded from an isolated mouse extensor digitorum longus muscle in the absence of γ-motoneuron activity. Specifically, we investigated the responses of individual sensory neurons to ramp-and-hold stretches and sinusoidal vibrations before and after the addition of the competitive and non-competitive AChR blockers d-tubocurarine and α-bungarotoxin, respectively. The presence of either drug did not affect the resting action potential discharge frequency. However, the action potential frequencies in response to stretch were increased. In particular, frequencies of the dynamic peak and dynamic index to ramp-and-hold stretches were significantly higher in the presence of either drug. Treatment of muscle spindle afferents with the high-affinity choline transporter antagonist hemicholinium-3 similarly increased muscle spindle afferent firing frequencies during stretch. Moreover, the firing rate during sinusoidal vibration stimuli at low amplitudes was higher in the presence of α-bungarotoxin compared to control spindles also indicating an increased sensitivity to stretch. Collectively these data suggest a modulation of the muscle spindle afferent response to stretch by AChRs in the central region of intrafusal fibres possibly fine-tuning muscle spindle sensitivity.

Adenylate Cyclase 1 modulates peripheral nerve branching patterns.

The Ca(2+)-stimulated adenylate cyclase 1 (AC1) is a key mediator of retinotopic map refinement and is required for the retraction response of retinal growth cones to the guidance cue ephrin-A5. We show here that AC1 is dynamically expressed in subpopulations of motor neurons in the spinal cord and sensory neurons of the dorsal root ganglia during development. AC1 was first detected around E12.5 in motoneurons of the medial aspect of the lateral motor column (LMCm) and the lateral region of the medial motor column (MMCl), which project to the ventral limb and body wall musculature, respectively. Expression levels gradually increased until they reached a maximum at a time when peripheral sensory and motor axons branch and establish connections with their targets. In barrelless mice, where a mutation inactivates the AC1 gene, sensory projections to the skin in the limbs and trunk region as well as innervations of the intercostal musculature provided by MMCl axons show increased branching. These results suggest a function of AC1 in the formation of peripheral nerve trajectories such as branching and pruning, after the initial projections have been laid down.

Semaphorin 3A-Neuropilin-1 signaling regulates peripheral axon fasciculation and pathfinding but not developmental cell death patterns.

In early development, an excess of neurons is generated, of which later about half will be lost by cell death due to a limited supply of trophic support by their respective target areas. However, some of the neurons die when their axons have not yet reached their target, thus suggesting that additional causes of developmental cell death exist. Semaphorin 3A (Sema3A), in addition to its function as a guidance cue and mediator of timing and fasciculation of motor and sensory axon outgrowth, can also induce death of sensory neurons in vitro. However, it is unknown whether Neuropilin-1 (Npn-1), its binding receptor in axon guidance, also mediates the death-inducing activity. We show here that abolished Sema3A-Npn-1 signaling does not influence the cell death patterns of motor or sensory neurons in mouse during the developmental wave of programmed cell death. The number of motor and sensory neurons was unchanged at embryonic day 15.5 when this wave is concluded. Interestingly, the defasciculation of early motor and sensory projections that is observed in the absence of Sema3A or Npn-1 persists to postnatal stages. Thus, Sema3A-Npn-1 signaling plays an important role in the guidance and fasciculation of motor and sensory axons but does not contribute to the developmental elimination of these neurons.

How axons see their way--axonal guidance in the visual system.

In humans up to 80% of the information received from the outside world is processed by the visual pathway. Therefore, understanding the molecular and cellular bases of the formation of the retinofugal projection has been in the focus of research during the last decades. Besides our interest in the development of the visual pathway per se this circuit is also an excellent model system to study axon guidance, midline crossing, and formation of topographic neuronal maps in general. The generation of genetic animal models as well as the design of in vitro loss- and gain-of-function paradigms have provided insight into transcriptional networks, identified signalling molecules, extracellular matrix components, morphogens, and activity patterns which are involved in the establishment of the visual pathway. To provide a picture as complete as possible, we will summarize molecular mechanisms involved in axon guidance and retinotopic mapping as well as neuronal activity shaping retinal and thalamocortical projections focusing on the mouse as a model system and highlight discoveries made in other organisms that contribute to our understanding.

The combined use of non-radioactive in situ hybridization and real-time RT-PCR to assess gene expression in cryosections.

Gene expression changes in pathophysiological states can be spatiotemporally monitored by in situ hybridization and reliably quantified by real-time RT-PCR. Here we developed a new method whereby adjacent slides of frozen sections can be used for gene expression analysis by in situ hybridization and real-time RT-PCR. We applied this method to assess the mRNA expression of connexin 43 (Cx43), the major astrocytic connexin, after kainate-induced seizures in rat hippocampus. Gap junction-building connexins play a role in the pathogenesis of several diseases of the brain, including epilepsy. The number of Cx43 mRNA-positive cells in the hippocampus of kainate-treated and control rats was automatically quantified by computerized image analysis of brain sections hybridized with DIG-labeled RNA probes. In parallel, real-time RT-PCR was used to examine the relative Cx43 mRNA levels in hippocampal tissue from adjacent brain sections. Applying these two very sensitive methods we showed that kainate induced seizures do not affect hippocampal connexin 43 mRNA expression.

Engrailed 1 mediates correct formation of limb innervation through two distinct mechanisms.

Engrailed-1 (En1) is expressed in the ventral ectoderm of the developing limb where it plays an instructive role in the dorsal-ventral patterning of the forelimb. Besides its well-described role as a transcription factor in regulating gene expression through its DNA-binding domain, En1 may also be secreted to form an extracellular gradient, and directly impact on the formation of the retinotectal map. We show here that absence of En1 causes mispatterning of the forelimb and thus defects in the dorsal-ventral pathfinding choice of motor axons in vivo. In addition, En1 but not En2 also has a direct and specific repulsive effect on motor axons of the lateral aspect of the lateral motor column (LMC) but not on medial LMC projections. Moreover, an ectopic dorsal source of En1 pushes lateral LMC axons to the ventral limb in vivo. Thus, En1 controls the establishment of limb innervation through two distinct molecular mechanisms.

Regulation of GABA transporter mRNA and protein after photothrombotic infarct in rat brain.

Animal models of focal ischemic infarcts reveal an impaired GABAergic (gamma-aminobutyric acid) neurotransmission. GABA, the main inhibitory neurotransmitter, is primarily taken up by specific sodium-dependent transporters. As these transporters play a crucial role in maintaining levels of GABA concentration, they may be functionally involved in ischemic processes. We investigated whether the mRNA and protein expression of GAT-1, the dominant neuronal GABA transporter, is altered after cortical infarct induced by photothrombosis in Wistar rats. In situ hybridization was performed to analyze GAT-1 mRNA-positive cells in cortical brain regions and the hippocampus. The lesion dramatically raised the number of GABA transporter mRNA-expressing cells in all investigated cortical regions. Double-labeling studies with a general neuronal marker and a marker for astrocytes revealed that cells expressing GAT-1 mRNA after photothrombosis are neurons. The mRNA expression pattern of all hippocampal subfields remained unchanged. In contrast, cortical GAT-1 protein density was only slightly affected and surprisingly in the opposite way. In the primary and secondary somatosensory cortex, density values were significantly reduced. Immunoreactivity was not altered in all investigated hippocampal areas. We found a marked discordance between the increased number of cells expressing GAT-1 mRNA in the cortex and the reduced tissue GAT-1 protein content. Focal brain ischemia obviously triggers mechanisms that interfere with GAT-1 transcriptional regulation and protein synthesis or turnover.

Up-regulation of Connexin43 in the glial scar following photothrombotic ischemic injury.

Several types of CNS injury and various diseases are associated with the development of a glial scar. Astrocytes are major components of the glial scar. They are interconnected by gap junctions, with connexin43 (Cx43) being the most prominent channel protein. We applied a model of focal cerebral ischemia to study the spatio-temporal expression of glial fibrillary acidic protein, as well as of Cx43 mRNA and protein in gliotic tissue up to 60 days after injury. Reactive astrocytes enveloping the lesion up-regulated their Cx43 mRNA and protein. A band of reactive astrocytes filling in the lesion exhibited elevated Cx43 and showed a high degree of proliferation. Because of these findings, we hypothesize a role for Cx43 in glial scar formation, specifically in the proliferation of astrocytes.