Fig 4 - uploaded by Constantino Sotelo
Content may be subject to copyright.
Olivocerebellar topography in control explant (8 DIV), retrograde axonal tracing. (A) Fluorescence micrograph of the left cerebellar plate of a control explant viewed with a u.v. filter, illustrating the injection sites. A DiA crystal (green-orange fluorescence) is inserted close to the anterior margin (a) of the cerebellar plate (blue), while a DiI crystal (red fluorescence) is placed close to its posterior margin (p). (B) After such injections, two complementary territories are labelled in the contralateral IO, a DiI-labelled rostromedian territory (thin arrows) and a DiAlabelled caudolateral territory (thick arrow). One region of the IO is unlabelled (asterisk, compare with Fig. 3B), and probably projects to the median cerebellum. (C) Schematic illustration of the organization of the olivocerebellar topography in control explants. The floor plate is represented by the grey line. IO, inferior olive; cer, cerebellum. Scale bars, 750 µm in A and 300 µm in B.
Source publication
The formation of the olivocerebellar projection is supposed to be regulated by positional information shared between pre- and postsynaptic neurons. However, experimental evidence to support this hypothesis is missing. In the chick, caudal neurons in the inferior olive project to the anterior cerebellum and rostral ones to the posterior cerebellum....
Contexts in source publication
Context 1
... glass micropipette and applied, under a dissecting microscope, on the posterior margin (originally anterior in rotation cases) of one (unilateral injection) or both (bilateral injection) cerebellar plates, whereas a crystal of DiA (4-(4-dihexadecylaminostyryl)-N- methylpyridinium iodide; Molecular Probes) was inserted in the anterior margin (Fig. 4A). In some cases, a drop of 2% fast-blue (Sigma) in GBSS was laid down the entire cerebellar plate with a micropipette, in addition to DiI and DiA crystals. The explants were allowed to survive for a further 24-36 hours before fixing in 4% PF for a minimum of 1 hour at RT. For short-survival time experiments, the explants (n=17) were ...
Context 2
... the anteroposterior axis of the cerebellum. As previously mentioned, large dye injections result in the labelling of the whole IO (Fig. 3B). Therefore, using control explants (n=62), we implanted (after 1 week in vitro) a small crystal of DiI close to the posterior margin of the cerebellar plate and a crystal of DiA close to its anterior margin (Fig. 4A). After 2 days, DiI-(93% of the cases) and DiA-(99% of the cases) labelled neurons were detected in the IO. Interestingly, the two labelled domains were not overlapping, but complementary: DiI- labelled neurons were found in an inverted L- shaped, median region, juxtaposed to the floor plate, almost extending on the entire rostrocau- ...
Context 3
... DiI-(93% of the cases) and DiA-(99% of the cases) labelled neurons were detected in the IO. Interestingly, the two labelled domains were not overlapping, but complementary: DiI- labelled neurons were found in an inverted L- shaped, median region, juxtaposed to the floor plate, almost extending on the entire rostrocau- dal length of the IO (see Fig. 4B). In a complementary fashion, DiA-labelled neurons were only detected in an ovoid region, more caudal and lateral than the DiI-labelled domain (Fig. 4B). Axonal guidance in the olivocerebellar system Some lateral and median IO neurons were not traced (compare Figs 3B and 4B) as they probably project to the median portion of the ...
Context 4
... but complementary: DiI- labelled neurons were found in an inverted L- shaped, median region, juxtaposed to the floor plate, almost extending on the entire rostrocau- dal length of the IO (see Fig. 4B). In a complementary fashion, DiA-labelled neurons were only detected in an ovoid region, more caudal and lateral than the DiI-labelled domain (Fig. 4B). Axonal guidance in the olivocerebellar system Some lateral and median IO neurons were not traced (compare Figs 3B and 4B) as they probably project to the median portion of the cerebellum, where no tracer was injected. The number of labelled IO neurons varied between explants, which could be due to a different neuronal survival, or ...
Context 5
... varied between explants, which could be due to a different neuronal survival, or more probably to a variability in dye crystal sizes and injection sites. In 1% of the cases no IO neurons were labelled, and in 7% of the cases only the caudolateral IO domain was labelled. The organization of this in vitro olivocerebellar map is summarized in Fig. 4C: to make it simpler we can conclude that, in vitro, neurons located in the caudolateral IO project to the anterior cerebellum, whereas rostromedian IO neurons project to the posterior cere- bellum. This map resembles the one that has been described in vivo by Furber (1983;see Introduction and Discussion). Inter- estingly, the map was ...
Similar publications
Although electromagnetic brain stimulation is a promising treatment in neurology and psychiatry, clinical outcomes are variable, and underlying mechanisms are ill-defined, which impedes the development of new effective stimulation protocols. Here, we show, in vivo and ex vivo, that repetitive transcranial magnetic stimulation at low-intensity (LI-r...
Citations
... We used vesicular glutamate transporter 2 (VGLUT2) immunohistochemistry to visualize any LI-rTMS-induced reinnervating climbing fibre terminals (as described) [11,12,14]. We then moved to our explant model of the olivocerebellar system in vitro, in which the cerebellum and brainstem are cultured "en bloc" to retain all cell populations and circuits [15,16]. These explants develop in vivo and can be manipulated to replicate the pedunculotomy lesion and PC reinnervation [16]. ...
Electromagnetic brain stimulation is a promising treatment in neurology and psychiatry. However, clinical outcomes are variable and underlying mechanisms remain ill-defined, impeding the development of new effective stimulation protocols. There is increasing application of repetitive transcranial magnetic stimulation (rTMS) to the cerebellum to induce forebrain plasticity through its long-distance cerebello-cerebral circuits. To better understand what magnetic stimulation does within the cerebellum, we have developed tools to generate defined low-intensity (LI) magnetic fields and deliver them in vivo, in 3D organotypic culture and in primary cultures, over a range of stimulation parameters. Here we show that low-intensity rTMS (LI-rTMS) to the cerebellum induces axon growth and synapse formation providing olivocerebellar reinnervation. This repair depends on stimulation pattern, with complex biomimetic patterns being most effective, and this requires the presence of a cellular magnetoreceptor, cryptochrome. To explain these reparative changes, we found that repair-promoting LI-rTMS patterns, but not ineffective ones, increased c-fos expression in Purkinje neurons, consistent with the production of reactive oxygen species by activated cryptochrome. Rather than activating neurons via induced electric currents, we propose that weak magnetic fields act through cryptochrome, activating intracellular signals that induce climbing fibre-Purkinje cell reinnervation. This information opens new routes to optimize cerebellar magnetic stimulation and its potential role as an effective treatment for neurological diseases.
... Sotelo and colleagues have argued that matching gene expression domains between the cerebellum and inferior olive contain cues that guide the formation of a precise topographical projection map (Wassef et al. 1992;Chédotal et al. 1997;Sotelo and Chédotal 2005). In support of this model, Nishida et al. (2002) demonstrated that overexpression of Ephrin-A2 by using retroviral vectors disrupts the general topography of the olivocerebellar projection. ...
Cerebellar architecture is organized around the Purkinje cell. Purkinje cells in the mouse cerebellum come in many different subtypes, organized first into four transverse zones and then further grouped into hundreds of reproducible topographical units – stripes. Stripes are identified by their functional properties, connectivity, and expression profiles. The molecular pattern of stripes is highly reproducible between individuals and is conserved through evolution. Pattern formation in the cerebellar cortex is a multistage process that begins with the generation of the Purkinje cells in the ventricular zone (VZ) of the fourth ventricle. During this stage, or shortly after, Purkinje cell subtypes are specified toward specific positions. Purkinje cells migrate from the VZ to form an array of clusters that form the framework for cerebellar topography. At around birth, these clusters begin to disperse, triggered by Reelin signaling pathway proteins, to form the adult stripe array.
The chapter will begin with a brief overview of adult cerebellar topography, primarily focusing on the mouse cerebellum, and then discuss the cellular and molecular mechanisms that establish these remarkable patterns. Considering how functionally diverse the cerebellum is despite its conserved organization of patterns, this chapter will end exploring how stripes might contribute to neuronal activity and the execution of cerebellar-dependent behaviors.
... Nevertheless, it is 61 unknown how these weak fields modify brain circuits, which are the structural basis of 62 human behaviour. The biological effects of magnetic stimulation depend on stimulation frequency, pattern, 65 duration and number of pulses delivered 1,5 . These are restricted in high intensity rTMS for 66 technical reasons (heating) and safety considerations (pain, seizures) 6 . ...
... An in silico search of the 22 genes regulated by LI-rMS showed that 8 (36%) 174 contained CLOCK or ARNTL binding sequences in their upstream promoter regions ( We also show the importance of stimulation frequency and pattern on post-lesion neural 194 repair. Although human high-intensity rTMS stimulation frequencies are broadly divided into 195 "excitatory" and "inhibitory", with iTBS and cTBS inducing the most consistent and long-196 lasting effects 1,5,6,27 , our data add a new dimension to this concept. We show that complexity 197 (iTBS vs. 10 Hz) and regularity (iTBS vs. random-iTBS) are more important than the number 198 preprint (which was not peer-reviewed) is the author/funder. ...
Magnetic brain stimulation is a promising treatment in neurology and psychiatry, but clinical outcomes are variable. Unfortunately, mechanisms underlying magnetic stimulation effects are ill-defined, which impedes the development of stimulation protocols appropriate for different neurological conditions. Here we show, in vivo and ex vivo, that repetitive transcranial magnetic stimulation at low-intensity (LI-rTMS) induces axon outgrowth and synaptogenesis to repair a neural circuit. This repair depends on stimulation pattern, with complex patterns being particularly effective, and its mechanism requires the presence of cryptochrome (Cry), a putative magneto-receptor. Effective LI-rTMS patterns altered expression of Cry target genes known to promote neuronal repair. Because LI-rTMS generates electric fields too weak to depolarise neurons, these findings indicate that the magnetic field itself induces the repair. Our data open a new framework for magnetic stimulation - cryptochrome-mediated molecular and structural neuroplasticity. This information suggests new routes to treatments specific for each neurological disease.
... The olivocerebellar path and its lesion has also been optimized in vitro using hindbrain organotypic explants which contain the whole circuit, are highly reproducible and readily manipulated (Chédotal et al., 1997;Letellier et al., 2009). Hindbrain explants were cultured from embryonic Swiss or Cryptochrome knock-out (Cry1 -/-::Cry2 -/-) mice at embryonic day 14 (E14) as previously described (Chédotal et al., 1997;Letellier et al., 2009). ...
... The olivocerebellar path and its lesion has also been optimized in vitro using hindbrain organotypic explants which contain the whole circuit, are highly reproducible and readily manipulated (Chédotal et al., 1997;Letellier et al., 2009). Hindbrain explants were cultured from embryonic Swiss or Cryptochrome knock-out (Cry1 -/-::Cry2 -/-) mice at embryonic day 14 (E14) as previously described (Chédotal et al., 1997;Letellier et al., 2009). E0 was the mating day. ...
... MargaretKennard (1899Kennard ( -1975: not a "principle" of brain plasticity but a founding mother of developmental neuropsychology. Cortex 46, 1043-1059 Dusart, I., Airaksinen, M.S., andSotelo, C. (1997). Purkinje cell survival and axonal regeneration are age dependent: an in vitro study. ...
Neuroplasticity is essential for the establishment and strengthening of neural circuits during the critical period of development, and are required for the brain to adapt to its environment. The mechanisms of plasticity vary throughout life, are generally more difficult to induce in the adult brain, and decrease with advancing age. Repetitive transcranial magnetic stimulation (rTMS) is commonly used to modulate cortical excitability and shows promise in the treatment of some neurological disorders. Low intensity magnetic stimulation (LI-rTMS), which does not directly elicit action potentials in the stimulated neurons, have also shown some therapeutic effects, and it is important to determine the biological mechanisms underlying the effects of these low intensity magnetic fields, such as would occur in the regions surrounding the central high-intensity focus of rTMS. We have used a focal low-intensity magnetic stimulation (10mT) to address some of these issues in the mouse cerebellum and olivocerebellar path. The cerebellum model is particularly useful as its development, structure, ageing and function are well described which allows us to easily detect eventual modifications. We assessed effects of in vivo or in vitro LI-rTMS on neuronal morphology, behavior, and post-lesion plasticity. We first showed that LI-rTMS treatment in vivo alters dendritic spines and dendritic morphology, in association with improved spatial memory. These effects were age dependent. To optimize stimulation parameters in order to induce post-lesion reinnervation we used our in vitro model of post-lesion repair to systematically investigate the effects of different LI-rTMS stimulation patterns and frequencies. We showed that the pattern of stimulation is critical for allowing repair, rather than the total number of stimulation pulses. Finally, we looked for potential underlying mechanisms participating in the effects of the LI-rTMS, using mouse mutants in vivo or in vitro. We found that the cryptochromes, which have magnetoreceptor properties, must be present for the response to magnetic stimulation to be transduced into biological effects. The ensemble of our results indicate that the effects of LI-rTMS depend upon the presence of magnetoreceptors, the stimulation protocol, and the age of the animal suggesting that future therapeutic strategies must be adapted to the neuronal context in each individual person.
... clusters also were found to receive BEN-ir olivocerebellar fibers ( [28]; Pourquié et al. [96]). Irrespective of reversal of the cerebellar plate, olivocerebellar fibers recognize polarity cues in their target region that organize their antero-posterior topography [27]. Ephrins and their receptors are distributed in parasagittal domains in chicken embryos [60]. ...
Sotelo stated in his introduction for a consensus paper on cerebellar development (Leto et al., Cerebellum 15:789–828, 2015) that “The work done in the late nineteenth century until the late 1970s provided substantial and significant information; however, it was only descriptive and barely addressed the mechanisms involved.” Observations and their description, the nomenclature that evolved from these studies, and the ideas they fostered, indeed, formed the basis for our understanding of the mechanisms that underlie the complex development of the cerebellum, to be reviewed in this volume. This chapter will highlight some of these early contributions to the origin of the cerebellum, its histogenesis, the migration of its neurons, the development of the longitudinal Purkinje cell zones, their target nuclei and their connections, and the folial pattern of the cerebellum.
... In the current study, we have used the olivocerebellar path ex vivo (Chedotal et al., 1997;) as a highly reproducible model with which to investigate mechanisms underlying BDNF-induced olivocerebellar reinnervation. We began by studying molecules downstream of BDNF signalling which are also expressed within the olivocerebellar system, in particular during the developmental period of active axonal growth and synaptogenesis. ...
... This is the first demonstration that Pax3 is involved in axonal outgrowth in the mammalian CNS. Hindbrain explant cultures were performed as described previously (Chedotal, Bloch-Gallego, & Sotelo, 1997; using Swiss mice at E14. Briefly, the embryos' heads were put into ice-cold Gey's balanced salt solution (Eurobio, Courtaboeuf, France) with 5 mg/ml glucose, and brains were quickly dissected out. ...
In the olivocerebellar pathway (OCP) the afferent climbing fibres (CFs), which are the terminal axon projections of the inferior olivary nucleus (ION), innervate cerebellar Purkinje cells (PCs). Following unilateral transection of mature OCP, the addition of the neurotrophic factor BDNF into the denervated cerebellum induces functional CF reinnervation of PCs. What mechanism underlies the BDNF-activated plastic window in the mature OCP and whether recapitulates developmental plasticity remains unknown. Using an optimized ex vivo model of the mouse OCP, we have found that the addition of BDNF into the de-afferented hemicerebellum induces both the outgrowth and elongation of transverse branches from intact CFs. This BDNF-induced plastic response is mediated by the up-regulation of the expression of transcription factor Pax3 in the intact ION. Increased pax3 gene in the ION up-regulates polysialic acid-neural cell adhesion molecule (PSA-NCAM), most likely in the olivocerebellar axons, which was found to be necessary and sufficient for CF reinnervation to PCs. We propose that the BDNF-activated plastic mechanism in the mature OCP involves the afferent Pax3 and PSA-NCAM, which underlies the sprouting of CFs and their appropriate recognition of denervated PCs. Early postnatal OCP shows a spontaneous plasticity following lesion that compensates anatomically and functionally for PC denervation. Using our ex vivo model of the OCP, we found that developmental post-lesion plasticity intrinsically activates and depends on the expression of Pax3 and PSA-NCAM. These results suggest that BDNF treatment in mature OCP reactivates some steps of developmental plasticity mechanisms.
... In low-intensity stimulation (LI-rMS) studies, solenoids (Di Loreto et al., 2009;Varro et al., 2009) or coils made "in house" have been applied to one-off experiments on cultured neurons/slices (Ahmed and Wieraszko, 2009;Rotem et al., 2014) or isolated nerves (Maccabee et al., 1993;Basham et al., 2009;RamRakhyani et al., 2013;Ahmed and Wieraszko, 2015) that do not permit ongoing stimulation sessions to model treatment-based protocols. Moreover, given that NIBS acts on complex neural circuits, stimulation parameters should ideally be assessed in culture models which retain some neural circuitry: e.g., organotypic hippocampal (Hausmann et al., 2001;Hogan and Wieraszko, 2004;Vlachos et al., 2012;Lenz et al., 2016) and cortico-striatal slices, hindbrain explants (Chedotal et al., 1997;Letellier et al., 2009) or microfluidic circuit cultures (Szelechowski et al., 2014). However, these diverse culture systems have unique dimensions and culture conditions, highlighting the need to establish a reliable and flexible LFMS/LI-rMS system that can be tailored to deliver a defined magnetic field that induces an electric field of predicted intensity and direction at a particular location within each culture. ...
... Here, we describe the design and construction of a generic stimulation device suitable for long-term LI-rMS in vitro that can be adapted to specific culture conditions. Specifically, we have built a device that applies LI-rMS at a range of frequencies to hindbrain explants containing a model of axonal injury and repair, the lesioned olivocerebellar path (Chedotal et al., 1997;Letellier et al., 2009). We use stimulation at an intensity that alters intracellular calcium and gene expression (Mattsson and Simkó, 2012;Grehl et al., 2015), provides neuroprotection (Yang et al., 2012) and reorganizes neural circuits in vivo (Rodger et al., 2012;Makowiecki et al., 2014). ...
... Our particular in vitro experiments required long-term culture and stimulation of multi-axial (L × W × H, 5 × 5 × 1 mm) organotypic mouse hindbrain explants cultured on 30 mm Millipore membranes (Millipore, USA) in six-well culture plates [Techno Plastic Products (TPP), Switzerland] and incubated at 35 • C in 95% air plus 5% CO 2 (Chedotal et al., 1997;Letellier et al., 2009). Details of dissection and culture procedures are provided below (Biological Validation). ...
Non-invasive brain stimulation (NIBS) by electromagnetic fields appears to benefit human neurological and psychiatric conditions, although the optimal stimulation parameters and underlying mechanisms remain unclear. Although, in vitro studies have begun to elucidate cellular mechanisms, stimulation is delivered by a range of coils (from commercially available human stimulation coils to laboratory-built circuits) so that the electromagnetic fields induced within the tissue to produce the reported effects are ill-defined. Here, we develop a simple in vitro stimulation device with plug-and-play features that allow delivery of a range of stimulation parameters. We chose to test low intensity repetitive magnetic stimulation (LI-rMS) delivered at three frequencies to hindbrain explant cultures containing the olivocerebellar pathway. We used computational modeling to define the parameters of a stimulation circuit and coil that deliver a unidirectional homogeneous magnetic field of known intensity and direction, and therefore a predictable electric field, to the target. We built the coil to be compatible with culture requirements: stimulation within an incubator; a flat surface allowing consistent position and magnetic field direction; location outside the culture plate to maintain sterility and no heating or vibration. Measurements at the explant confirmed the induced magnetic field was homogenous and matched the simulation results. To validate our system we investigated biological effects following LI-rMS at 1 Hz, 10 Hz and biomimetic high frequency, which we have previously shown induces neural circuit reorganization. We found that gene expression was modified by LI-rMS in a frequency-related manner. Four hours after a single 10-min stimulation session, the number of c-fos positive cells increased, indicating that our stimulation activated the tissue. Also, after 14 days of LI-rMS, the expression of genes normally present in the tissue was differentially modified according to the stimulation delivered. Thus we describe a simple magnetic stimulation device that delivers defined stimulation parameters to different neural systems in vitro. Such devices are essential to further understanding of the fundamental effects of magnetic stimulation on biological tissue and optimize therapeutic application of human NIBS.
... 72), whereas VGLUT1 and VGLUT2 are differentially expressed in bands of mossy fibres 69 . A body of evidence has also accumulated to indicate that the terminals of climbing fibres and mossy fibres arising from different sources align with cerebellar stripes of various molecular markers of Purkinje cells, most notably zebrin II 15,[76][77][78][79][80][81] . Specifically, there is a correspondence between individual Purkinje cell zones (defined by their climbing fibre input, see above) and zebrin II expression. ...
The adult mammalian cerebellar cortex is generally assumed to have a uniform cytoarchitecture. Differences in cerebellar function are thought to arise, in the main, through distinct patterns of input and output connectivity, rather than as a result of variations in cortical microcircuitry. However, evidence from anatomical, physiological and genetic studies is increasingly challenging this orthodoxy and there are now various lines of evidence that the cerebellar cortex is non uniform. Here we develop the hypothesis that regional differences in cerebellar cortical microcircuit properties lead to important differences in information processing.
... 72), whereas VGLUT1 and VGLUT2 are differentially expressed in bands of mossy fibres 69 . A body of evidence has also accumulated to indicate that the terminals of climbing fibres and mossy fibres arising from different sources align with cerebellar stripes of various molecular markers of Purkinje cells, most notably zebrin II 15,[76][77][78][79][80][81] . Specifically, there is a correspondence between individual Purkinje cell zones (defined by their climbing fibre input, see above) and zebrin II expression. ...
The adult mammalian cerebellar cortex is generally assumed to have a uniform cytoarchitecture. Differences in cerebellar function are thought to arise primarily through distinct patterns of input and output connectivity rather than as a result of variations in cortical microcircuitry. However, evidence from anatomical, physiological and genetic studies is increasingly challenging this orthodoxy, and there are now various lines of evidence indicating that the cerebellar cortex is not uniform. Here, we develop the hypothesis that regional differences in properties of cerebellar cortical microcircuits lead to important differences in information processing.
... In a second series of experiments, we also tested whether magnetic stimulation parameters can promote PC reinnervation post-lesion. These experiments were undertaken using our explant model of the olivocerebellar system in vitro [13,14], in which the cerebellum and brainstem are cultured "en bloc" to retain all cell populations and circuits. These explants develop as in vivo and can be manipulated to replicate the pedunculotomy lesion and PC reinnervation [14]. ...
Non-invasive stimulation of the human cerebellum, such as by transcranial magnetic stimulation (TMS), is increasingly used to investigate cerebellar function and identify potential treatment for cerebellar dysfunction. However, the effects of TMS on cerebellar neurons remain poorly defined. We applied low-intensity repetitive TMS (LI-rTMS) to the mouse cerebellum in vivo and in vitro and examined the cellular and molecular sequelae. In normal C57/Bl6 mice, 4 weeks of LI-rTMS using a complex biomimetic high-frequency stimulation (BHFS) alters Purkinje cell (PC) dendritic and spine morphology; the effects persist 4 weeks after the end of stimulation. We then evaluated whether LI-rTMS could induce climbing fibre (CF) reinnervation to denervated PCs. After unilateral pedunculotomy in adult mice and 2 weeks sham or BHFS stimulation, VGLUT2 immunohistochemistry was used to quantify CF reinnervation. In contrast to sham, LI-rTMS induced CF reinnervation to the denervated hemicerebellum. To examine potential mechanisms underlying the LI-rTMS effect, we verified that BHFS could induce CF reinnervation using our in vitro olivocerebellar explants in which denervated cerebellar tissue is co-cultured adjacent to intact cerebella and treated with brain-derived neurotrophic factor (BDNF) (as a positive control), sham or LI-rTMS for 2 weeks. Compared with sham, BDNF and BHFS LI-rTMS significantly increased CF reinnervation, without additive effect. To identify potential underlying mechanisms, we examined intracellular calcium flux during the 10-min stimulation. Complex high-frequency stimulation increased intracellular calcium by release from intracellular stores. Thus, even at low intensity, rTMS modifies PC structure and induces CF reinnervation.
