Impairment of cerebellar long-term depression and GABAergic transmission in prion protein deficient mice ectopically expressing PrPLP/Dpl

Prion protein (PrPC) knockout mice, named as the “Ngsk” strain (Ngsk Prnp0/0 mice), show late-onset cerebellar Purkinje cell (PC) degeneration because of ectopic overexpression of PrPC-like protein (PrPLP/Dpl). Our previous study indicated that the mutant mice also exhibited alterations in cerebellum-dependent delay eyeblink conditioning, even at a young age (16 weeks of age) when neurological changes had not occurred. Thus, this electrophysiological study was designed to examine the synaptic function of the cerebellar cortex in juvenile Ngsk Prnp0/0 mice. We showed that Ngsk Prnp0/0 mice exhibited normal paired-pulse facilitation but impaired long-term depression of excitatory synaptic transmission at synapses between parallel fibres and PCs. GABAA-mediated inhibitory postsynaptic currents recorded from PCs were also weakened in Ngsk Prnp0/0 mice. Furthermore, we confirmed that Ngsk Prnp0/0 mice (7–8-week-old) exhibited abnormalities in delay eyeblink conditioning. Our findings suggest that these alterations in both excitatory and inhibitory synaptic transmission to PCs caused deficits in delay eyeblink conditioning of Ngsk Prnp0/0 mice. Therefore, the Ngsk Prnp0/0 mouse model can contribute to study underlying mechanisms for impairments of synaptic transmission and neural plasticity, and cognitive deficits in the central nervous system.


Scientific RepoRtS
| (2020) 10:15900 | https://doi.org/10.1038/s41598-020-72753-6 www.nature.com/scientificreports/ PrP C is highly expressed in normal cerebellar Purkinje cells (PCs) and granule cells 11,12 , indicating that the protein plays a role in normal cerebellar synaptic function and neuronal plasticity. Although electrophysiological studies of cerebellar function have been already performed in ZrchI Prnp 0/0 mice, the physiological properties of cerebellar PCs in Ngsk Prnp 0/0 mice have not been characterized [13][14][15][16][17][18][19][20] . Therefore, in the present study, we examined whether a deficiency in PrP C and ectopic expression of PrPLP/Dpl in Ngsk Prnp 0/0 mice affect cerebellar physiological functions by evaluating the basic excitatory and inhibitory synaptic transmission to PCs and long-term depression (LTD) of excitatory synaptic transmission at parallel fibre (PF)-PC synapses. We found that Ngsk Prnp 0/0 mice showed not only weakened GABA A -mediated inhibitory postsynaptic currents in PCs but also impaired LTD, suggesting that PrPLP/Dpl expression can induce cerebellar dysfunctions by impairing cerebellar synaptic transmission.
Altered delay eyeblink conditioning in Ngsk Prnp 0/0 mice at 7-8 weeks of age. Finally, we examined delay eyeblink conditioning, a form of cerebellum-dependent discrete motor learning, in Ngsk Prnp 0/0 mice aged 7-8 weeks, which were almost the age of mice used for electrophysiological studies. With conditioning, the animals learn the adaptive timing of eye blinking; in our study, the conditioned response frequency (CR%) for the control mice progressively and significantly increased to over 70% on day 7 (Fig. 4a). On the contrary, CR% for Ngsk Prnp 0/0 mice did not reach 60%. However, repeated measures ANOVA failed to reveal significant differences between the two groups (session × group interaction, F(6, 84) = 0.58, p = 0.57, a genotypic effect, F(1, 14) = 2.75, p = 0.12). The normalized electromyographic (EMG) amplitude on day 7 for Ngsk Prnp 0/0 mice seemed to be lower than that for the control mice (Fig. 4b). Indeed, CR amplitude on day 7 was significantly reduced in Ngsk Prnp 0/0 mice (p = 0.023, Fig. 4c, right panel). Peak latency was also significantly decreased in Ngsk Prnp 0/0 mice compared to that in the control mice (p = 0.031, Fig. 4c, left panel). These results confirmed www.nature.com/scientificreports/ that the timing and amplitude of conditioned eyeblink response were altered in juvenile Ngsk Prnp 0/0 mice that had not yet undergone PC degeneration.

Discussion
Several Prnp null mutant mouse strains, including Ngsk, have been questioned in the field as to whether their phenotype is physiological 24 . Indeed, given that many different PrP knockout mouse strains produce diverse physiological phenotypes, most of the PrP knockout mouse strains have disparate findings because the Prnp −/− locus is surrounded by genes other than Prnp itself ("flanking genes"), which it is thought to be indicative of 2,5,7 . Nevertheless, the present study aimed to specifically characterize the electrophysiological properties of Ngsk Prnp 0/0 mice strain, which exhibits cerebellar PC degeneration and motor learning deficits. The present electrophysiological study shows that cerebellar LTD is significantly impaired in Ngsk Prnp 0/0 mice (Fig. 3). Furthermore, our behavioral study indicated that Ngsk Prnp 0/0 mice (7-8-week-old) exhibited abnormalities in CR amplitude and CR timing, without a significant difference in CR probabilities (Fig. 4). These behavioral results replicate those obtained in experiments with Ngsk Prnp 0/0 mice at 16 weeks old 10 ; however, the extent of their learning disability appears to be more pronounced. The reason for the severe impairments of 7-8-week-old mice is unclear. In Ngsk Prnp 0/0 mice, GFAP begins to increase gradually from 7 to 8 weeks of age 25 . GFAP is thought to be a significant factor needed for proper communication between Bergmann glia and PC, enabling occurrence of LTD 26 . Indeed, GFAP KO mice exhibited the impairments of cerebellar LTD and eyeblink conditioning 27 . Hence, one possibility is that in 16-week-old Ngsk mutants, improved LTD due to increased GFAP may have had a milder impact on the motor learning disabilities. Regardless, the present results are consistent with those of previous studies suggesting parallelism between impaired cerebellar LTD and altered delay eyeblink conditioning [28][29][30][31][32] , although the extent of impairment in both conditioned eyeblink response and cerebellar LTD appeared to be less than that observed in the previous reports [28][29][30]32 . As in the past, the results of the present study do not demonstrate a direct causal relationship between LTD and eyeblink conditioning, but suggest that there is a common molecular basis for both.
In addition, the effects of molecular layer interneuron-PC feed-forward inhibition (FFI) and absence of FFI on LTD formation have been studied in mice that are genetically deficient in inhibitory synaptic inputs to the PC 33 . The excitatory/inhibitory (E/I) ratio of PCs in these mice appears to be imbalanced, resulting in smaller action potential variability and loss of temporal fidelity of PC responses to parallel fiber stimulation. Furthermore, although LTD formation was normal in these mice, the vestibulo ocular reflex was impaired 33 . Eyeblink conditioning was also found to be impaired in the same mice 34 . Ngsk Prnp 0/0 mice exhibited not only impaired LTD but also weakened GABA A receptor-mediated inhibition in the cerebellum. Thus, alternatively, the dysfunction of inhibitory synaptic transmission in the molecular layer could be also responsible for the impairment of CR acquisition or timing 35 . IPSCs and long-term potentiation in hippocampal CA1 pyramidal cells of ZrchI Prnp 0/0 mice have been reported to be abnormalities 14 , but the cerebellar LTD has not been clarified in the ZrchI Prnp 0/0 mice. Previous studies suggested PrP-mediated several possible mechanisms underlying the regulation of cerebellar LTD and cellular toxicity 19,20,[36][37][38][39] , e.g., PrP can bind to mGluR1 and modulate its function to prevent irregular Ca 2+ signalling 39 . Because mGluR1 expressed in PCs is essential for both LTD induction and eyeblink www.nature.com/scientificreports/ conditioning 28,40,41 , the deficiency in PrP-mediated regulation of mGluR1 may be responsible for the impaired cerebellar plasticity in Ngsk Prnp 0/0 mice. Histological changes in Ngsk Prnp 0/0 mice occur at an age of approximately 40 weeks 42 , and a molecular mechanism underlying neuronal degeneration induced by ectopic expression of PrPLP/Dpl has not been identified, although some hypotheses have been suggested [43][44][45][46] . In the present study, we found that IPSCs in cerebellar PCs were altered in Ngsk Prnp 0/0 mice, whereas a previous report showed different results using the ZrchI Prnp 0/0 mouse cerebellum 15 . The discrepancies in IPSCs between the two types of the mutant mice can be explained by ectopic expression of PrPLP/Dpl in Ngsk Prnp 0/0 mice. Our result is rather similar to that the ZrchI Prnp 0/0 mouse hippocampus exhibits a reduction in GABA A receptor-mediated fast inhibition, suggesting that PrP C plays a key role in normal inhibitory postsynaptic function 14 and implies that excessive excitement of PCs induced by suppressing inhibitory inputs via ectopic overexpression of PrPLP/Dpl inhibits the maintenance of PCs in these Ngsk Prnp 0/0 mice. Thus, impairment of CR acquisition in old Ngsk Prnp 0/0 mice could be caused by a www.nature.com/scientificreports/ secondary effect of PrPLP/Dpl overexpression, particularly the loss of cerebellar PCs. Figure 5 illustrates such a schematic model for age-dependent alterations of delay eyeblink conditioning in Ngsk Prnp 0/0 mice, from the present study and previous reports 10 .
There is at least one other possible explanation for alteration of eyeblink conditioning in young Ngsk Prnp 0/0 mice. Since normal prion protein is expressed on all neuronal cell types in the cerebellum 11 , alterations in GABA A receptor-mediated IPSCs could be true in other types of cerebellar neurons other than Purkinje cells. Thus, GABAergic transmission at Golgi-granule cell synapses may be modified in PrP C -null mice. Considering the timing model described in previous studies 47,50,51 , functional alterations in synaptic transmission between Golgi cells and granule cells may be a factor causing changes in learning-dependent CR timing in young Ngsk and ZrchI Prnp 0/0 mice. Their altered CR timing may be attributed to abnormal regulation of the granule cells by Golgi cells (Fig. 5b). This explanation is consistent with a previous immunohistochemical study indicating that PrP C is most highly expressed in the axon terminals of granule cells in the cerebellum 52 . Therefore, the physiological properties of granule cells in Ngsk Prnp 0/0 mice should be further examined. Furthermore, because aberrant timing of eyeblink conditioning was also observed in Zrch I mice 10 , it is important to investigate the physiology of granule cells in Zrch III mice on a pure C57BL/6J genetic background for future studies 53 .
An appropriate E/I balance is essential for normal adult brain function 54,55 . Thus E/I imbalance affects the normal function and disrupts synchronization between various circuit elements, and can cause autism spectrum disorder (ASD) 56,57 , schizophrenia 58,59 , and Alzheimer's disease (AD) 60 . Indeed, several studies on their model mice have elaborated on the correlation between decreased IPSCs and neuronal death 61,62 . In most human prion diseases, including Creutzfeldt-Jakob disease (CJD), neuronal loss in the cerebellum and abnormal PrP deposition are major neuropathological findings. Furthermore, epileptic-like symptoms or abnormal waves in EEG are often observed in patients with CJD [63][64][65] . Therefore, the Ngsk Prnp 0/0 mouse model may help in studying the mechanisms underlying synaptic loss and neurodegeneration in the cerebellum resulting from the loss of PrP c and ectopic expression of PrPLP/Dpl 66 .

Materials and methods
Subjects. Ngsk Prnp 0/0 mice were obtained as described previously 36 . Male F3 Ngsk Prnp 0/0 mice were crossed with female C57BL/6J mice (purchased from CLEA Japan, Tokyo, Japan), producing F4 heterozygous mice (Prnp +/0 mice). The mutant mice (Ngsk Prnp 0/0 ) and their littermate controls (Ngsk Prnp +/+ ) were derived by inter-crossing F4 Ngsk Prnp +/0 male and female mice. Their genotypes were confirmed by polymerase chain reaction (PCR) amplification of genomic DNA extracted from the tail of each mouse using specific primers for the mouse PrP gene in a 346-base pair PCR fragment (5′-CCG CTA CCC TAA CCA AGT GT-3′ and 5′-CCT AGA CCA CGA GAA TGC GA-3′) and neomycin-resistant gene (5′-GGT GCC CTG AAT GAA CTG CA-3′ and 5′-GGT www.nature.com/scientificreports/ AGC CGG ATC AAG CGT AT-3′), resulting in a 227-base pair PCR fragment). All subjects were maintained on a 12-h:12-h dark: light cycle with food and water available ad libitum. All animal procedures were performed in accordance with the guidelines for animal experimentation from the ethical committee of The University of Tokyo and Tokushima Bunri University. The experimental protocol was approved by the guidelines for the care and use of experimental animals in the animal investigation committee at Tokushima Bunri University, and the animal welfare committees of The University of Tokyo. In addition, the minimum number of required animals was used for these experiments, and efforts were made to minimize pain.
Electrophysiology. Cerebellar slices from 3-6-week-old Ngsk Prnp +/+ (control) and Ngsk Prnp 0/0 mice were prepared as described previously 67  Eyeblink conditioning. For the behavioural study, 7-8-week-old Ngsk Prnp +/+ (control) and Ngsk Prnp 0/0 male mice were used. The surgery was performed as described previously 32,68,69 . The mice were anesthetized with ketamine (80 mg/kg, i.p. Sankyo, Tokyo, Japan) and xylazine (20 mg/kg, i.p. Bayer, Tokyo, Japan). Four Tefloncoated stainless-steel wires (100 µm in diameter, A-M Systems, WA, USA) were implanted subcutaneously under the left eyelid. Two of the wires were used to deliver the US and the remaining two to record an electromyogram (EMG) from the musculus orbicularis oculi, which is responsible for eyelid closure. Here, we modified the conventional EMG procedure to improve the sensitivity for detecting MOO activities. The mice were trained in delay eyeblink conditioning, in which the CS overlaps and coterminates with the US, for seven days. A tone of 352 ms duration (1 kHz, 80 dB) was used as CS and electrical shock with 100 ms duration (100 Hz square pulses) as US. The US intensity was carefully determined, and the minimal current amplitude required to elicit an eyeblink response with constant amplitude was adjusted daily for each animal (less than 0.5 mA). Experiments were conducted during the light phase of the LD cycle in a container (10 cm in diameter) placed in a sound-and lightattenuating chamber. Daily acquisition training consisted of 100 trials grouped in 10 blocks. Conditioning sessions consisted of 10 CS-only (every 10th trial) and 90 CS-US paired trials. The CR amplitude was calculated as the average amplitude over the 50 ms period just before the US. Data were analyzed as described previously 10,30 .
Statistical analysis. All data and samples were analyzed by an individual blinded to the genotype. Unpaired t-tests or the Mann-Whitney test were used. Data for eyeblink conditioning were analyzed by the two-way repeated measures ANOVA to assess the effects of genotype and/or session. The difference was considered significant when the P value was less than 0.05. Tests were performed using Excel or GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA). All data are displayed as mean ± standard error of the mean (SEM).