The effect of SK channel modulators on the simple spike firing frequency in discharge of cerebellar Purkinje cells in laboratory mice

The activity of cerebellar Purkinje cells is studied as affected by CyPPA, a positive modulator of small-conductance calcium-activated potassium channels type 3 and 2 (SK3/SK2), and NS309, an activator of small- and intermediate-conductance calcium-activated potassium channels (IK/SK), in male two-month-old laboratory mice. CyPPA decreases the simple spike firing frequency in the discharge of Purkinje cells by an average of 25% 1 hour after application of 1mM of the compound. An application of 100 μM of NS309 reduces the simple spike firing frequency by an average of 47% during the same period. These results confirm the hypothesis that SK channels may be involved in the downregulation of simple spike firing frequency in Purkinje cells. The frequency-regulating effect of NS309 is stronger, suggesting that IK/SK channels play a decisive role in the regulation of Purkinje cell spiking activity. Since an increase of simple spike firing frequency in these cells is symptomatic of many locomotor activity disorders, e.g., spinocerebellar ataxia, the substances studied or their functional analogues might be of medicinal interest.     

Changes in dendritic axial resistance alter synaptic integration in cerebellar Purkinje cells

The ability of neurons to process synaptic inputs depends critically on their passive electrical properties. The intracellular resistivity, R_i, is one of the parameters that determine passive properties, yet few experiments have explored how changes in R_i might affect synaptic integration. In this work, I addressed this issue by using targeted dendritic occlusion to locally increase R_i in cerebellar Purkinje cells and examining the consequences of this manipulation for the summation of synaptic inputs. To achieve dendritic occlusion, I used two glass micropipettes to gently pinch the dendritic trunk close to the soma. This pinching produced stereotypical changes in the responses to test pulses applied at the soma under voltage and current clamp. A simple model confirmed that these changes were due to increases in R_i in the dendritic trunk. These localized increases in R_i produced striking alterations in the shapes of postsynaptic potentials at the soma, increasing their amplitude and accelerating their decay kinetics. As a consequence, dendritic occlusion sharpened temporal precision during the summation of synaptic inputs. These findings highlight the importance of local changes in intracellular resistivity for the passive electrical properties of neurons, with implications for their ability to process synaptic information.     

Signal processing in cerebellar Purkinje cells

Mechanisms and functional implications of signal processing in cerebellar Purkinje cells have been the subject of recent extensive investigations. Complex patterns of their planar dendritic arbor are analysed with computer-aided reconstructions and also topological analyses. Local computation may occur in Purkinje cell dendrites, but its extent is not clear at present. Synaptic transmission and electrical and ionic activity of Purkinje cell membrane have been revealed in detail, and related biochemical processes are being uncovered. A special type of synaptic plasticity is present in Purkinje cell dendrites; long-term depression (LTD) occurs in parallel fiber-Purkinje cell transmission when the parallel fibers are activated with a climbing fiber innervating that Purkinje cell. Evidence indicates that synaptic plasticity in Purkinje cells is due to sustained desensitization of Purkinje dendritic receptors to glutamate, which is a putative neurotransmitter of parallel fibers, and that conjunctive activation of a climbing fiber and parallel fibers leads to desensitization through enhanced intradendritic calcium concentration. A microzone of the cerebellar cortex is connected to an extracerebellar neural system through the inhibitory projection of Purkinje cells to a cerebellar or vestibular nuclear cell group. Climbing fiber afferents convey signals representing control errors in the performance of a neural system, and evoke complex spikes in Purkinje cells of the microzone connected to the neural system. Complex spikes would modify the performance of the microzone by producing LTD in parallel fiber-Purkinje cell synapses, and consequently would improve the overall performance of the neural system. The primary function of the cerebellum thus appears to be endowing adaptability to numerous neural control systems in the brain and spinal cord through error-triggered reorganization of the cerebellar cortical circuitry.     

Aging of cerebellar Purkinje cells

Cerebellar Purkinje cells (PCs), the sole output neurons in the cerebellar cortex, play an important role in the cerebellar circuit. PCs appear to be rather sensitive to aging, exhibiting significant changes in both morphology and function during senescence. This article reviews such changes during the normal aging process, including a decrease in the quantity of cells, atrophy in the soma, retraction in the dendritic arborizations, degeneration in the subcellular organelles, a decline in synapse density, disorder in the neurotransmitter system, and alterations in electrophysiological properties. Although these deteriorative changes occur during aging, compensatory mechanisms exist to counteract the impairments in the aging PCs. The possible neural mechanisms underlying these changes and potential preventive treatments are discussed.     

Cellular and molecular control of dendritic growth and development of cerebellar Purkinje cells

Purkinje cells are the principal neurons of the cerebellar cortex and are characterized by a large and highly branched dendritic tree. For this reason, they have for a long time been an attractive model system to study the regulation of dendritic growth and differentiation. In this article, I will first review studies on different aspects of Purkinje cell dendritic development and then go on to present studies which have aimed at experimentally altering Purkinje cell dendritic development. Some of the cellular and molecular mechanisms which have been shown by these studies to be important determinants of Purkinje cell dendritic development will be discussed, in particular the role of the parallel fiber input, of hormones, and of neuronal growth factors. The organotypic slice culture method will be introduced as an important experimental tool to study Purkinje cell dendritic development under controlled conditions. Using cerebellar slice cultures, protein kinase C (PKC) has been identified as a major determinant of Purkinje cell dendritic development and the contribution of specific isoforms of PKC will be discussed. Finally, it will be shown that Purkinje cell dendritic development in slice cultures does not depend on the activation of glutamate receptors and appears to be independent of the presence of the neurotrophin BDNF. These studies indicate that the initial outgrowth of the Purkinje cell dendritic tree can occur in the absence of signals derived from afferent fibers, but is under control of PKC signaling.     

Cre recombinase expression in cerebellar Purkinje cells

The cerebellar cortex and its sole output, the Purkinje cell, have been implicated in motor coordination, learning and cognitive functions. Therefore, the ability to generate Purkinje cell-specific mutations in physiologically relevant genes is of particular neurobiological interest. A suitable approach is the Cre/loxP strategy that allows temporally and spatially controlled gene inactivation. Here, we present the characterization of transgenic mouse strains expressing Cre recombinase controlled by the L7/pcp-2 gene. Endogenous L7/pcp-2 protein is expressed exclusively in Purkinje cells and retinal bipolar neurones. Recombination was detected by beta-galactosidase histochemistry in tissues from crosses of the L7/pcp-2:Cre transgenic lines with two different indicator strains, GtROSA26 and ACZL. Purkinje cells in all folia of the cerebellum displayed intense beta-galactosidase staining, whereas only few blue cells were observed in the retina and other parts of the CNS. Thus, these transgenic lines are potentially of great importance for genetic manipulations in cerebellar Purkinje cells.     

Junctophilin-mediated channel crosstalk essential for cerebellar synaptic plasticity

Functional crosstalk between cell-surface and intracellular ion channels plays important roles in excitable cells and is structurally supported by junctophilins (JPs) in muscle cells. Here, we report a novel form of channel crosstalk in cerebellar Purkinje cells (PCs). The generation of slow afterhyperpolarization (sAHP) following complex spikes in PCs required ryanodine receptor (RyR)-mediated Ca(2+)-induced Ca(2+) release and the subsequent opening of small-conductance Ca(2+)-activated K(+) (SK) channels in somatodendritic regions. Despite the normal expression levels of these channels, sAHP was abolished in PCs from mutant mice lacking neural JP subtypes (JP-DKO), and this defect was restored by exogenously expressing JPs or enhancing SK channel activation. The stimulation paradigm for inducing long-term depression (LTD) at parallel fiber-PC synapses adversely established long-term potentiation in the JP-DKO cerebellum, primarily due to the sAHP deficiency. Furthermore, JP-DKO mice exhibited impairments of motor coordination and learning, although normal cerebellar histology was retained. Therefore, JPs support the Ca(2+)-mediated communication between voltage-gated Ca(2+) channels, RyRs and SK channels, which modulates the excitability of PCs and is fundamental to cerebellar LTD and motor functions.     

RGS8 expression in developing cerebellar Purkinje cells

The regulators of G protein signaling (RGS) proteins modulate heterotrimeric G protein signaling. RGS8 belongs to B/R4 subfamily of RGS proteins and is specifically expressed in Purkinje cells of adult cerebellum. Here, to examine the expression of RGS8 mRNA in developing cerebellum, we performed in situ hybridization. Apparent signals for expression of RGS8 mRNA were first detected on day 9 after birth, then RGS8 mRNA expression in Purkinje cells increased up to day 21, and its levels decreased to some extent in adult Purkinje cells. We also studied the expression of RGS7, which is expressed in Golgi cells in the granule cell layer of adult cerebellum. The expression of RGS7 mRNA was recognized in 7 day neonatal cerebellum. When examined with anti-RGS8 antibody, the RGS8 protein was already excluded from nucleus on day 9, and was distributed in cell body and dendrites in differentiating Purkinje cells of 14 day neonates.     

Electrical activity in cerebellar cultures determines Purkinje cell dendritic growth patterns.

In primary dissociated cultures of mouse cerebellum a number of Purkinje cell-specific marker proteins and characteristic ionic currents appear at the appropriate developmental time. During the first week after plating, Purkinje cell dendrites elongate, but as electrical activity emerges the dendrites stop growing and branch. If endogenous electrical activity is inhibited by chronic tetrodotoxin or high magnesium treatment, dendrites continue to elongate, as if they were still immature. At the time that branching begins, intracellular calcium levels become sensitive to tetrodotoxin, suggesting that this cation may be involved in dendrite growth. Even apparently mature Purkinje cells alter their dendritic growth in response to changes in activity, suggesting long-term plasticity.     

Axonal and dendritic transport in Purkinje cells of cerebellar slice cultures studied by microinjection of horseradish peroxidase

Axonal and dendritic transport in single Purkinje neurons of cerebellar slice cultures was quantified as single transport distances. Examination of the cells within a vital tissue was regarded as being an approach to the in situ condition. The Purkinje cells were organotypically integrated in the in vitro tissues and extended long axonal projections connecting synapses to the target neurons. The tracer horseradish peroxidase (HRP) was applied via microinjection to the somata of the Purkinje cells and the injected neurons were incubated thereafter for defined time-intervals. The tracer was transported anterogradely into the neuron processes. The measurements on both the axonal and the dendritic transport of microinjected HRP revealed continuous transportation with increasing times of postincubation. This transport was reduced by the use of microtubule-depolymerizing drugs. The axonal transport of the tracer was either retarded in colchicine-treated cells or continuously reduced for up to 50% in vinblastine-treated neurons. Thus, a correlation of axonal transport to the microtubules was demonstrated. The dendrites were filled with the tracer after 60 min of postincubation. Dendritic transport was reduced by the use of vinblastine, and not significantly by colchicine. The results strongly support the dependence of neuronal transport on microtubules as a component of the cytoskeleton. Received: 26 April 1998 / Accepted: 18 August 1998     

Abnormal features in mutant cerebellar Purkinje cells lacking junctophilins

Junctional membrane complexes (JMCs) generated by junctophilins are required for Ca(2+)-mediated communication between cell-surface and intracellular channels in excitable cells. Knockout mice lacking neural junctophilins (JP-DKO) show severe motor defects and irregular cerebellar plasticity due to abolished channel crosstalk in Purkinje cells (PCs). To precisely understand aberrations in JP-DKO mice, we further analyzed the mutant PCs. During the induction of cerebellar plasticity via electrical stimuli, JP-DKO PCs showed insufficient depolarizing responses. Immunochemistry detected mild impairment in synaptic maturation and hyperphosphorylation of protein kinase Cgamma in JP-DKO PCs. Moreover, gene expression was slightly altered in the JP-DKO cerebellum. Therefore, the mutant PCs bear marginal but widespread abnormalities, all of which likely cause cerebellar motor defects in JP-DKO mice.     

Cloning and characterization of SK2 channel from chicken short hair cells

In the inner ear of birds, as in mammals, reptiles and amphibians, acetylcholine released from efferent neurons inhibits hair cells via activation of an apamin-sensitive, calcium-dependent potassium current. The particular potassium channel involved in avian hair cell inhibition is unknown. In this study, we cloned a small-conductance, calcium-sensitive potassium channel (gSK2) from a chicken cochlear library. Using RT-PCR, we demonstrated the presence of gSK2 mRNA in cochlear hair cells. Electrophysiological studies on transfected HEK293 cells showed that gSK2 channels have a conductance of approximately 16 pS and a half-maximal calcium activation concentration of 0.74±0.17 M. The expressed channels were blocked by apamin (IC₅₀=73.3±5.0 pM) and d-tubocurarine (IC₅₀=7.6±1.0 M), but were insensitive to charybdotoxin. These characteristics are consistent with those reported for acetylcholine-induced potassium currents of isolated chicken hair cells, suggesting that gSK2 is involved in efferent inhibition of chicken inner ear. These findings imply that the molecular mechanisms of inhibition are conserved in hair cells of all vertebrates.     

Lurcher GRID2-induced death and depolarization can be dissociated in cerebellar Purkinje cells

The Lurcher mutation transforms the GRID2 receptor into a constitutively opened channel. In Lurcher heterozygous mice, cerebellar Purkinje cells are permanently depolarized, a characteristic that has been thought to be the primary cause of their death, which occurs from the second postnatal week onward. The more dramatic phenotype of Lurcher homozygotes is thought to be due to a simple gene dosage effect of the mutant allele. We have analyzed the phenotype of Lurcher/hotfoot heteroallelic mutants bearing only one copy of the Lurcher allele and no wild-type Grid2. Our results show that the absence of wild-type GRID2 receptors in these heteroallelic mutants induces an early and massive Purkinje cell death that is correlated with early signs of autophagy. This neuronal death is independent of depolarization and can be explained by the direct activation of autophagy by Lurcher GRID2 receptors through the recently discovered signaling pathway formed by GRID2, n-PIST, and Beclin1.     

Expression of the yes proto-oncogene in cerebellar Purkinje cells.

To identify the kinds of cells in the brain that express the yes proto-oncogene, we examined chicken brains by using immunofluorescent staining and in situ hybridization. Both approaches showed that the highest level of the yes gene product was in cerebellar Purkinje cells. In addition, we analyzed Purkinje cell degeneration (pcd) mutant mice. The level of yes mRNA in cerebella of pcd mutants was four times lower than that found in cerebella of normal littermates. Our studies point to Purkinje cells as an attractive model for functional studies of the yes protein.     

Cell lineage analysis of cerebellar Purkinje cells in mouse chimeras

Murine chimeras provide an experimental system in which cell lineage analysis of the mammalian central nervous system (CNS) can be accomplished. Utilizing a cell marker system that permits the identification of cells of each genotype in various cell populations present in histologic sections of the CNS at different developmental periods, fate maps of the mammalian CNS can be constructed. Thus, the presence or persistence of clones of cells can be readily visualized in simply organized CNS regions, like the cerebellar cortex. The electrophoretic variants of the glycolytic enzyme, glucosephosphate isomerase (GPI, E.C. 5.3.1.9; GPI-1A, GPI-1B), are the genotype-specific cell markers most commonly used by experimental mammalian embryologists in studies of cell lineage utilizing mammalian chimeras. We have adapted this cell marker system to permit the visualization and unequivocal identification of cells containing the GPI-1B variant throughout the CNS of adult $\text{BALB}\text{cBy}{}^{\mathrm{J}}\text{a3}\text{C57BL}\text{6J}$ chimeric mice. Utilizing allozyme-specific anti-GPI-1B antisera in immunocytochemical (PAP) staining techniques, we can score small as well as large cell populations, neurons as well as glia. We have reconstructed and statistically analyzed the location and distribution of chimerism present in the Purkinje cell population of four of these chimeric mice. We found the Purkinje cells in each of these animals existed as small (3–8) cell patches of like genotype that were not randomly arranged. This suggests that clones of cells may persist as contiguous groups of cells throughout mammalian cerebellar development.     

The acid-activated ion channel ASIC contributes to synaptic plasticity, learning, and memory

Many central neurons possess large acid-activated currents, yet their molecular identity is unknown. We found that eliminating the acid sensing ion channel (ASIC) abolished H(+)-gated currents in hippocampal neurons. Neuronal H(+)-gated currents and transient acidification are proposed to play a role in synaptic transmission. Investigating this possibility, we found ASIC in hippocampus, in synaptosomes, and in dendrites localized at synapses. Moreover, loss of ASIC impaired hippocampal long-term potentiation. ASIC null mice had reduced excitatory postsynaptic potentials and NMDA receptor activation during high-frequency stimulation. Consistent with these findings, null mice displayed defective spatial learning and eyeblink conditioning. These results identify ASIC as a key component of acid-activated currents and implicate these currents in processes underlying synaptic plasticity, learning, and memory.     

The hyperpolarization-activated HCN1 channel is important for motor learning and neuronal integration by cerebellar Purkinje cells

In contrast to our increasingly detailed understanding of how synaptic plasticity provides a cellular substrate for learning and memory, it is less clear how a neuron's voltage-gated ion channels interact with plastic changes in synaptic strength to influence behavior. We find, using generalized and regional knockout mice, that deletion of the HCN1 channel causes profound motor learning and memory deficits in swimming and rotarod tasks. In cerebellar Purkinje cells, which are a key component of the cerebellar circuit for learning of correctly timed movements, HCN1 mediates an inward current that stabilizes the integrative properties of Purkinje cells and ensures that their input-output function is independent of the previous history of their activity. We suggest that this nonsynaptic integrative function of HCN1 is required for accurate decoding of input patterns and thereby enables synaptic plasticity to appropriately influence the performance of motor activity.     

Responses of cerebellar Purkinje cells to mechanical stimulation of the Achilles' tendon

Responses of cerebellar Purkinje cells to mechanical stimulation of the Achilles' tendon were studied in unanesthetized decerebrate cats. Approximately two-thirds of the Purkinje cells tested were activated in response to stimulation through climbing fibers, i.e., they generated a complex spike. In half of these cells (group A) the probability of appearance of a complex spike to a blow on the tendon was from 0.5 to 0.9 and the latent period of response from 20 to 25 msec. Purkinje cells with a latent period of response of over 35 msec were characterized by low probability of response (under 0.5) to a tap (group B). Responses of Purkinje cells to excitation of mossy fibers were weaker and more varied.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 13, No. 2, pp. 159–167, March–April, 1981.