Calcium extrusion mechanisms and dendritic development of cerebellar Purkinje cells

The cerebellum plays a vital role in motor learning and refining the motor activity. Many cell components in the cerebellum synchronise and process motor function, with Purkinje cells and granule cells being the key players in cerebellar circuitry. In particular, Purkinje cells are the principal neurons of the cerebellar cortex and are well known for their profuse and elaborate dendritic arbour.
Previously, our lab has shown that the activity of Protein kinase C and the metabotropic glutamate receptor mGluR1 severely inhibits growth and development of Purkinje cell dendritic arbour (Metzger and Kapfhammer, 2000; Schrenk et al., 2002; Sirzen-Zelenskaya et al., 2006). The dendritic reduction caused by the activation of mGluR1 and PKC is partially rescued by the blockade of P⁄Q and T-type of Ca2+ channels (Gugger et al., 2012). Besides the Ca2+ influx, Ca2+ extrusion mechanisms also play an important role in Purkinje cell dendritic development (Huang et. al., 2010; Kim et al., 2007; Chris et al., 2013).
In my thesis, I have studied two plasma membrane antiporters that are involved in modulating calcium equilibrium in Purkinje cell dendritic development. The plasma membrane Ca2+-ATPase2 (PMCA2) is involved in the extrusion of calcium and cerebellar synapse function. Of the 4 known PMCA variants, PMCA1 and PMCA4 are expressed ubiquitously whereas PMCA2 and PMCA3 are expressed prevalently in the central nervous systems. The PMCA2 isoform is highly expressed in the cerebellum, particularly in Purkinje cell dendrites and dendritic spines. By immunohistochemistry, we confirmed that PMCA2 immunoreactivity (IR) was strongly expressed at the dendritic plasma membrane and in dendritic spines of Purkinje cells. The chronic functional inhibition of PMCA2 by carboxyeosin in cerebellar slice cultures resulted in a slight reduction of the Purkinje cell dendritic arbor. On the other hand, chronic activation of mGluR1 by DHPG induced a strong reduction of the Purkinje cell dendritic tree. With co-treatment of an mGluR1 agonist and PMCA2 antagonist, PMCA2 functional inhibition surprisingly had a partial rescuing effect for the DHPG induced reduction of Purkinje cell dendritic development, indicating that PMCA2 plays an important role in calcium homeostasis controlling Purkinje cell dendritic growth and development. These finding suggest that PMCA2 is important for the maintenance and control of the calcium equilibrium in developing Purkinje cell dendrites and that this equilibrium is critical for the control of the dendritic growth and expansion.
The Na+/Ca2+ exchanger (NCX) is another calcium extrusion mechanism in Purkinje cells that mediates Ca2+ and Na+ fluxes across the plasma membrane in a so-called bi-directional mode; the forward mode and the reverse mode. NCX has three isoforms, NCX1–3, and all of them are expressed in Purkinje cells. NCX2 and NCX3 are predominantly expressed in the Purkinje cell soma, but NCX1 being the most abundant was expressed in the cell soma, stem dendrite and distal dendrites. The expression of NCX was not only restricted to Purkinje cells, but ubiquitous in the cerebellum particularly in the cytoplasm of cerebellar granule cells and molecular interneurons like basket cells and stellate cells.
The pharmacological blockade of the forward mode of NCX (Ca2+ efflux mode) by bepridil moderately inhibited growth and development of the Purkinje cell dendritic arbor in cerebellar slice cultures. However, the blockade of the reverse mode (Ca2+ influx mode) by KB-R7943 severely reduced the dendritic arbour and induced a morphological change with thickened distal dendrites. We used a number of additional NCX inhibitors like CB-DMB, ORM-10103, SEA0400, YM-244769 and SN-6 which have higher specificity for NCX isoforms and target either the forward, reverse or both modes. All these inhibitors produced a strong dendritic reduction similar to that seen with KB-R7943 without producing thickened distal dendrites. This indicates that effect of KB-R7943 on the thickened distal dendritic morphology was fairly unspecific and unrelated to the function of NCX. When KB-R7943 was combined with the antagonists of voltage gated calcium channels, the dendritic reduction was consistent and also apparent in the absence of bioelectrical activity indicating that it was mediated by NCX expressed in Purkinje cells. The pharmacological treatments also seem to have affected the morphology and number of dendritic spines on the Purkinje cell dendritic arbor.
Our findings indicate that the disturbance of the NCX-dependent calcium transport in Purkinje cells induces changes in the calcium handling of Purkinje cells causing dendritic reduction. Further, they underline the importance of the calcium equilibrium for the development and growth of the dendritic arbour in cerebellar Purkinje cells.