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Purkinje cells in alert wildtype and L7-PKCI mutant mice show similar mean firing rates and coefficients of variation. A, Interspike interval histograms of simple spike (SS) and complex spike (CS) activity of a wildtype (L7-PKCI/) Purkinje cell. Mean firing rates, f ss 78 spikes/sec and f cs 1.2 spikes/sec. Coefficients of variation, C Vss 0.39 and C Vcs 0.81. Bin width is 0.5 msec in A 1 and 50 msec in A 2. B, Interspike interval histograms of a mutant (L7-PKCI/) Purkinje cell. Mean firing rates, f ss 58 spikes/sec and f cs 1.2 spikes/sec. Coefficients of variation, C Vss 0.64 and C Vcs 1.09. C, D, Population average of the mean simple spike rates ( C) and the mean complex spike rates ( D) in wild-type and mutant Purkinje cells. Error bars indicate 1 SD. Mean SD in wild types (n 44) (f ss 59 26 spikes/sec and f cs 0.9 0.4 spikes/sec) and in mutants (n 51) (f ss 58 21 spikes/sec and f cs 1.0 0.4 spikes/sec). Neither simple spike nor complex spike firing rates are significantly different. E, Scatter plot of simple spike versus complex spike mean firing rates for wild-type and mutant Purkinje cells. Note the considerable yet very similar cell-tocell variability in wild-type (E) and mutant (F) mice. F, Scatter plot of simple spike versus complex spike coefficients of variation for both cell populations. Typically, C Vss and C Vcs are 1, and the distributions largely overlap. Mean SD in wild types, C Vss 0.7 0.4 and C Vcs 0.9 0.2; in mutants, C Vss 0.7 0.3 and C Vcs 0.9 0.2.
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A longstanding but still controversial hypothesis is that long-term depression (LTD) of parallel fiber-Purkinje cell synapses in the cerebellum embodies part of the neuronal information storage required for associative motor learning. Transgenic mice in which LTD is blocked by Purkinje cell-specific inhibition of protein kinase C (PKC) (L7-PKCI mut...
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... examine whether inhibition of various PKC isoforms and/or the consequent blockage of LTD induction affected the short- term stochastics of Purkinje cell firing, we plotted ISI histograms. Figure 2 shows the simple spike and complex spike ISI histograms obtained from a wild-type (L7-PKCI/) ( Fig. 2 A 1 , A 2 ) and a mutant Purkinje cell (L7-PKCI/) ( Fig. 2 B 1 , B 2 ). The mean firing rate, f, and the coefficient of variation, C V , of the simple spike and complex spike discharge were quantified for each cell. ...
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... examine whether inhibition of various PKC isoforms and/or the consequent blockage of LTD induction affected the short- term stochastics of Purkinje cell firing, we plotted ISI histograms. Figure 2 shows the simple spike and complex spike ISI histograms obtained from a wild-type (L7-PKCI/) ( Fig. 2 A 1 , A 2 ) and a mutant Purkinje cell (L7-PKCI/) ( Fig. 2 B 1 , B 2 ). The mean firing rate, f, and the coefficient of variation, C V , of the simple spike and complex spike discharge were quantified for each cell. Figure 2, C and D, shows comparisons of the population averages of the mean simple spike and mean complex spike rates for the ...
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... whether inhibition of various PKC isoforms and/or the consequent blockage of LTD induction affected the short- term stochastics of Purkinje cell firing, we plotted ISI histograms. Figure 2 shows the simple spike and complex spike ISI histograms obtained from a wild-type (L7-PKCI/) ( Fig. 2 A 1 , A 2 ) and a mutant Purkinje cell (L7-PKCI/) ( Fig. 2 B 1 , B 2 ). The mean firing rate, f, and the coefficient of variation, C V , of the simple spike and complex spike discharge were quantified for each cell. Figure 2, C and D, shows comparisons of the population averages of the mean simple spike and mean complex spike rates for the two genotypes. Note that the average values of the simple ...
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... mean firing rate, f, and the coefficient of variation, C V , of the simple spike and complex spike discharge were quantified for each cell. Figure 2, C and D, shows comparisons of the population averages of the mean simple spike and mean complex spike rates for the two genotypes. Note that the average values of the simple spike rates in L7-PKCI/ and L7-PKCI/ mice are not significantly different (f ss 60 spikes/sec for both genotypes; Student's t test; p 0.8) (Fig. 2C). ...
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... quantified for each cell. Figure 2, C and D, shows comparisons of the population averages of the mean simple spike and mean complex spike rates for the two genotypes. Note that the average values of the simple spike rates in L7-PKCI/ and L7-PKCI/ mice are not significantly different (f ss 60 spikes/sec for both genotypes; Student's t test; p 0.8) (Fig. 2C). The average values of the complex spike rates are also very similar for the two genotypes (f cs 1.0 spikes/sec in both cases; Student's t test; p 0.3) (Fig. 2 D). It should be noted, however, that there is considerable cell-to-cell variability in the mean firing rates and that this variability rather than the popu- lation average ...
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... Note that the average values of the simple spike rates in L7-PKCI/ and L7-PKCI/ mice are not significantly different (f ss 60 spikes/sec for both genotypes; Student's t test; p 0.8) (Fig. 2C). The average values of the complex spike rates are also very similar for the two genotypes (f cs 1.0 spikes/sec in both cases; Student's t test; p 0.3) (Fig. 2 D). It should be noted, however, that there is considerable cell-to-cell variability in the mean firing rates and that this variability rather than the popu- lation average might be different in the two genotypes. To over- come this potential pitfall, we also compared the distributions of mean firing rates. It turned out, however, that ...
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... To over- come this potential pitfall, we also compared the distributions of mean firing rates. It turned out, however, that the distribution of both simple spike rates and complex spike rates is indistinguish- able (KS test; p 0.6). Even when the mean complex spike rate of individual Purkinje cells is plotted against their mean simple spike rate (Fig. 2 E, scatter plot), it is observed that the two- dimensional (2D) distribution of the PKCI/ data (Fig. 2 E, E) overlaps the distribution of the PKCI/ data (Fig. 2 E, F), indicating that there is no significant difference (2D KS test; p 0.7). Similar results were obtained with regard to the temporal jitter in simple spikes and complex spikes, as ...
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... out, however, that the distribution of both simple spike rates and complex spike rates is indistinguish- able (KS test; p 0.6). Even when the mean complex spike rate of individual Purkinje cells is plotted against their mean simple spike rate (Fig. 2 E, scatter plot), it is observed that the two- dimensional (2D) distribution of the PKCI/ data (Fig. 2 E, E) overlaps the distribution of the PKCI/ data (Fig. 2 E, F), indicating that there is no significant difference (2D KS test; p 0.7). Similar results were obtained with regard to the temporal jitter in simple spikes and complex spikes, as quantified by the C V . This is shown in the scatter plot of Figure 2 F, in which the complex spike ...
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... rates and complex spike rates is indistinguish- able (KS test; p 0.6). Even when the mean complex spike rate of individual Purkinje cells is plotted against their mean simple spike rate (Fig. 2 E, scatter plot), it is observed that the two- dimensional (2D) distribution of the PKCI/ data (Fig. 2 E, E) overlaps the distribution of the PKCI/ data (Fig. 2 E, F), indicating that there is no significant difference (2D KS test; p 0.7). Similar results were obtained with regard to the temporal jitter in simple spikes and complex spikes, as quantified by the C V . This is shown in the scatter plot of Figure 2 F, in which the complex spike coefficient of variation is plotted versus the simple spike ...
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... results were obtained with regard to the temporal jitter in simple spikes and complex spikes, as quantified by the C V . This is shown in the scatter plot of Figure 2 F, in which the complex spike coefficient of variation is plotted versus the simple spike coefficient of variation for wild-type ( Fig. 2 F, E) and mutant ( Fig. 2 F, F) Purkinje cells. Note that the simple spike coefficient of variation was 1 for the vast majority of cells, indicating that the simple spike ISI distributions are typically more regular than the distributions associated with a pure Pois- son process (for which C V equals 1). ...
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... difference (2D KS test; p 0.7). Similar results were obtained with regard to the temporal jitter in simple spikes and complex spikes, as quantified by the C V . This is shown in the scatter plot of Figure 2 F, in which the complex spike coefficient of variation is plotted versus the simple spike coefficient of variation for wild-type ( Fig. 2 F, E) and mutant ( Fig. 2 F, F) Purkinje cells. Note that the simple spike coefficient of variation was 1 for the vast majority of cells, indicating that the simple spike ISI distributions are typically more regular than the distributions associated with a pure Pois- son process (for which C V equals 1). The complex spike coeffi- ...
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... KS test; p 0.7). Similar results were obtained with regard to the temporal jitter in simple spikes and complex spikes, as quantified by the C V . This is shown in the scatter plot of Figure 2 F, in which the complex spike coefficient of variation is plotted versus the simple spike coefficient of variation for wild-type ( Fig. 2 F, E) and mutant ( Fig. 2 F, F) Purkinje cells. Note that the simple spike coefficient of variation was 1 for the vast majority of cells, indicating that the simple spike ISI distributions are typically more regular than the distributions associated with a pure Pois- son process (for which C V equals 1). The complex spike coeffi- cients of variation were also 1 for ...
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... Purkinje cells, respectively. Note that the conditional firing rates in Figure 4, A 1 and B 1 , peak at approximately 11 msec, whereas peaks occur at approximately 14, 28, and 42 msec in Figure 4, A 2 and B 2 . For the vast majority of wild-type and mutant Purkinje cells, these findings reflected the regularity of the ISI distribution (compare Fig. 2, A 1 and B 1 , for the ISI histograms of the two cells in Fig. 4, A 1 and B 1 ). This is illustrated in Figure 4, A 1,2 and B 1,2 , by superimposing the renewal density histograms, R () (solid curves), computed from the correspond- ing ISI histograms (see Materials and Methods). Note that the predicted oscillatory patterns correspond ...
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... duration was approximately 11 msec for both genotypes (Student's t test; p 0.7). Because the climbing fiber pause reflects only a single event (i.e., the shortest interval occurring between a complex spike and a subsequent simple spike within a recording epoch), we also made histograms of the complex spike- simple spike intervals for each cell ( Fig. 5 A 2 , B 2 ) and quantified the mean and variance of these intervals. As shown in Figure 5D, the mean duration of the complex spike-simple spike interval strongly depends on the mean simple spike ISI (Pearson's corre- lation coefficient, 0.9) in both the PKCI/ (Fig. 5D, F) and PKCI/ (Fig. 5D, E) cell population (2D KS test; p 0.2). The mean ...
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... increase in complex spike rate to 1.4 0.7 spikes/sec (mean SD; p 0.001) in the mutants (using Monte Carlo simulation, zaq;/assuming that 50% of the PKCI/ cells are excited by two climbing fibers). Clearly, the increase in both mean and SD should have emerged as a change in the distribution of complex spike rates, but this was not the case either (Fig. 2 E). Although two distinctly different complex spike waveforms were occasionally identified during spike discrimination, these differ- ent complex spikes were never picked up from one and the same Purkinje cell (as inferred from analysis of the climbing fiber pause) and were therefore not included in the analysis. These findings raised the ...
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... that these curves resemble the data remarkably well in all four cases. The ISI histograms used to compute the curves in A 1 and B 1 are plotted in Figure 2, A 1 , and B 1 , respectively (same cell recordings). C, Simple spike oscillation period versus the most likely simple spike ISI for wild-type (E) and mutant (F) Purkinje cells. ...
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... As shown in Figure 6, multiple climbing fiber innervation was not found in either L7-PKCI/ or L7- PKCI/ Purkinje cells. Thus, stimulation of the granular layer or the molecular layer near the recorded Purkinje cell (50 -70 m away) evoked typical all-or-none climbing fiber-mediated EPSCs in the Purkinje cells obtained from slices of L7-PKCI/ mice (Fig. 6 A1,A2) (n 34; 10 cells studied blind) or their wild-type littermates (Fig. 6 B) (n 18; 16 cells studied blind). In the current-clamp mode, these responses recorded at 70 mV con- sisted of an initial full spike followed by a plateau of depolariza- tion on which partial spikes were superimposed in both L7- PKCI/ (n 5) and L7-PKCI/ (n 5) cells ...
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Citations
... To test this hypothesis, we designed a complex foraging task, where mice spontaneously developed different foraging strategies to obtain a reward after visiting a subset of hidden locations. We used L7-PKCI transgenic mice, which exhibit altered PKC-dependent mechanisms in cerebellar Purkinje cells 14,15 and compared their behavior to that of littermate controls. ...
... It was previously shown that mice with altered PKC-dependent cerebellar functions (L7-PKCI) 14,15 exhibit deficits in using selfmotion and external cues information in a goal-directed task 9,11 . However, it is unclear how PKC-dependent cerebellar functions contribute to the spatial optimization of the animal's trajectory in a task where the reward can be obtained using different strategies. ...
The cerebellum contributes to goal-directed navigation abilities and place coding in the hippocampus. Here we investigated its contribution to foraging strategies. We recorded hippocampal neurons in mice with impaired PKC-dependent cerebellar functions (L7-PKCI) and in their littermate controls while they performed a task where they were rewarded for visiting a subset of hidden locations. We found that L7-PKCI and control mice developed different foraging strategies: while control mice repeated spatial sequences to maximize their rewards, L7-PKCI mice persisted to use a random foraging strategy. Sequential foraging was associated with more place cells exhibiting theta-phase precession and theta rate modulation. Recording in the dark showed that PKC-dependent cerebellar functions controlled how self-motion cues contribute to the selectivity of place cells to both position and direction. Thus, the cerebellum contributes to the development of optimal sequential paths during foraging, possibly by controlling how self-motion and theta signals contribute to place cell coding.
... The experimenters were blind in respect to animal genotype. All synapses (Goossens et al., 2001;De Zeeuw et al., 1998). Control mice for L7-PKCI groups were their wild-type littermates. ...
Head direction (HD) cells, key neuronal elements in the mammalian's navigation system, are hypothesized to act as a continuous attractor network, in which temporal coordination between cell members is maintained under different brain states or external sensory conditions, resembling a unitary neural representation of direction. Whether and how multiple identified HD signals in anatomically separate HD cell structures are part of a single and unique attractor network is currently unknown. By manipulating the cerebellum, we identified pairs of thalamic and retrosplenial HD cells that lose their temporal coordination in the absence of external sensory drive, while the neuronal coordination within each of these brain regions remained intact. Further, we show that distinct cerebellar mechanisms are involved in the stability of direction representation depending on external sensory conditions. These results put forward a new role for the cerebellum in mediating stable and coordinated HD neuronal activity toward a unitary thalamocortical representation of direction.
... PC morphology and firing properties appear normal in young adult L7-PKCI mice (52,62); however, a larger fraction of L7-PKCI PCs (47% in L7-PKCI vs. <10% in wild-type mice) are innervated by two climbing fibers (19). This double innervation has only a nominal effect in SS and CS modulation as indicated by the lack of detectable effects on SS and CS responses in the cerebellar vermis, paramedian lobe, and flocculus of L7-PKCI mice (52,62). ...
... PC morphology and firing properties appear normal in young adult L7-PKCI mice (52,62); however, a larger fraction of L7-PKCI PCs (47% in L7-PKCI vs. <10% in wild-type mice) are innervated by two climbing fibers (19). This double innervation has only a nominal effect in SS and CS modulation as indicated by the lack of detectable effects on SS and CS responses in the cerebellar vermis, paramedian lobe, and flocculus of L7-PKCI mice (52,62). Double innervation may affect normal climbing fiberdependent synaptic plasticity, particularly if the two climbing fibers carry different information. ...
Significance
For spatial navigation, self-centered information sensed by sensory organs is transformed into word-centered information about our motion and orientation in space. The cerebellar posterior vermis helps transform vestibular afferent cues into translation and tilt information, but empirical evidence on the neuronal computations involved is lacking. We investigate these computations using a modeling approach and transgenic mice where protein kinase C (PKC) is inhibited at cerebellar cortex output neurons (Purkinje cells [PCs]). We demonstrate that most PCs have tilt and translation information and that the gain and timing of translation information are modulated by PKC-dependent processes. Hence, translation and tilt information are independently regulated in PCs. Moreover, PKC-dependent processes at PCs participate in generating world-centered motion information.
... A variety of recording, stimulation and perturbation approaches have yielded considerable, convergent evidence for a role of LTD in learning [10,11,15,18,[31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46] but also yielded results that call into question the necessity of parallel fiber-to-Purkinje cell LTD for learning [13,40,41,[47][48][49]. One of the most widely used experimental approaches has been to employ pharmacological or molecular-genetic techniques to perturb LTD, and then test the effects on one or more cerebellum-dependent learning tasks. ...
... The time between stimuli is not drawn to scale. The following columns describe the parameters of the parallel fiber and climbing fiber stimuli and the pairings between them, the region of the cerebellum where the investigation was conducted, and the presence or absence of blockers of inhibition [5,13,31,34, strikingly different from the timing requirements in the wellstudied vermis. In the flocculus, LTD is only induced if the delay between parallel fiber and climbing fiber activation is matched to the feedback delay for climbing fibers to signal errors in vivo during oculomotor learning, which is approximately 120 ms ( Fig. 1) [52,80,81]. ...
Climbing fiber-driven long-term depression (LTD) of parallel fiber synapses onto cerebellar Purkinje cells has long been investigated as a putative mechanism of motor learning. We recently discovered that the rules governing the induction of LTD at these synapses vary across different regions of the cerebellum. Here, we discuss the design of LTD induction protocols in light of this heterogeneity in plasticity rules. The analytical advantages of the cerebellum provide an opportunity to develop a deeper understanding of how the specific plasticity rules at synapses support the implementation of learning.
... Other research showed that LTD requires clathrin-mediated endocytosis, cerebellar LTD is blocked by loading the PCs with a peptide that disrupts dynamin function (Wang and Linden, 2000). Postsynaptic CF-dependent PF LTD is loss upon knockdown of mGluR1 (Aiba et al., 1994), inhibition of PKC (De Zeeuw et al., 1998;Goossens et al., 2001) or in GluA2K882A knock-in mice (with a point mutation that prevents phosphorylation at S880 of GLUA2 by PKC; (Schonewille et al., 2011;Steinberg et al., 2006)). These data confirm that CF-dependent PF-LTD is dependent on GluA2 phosphorylation at S880 by PKC to facilitate PICK1 interaction and promote internalization of GluA2-containing AMPARs. ...
The establishment and maturation of functional synapses underlies the proper function of the Central Nervous System. A large number of secreted and transmembrane molecules are needed to control the mechanisms ensuring synaptic development, maturation and plasticity during the life of the organism. Sushi domain-containing proteins are evolutionary conserved in synapses and mutations in synaptic Sushi domain proteins have been related to neurological and psychiatric disorders in humans. I used the olivocerebellar network as a model to study the potential role of several Sushi domain-containing proteins in excitatory synapse formation and function. Based on previous data from our laboratory, I decided to study two genes, one coding the transmembrane Sushi domain containing protein 4 (SUSD4) and another gene coding four isoforms of Masp1/3 (Mannan-binding lectin serine protease 1/3). First, I identified that the third isoform of Masp1/3, that produces the protein MAP44 (Mannose-binding lectin-associated protein of 44 kDa) lacking the serine protease domain, is the most abundantly expressed isoform in the cerebellum and in the inferior olive. The data obtained by analyzing the Masp1/3 knockout mice suggest that the cytoarchitecture of the cerebellum and excitatory synaptogenesis on cerebellar Purkinje cells is not grossly affected in the absence of Masp1/3. Second, I found that Susd4 is also expressed in the olivocerebellar network during postnatal development and in the adult. Using Susd4 knock-out mice, I found that the absence of SUSD4 leads to deficits in motor learning, lack of climbing fiber-dependent parallel fiber long-term depression and facilitation of parallel fiber long-term potentiation. Climbing fiber transmission is increased transiently during maturation and is associated with misregulation of AMPA receptors content at synapses. Affinity purification followed by mass spectrometry revealed that E3 ubiquitin ligases of the HECT family bind to the cytoplasmic tail of SUSD4. Finally, I provide evidence for a regulation of AMPA receptor degradation by the SUSD4/HECT ligase complex. While molecular mechanisms controlling synaptic incorporation of glutamate receptors are well described, what controls their specific removal from synapses remains to be found. SUSD4 function could be a general mechanism allowing precise spatiotemporal control of the turnover of specific target proteins in cells by HECT ubiquitin ligases. These results provide novel insights into the role of Sushi domain proteins in the molecular mechanisms controlling synaptic plasticity and function in the CNS.
... Additionally, aberrant MAPK signaling via reduced ERK phosphorylation and compromised nuclear translocation of ERK was shown to underlie SCA14 [79]. Given the essential role of PKC in cerebellar LTD [80,81], SCA14 PCs showed impaired pruning of CF synapses and failure of LTD expression affecting synaptic plasticity [82]. Increased phosphorylation levels of ERKs were detected brain tissue of mouse lacking all four PTPRR isoforms which are predominantly expressed in the PCs [83]. ...
The genetically heterozygous spinocerebellar ataxias are all characterized by cerebellar atrophy and pervasive Purkinje Cell degeneration. Up to date, more than 35 functionally diverse spinocerebellar ataxia genes have been identified. The main question that remains yet unsolved is why do some many genetic insults lead to Purkinje Cell degeneration and spinocerebellar ataxia? To address this question it is important to identify intrinsic pathways important for Purkinje Cell function and survival. In this review, we discuss the current consensus on shared mechanisms underlying the pervasive Purkinje Cell loss in spinocerebellar ataxia. Additionally, using recently published cell type specific expression data, we identified several Purkinje Cell-specific genes and discuss how the corresponding pathways might underlie the vulnerability of Purkinje Cells in response to the diverse genetic insults causing spinocerebellar ataxia.
... In this section we provide a detailed protocol for the surgi- cal preparation of a craniotomy to access the cerebellum, followed by an overview of the experimental setup and the protocol for in vivo electrophysiological recording in an awake mouse and the data analysis. These methods have been successfully developed and applied in our laboratory over the last decade to record neuronal activities in an anesthetized [19,48], alert [81,83,84], or behav- ing mouse [85,86]. ...
... The absence of a climbing fiber pause, the minimum duration between a complex spike and the following simple spike, is taken to indicate that the isolation is imperfect and there is a second unit present. For each cell several parameters for simple and complex spikes can be calculated, including firing rate, CV and mean CV2, as well as the climbing fiber pause [84]. CV is the standard deviation of inter-spike intervals (ISI) divided by the mean; the mean CV2 is calculated as the mean of 2·|(ISI)n + 1 − ISIn|/(ISIn + 1 + ISIn) [99]. ...
Single-unit recordings in vivo are the unitary elements in the processing of the brain and as such essential in systems physiology to understand brain functioning. In the cerebellum, a structure with high levels of intrinsic activity, studying these elements in vivo in an awake animal is imperative to obtain information regarding the processing features of these units in action. In this chapter we address the rationale and the approach of recording electrophysiological activity in the cerebellum, particularly that of Purkinje cells, in vivo in the awake, active animal. In line with the developing appreciation for the diversity within populations of the cells of the same type, there is a growing interest in the differentiation within the population of Purkinje cells. Here we describe a successful approach to analyzing the activity of two populations of Purkinje cells, which differ in connectivity and the expression of several genes. By driving the expression of a fluorescent marker with the promotor of one of the differentiating genes, the presence of a fluorescence signal could be used to recognize and approach Purkinje cells, while the intensity of the signal can be used as a marker to identify the two subpopulations. Finally, the drawbacks and the advantages of this technique are discussed and placed into a future perspective.
... [9][10][11][12][13] In particular, mice with Purkinje cell-specific gene modifications played a pivotal role. [14][15][16][17][18][19] Purkinje cell-specific gene expression in most transgenic mice has used the Purkinje cell protein 2 (PCP2)/L7 promoter (henceforth L7), which drives the transcription of the L7 gene to produce the Purkinje cell-specific L7 protein. [20][21][22] The L7 structural gene contains four exons that extend over about 2 kb in the genome. ...
... [9][10][11][12][13] In particular, mice with Purkinje cell-specific gene modifications played a pivotal role. [14][15][16][17][18][19] Purkinje cell-specific gene expression in most transgenic mice has used the Purkinje cell protein 2 (PCP2)/L7 promoter (henceforth L7), which drives the transcription of the L7 gene to produce the Purkinje cell-specific L7 protein. [20][21][22] The L7 structural gene contains four exons that extend over about 2 kb in the genome. ...
Cell type-specific promoters in combination with viral vectors and gene editing technology permit efficient gene manipulation in specific cell populations. Cerebellar Purkinje cells play a pivotal role in cerebellar functions. Although the Purkinje cell-specific L7 promoter is widely used for the generation of transgenic mice, it remains unsuitable for viral vectors because of its large size (3 kb) and exceedingly weak promoter activity. Here, we found that the 0.8-kb region (named here as L7-6) upstream of the transcription initiation codon in the first exon was alone sufficient as a Purkinje cell-specific promoter, presenting a far stronger promoter activity over the original 3-kb L7 promoter with a sustained significant specificity to Purkinje cells. Intravenous injection of adeno-associated virus vectors that are highly permeable to the blood brain barrier confirmed the Purkinje cell specificity of the L7-6 in the central nervous system. The features of the L7-6 were also preserved in the marmoset, a non-human primate. The high sequence homology of the L7-6 among mouse, marmoset, and human suggests the preservation of the promoter strength and Purkinje cell-specificity features also in humans. These findings suggest that L7-6 will facilitate the cerebellar research targeting the pathophysiology and gene therapy of cerebellar disorders.
... More importantly, we observed deficits of chemically induced long-term depression (DHPG-LTD) in Rbfox3 -/mice (Fig 5d), consistent with previous findings of electrically induced long-term depression (LFS-LTD) in Rbfox3 -/mice [6], although both types of LTD use different receptors and upstream mechanisms [22]. Activation of protein kinase C (PKC) is necessary for induction of LTD [23][24][25]. However, LTD deficits in Rbfox3 -/mice could not be rescued by applying the protein kinase C agonist, bryostatin-1 (Fig 5e). ...
Dysfunction of RBFOX3 has been identified in neurodevelopmental disorders such as autism spectrum disorder, cognitive impairments and epilepsy and a causal relationship with these diseases has been previously demonstrated with Rbfox3 homozygous knockout mice. Despite the importance of RBFOX3 during neurodevelopment, the function of RBFOX3 regarding neurogenesis and synaptogenesis remains unclear. To address this critical question, we profiled the developmental expression pattern of Rbfox3 in the brain of wild-type mice and analyzed brain volume, disease-relevant behaviors, neurogenesis, synaptic plasticity, and synaptogenesis in Rbfox3 homozygous knockout mice and their corresponding wild-type counterparts. Here we report that expression of Rbfox3 differs developmentally for distinct brain regions. Moreover, Rbfox3 homozygous knockout mice exhibited cold hyperalgesia and impaired cognitive abilities. Focusing on hippocampal phenotypes, we found Rbfox3 homozygous knockout mice displayed deficits in neurogenesis, which was correlated with cognitive impairments. Furthermore, RBFOX3 regulates the exons of genes with synapse-related function. Synaptic plasticity and density, which are related to cognitive behaviors, were altered in the hippocampal dentate gyrus of Rbfox3 homozygous knockout mice; synaptic plasticity decreased and the density of synapses increased. Taken together, our results demonstrate the important role of RBFOX3 during neural development and maturation. In addition, abnormalities in synaptic structure and function occur in Rbfox3 homozygous knockout mice. Our findings may offer mechanistic explanations for human brain diseases associated with dysfunctional RBFOX3.
