The heterogeneity of neural gene expression and the spatially limited expression of many low-abundance messenger RNAs in the brain has made cloning and analysis of such messages difficult. To generate amounts of nucleic acids sufficient for use in standard cloning strategies, we have devised a method for producing amplified heterogeneous populations of RNA from limited quantities of cDNA. Whole cerebellar RNA was primed with a synthetic oligonucleotide containing the T7 RNA polymerase promoter sequence 5' to a polythymidyate region. After second-strand cDNA synthesis,

Voltage-sensitive K+ channels were studied in rat cerebellar Purkinje neurons in culture using the single-channel recording technique. Recordings in the cell-attached and outside-out configuration revealed multiple voltage-sensitive K+ channel types in patches from both the somatic and the dendritic regions. K+ channel types were present in all patches studied. The same channel types were observed in somatic and dendritic recordings. Channel types were identified by reversal potential, single-channel conductance, voltage sensitivity, and patterns of activity. In cell-attached patches recorded under physiological conditions, 3 channel types were identified. Mean single-channel conductances were 92, 57, and 12 pS. All 3 channel types were activated by membrane depolarization. Similar channel types were identified in inside-out and outside-out patches recorded under physiological conditions. Two additional channel types were identified in the outside-out patches, with mean single-channel conductances of 41 and 26 pS. In cell-attached recordings under symmetrical K+ conditions, 6 channel types were identified. Mean single-channel conductances were 222, 134, 39, 25, 14, and 15 pS. Channel types with mean conductances of 222, 134, and 39 pS required membrane depolarization for activation. A comparison of channel properties indicated that these channel types correlated with the 3 channel types observed in cell-attached patches under physiological conditions. The 3 smaller-conductance channel types (25, 14, and 15 pS) were active at potentials around rest or at hyperpolarized membrane potentials. Two K+ channel types (39 and 25 pS) were commonly associated with the late phase of extracellularly recorded spontaneous spike events, suggesting a functional role in the repolarizing phase of somatic and dendritic action potentials. These results demonstrate that voltage-sensitive K+ channels are a prominent component of both the somatic and the dendritic membrane of the cerebellar Purkinje neuron and support the view that multiple voltage-sensitive K+ channel types contribute to the membrane functions of both cellular regions in this CNS neuronal type.

The relationship between calcium conductances and developmental changes in the active and passive membrane properties of cerebellar Purkinje neurons from rats was studied in a culture model system by using current-clamp and voltage-clamp techniques. These cultures, at 6–21 d of age, represented the main period of morphological and physiological development of the Purkinje neuron. In the current-clamp studies, input resistance decreased and the current-voltage curve became more S-shaped as the neurons matured in culture. Spike-generating properties also changed. Immature Purkinje neurons without dendritic structure produced repetitive, fast TTX-sensitive simple spikes when stimulated electrically. The simple spike frequency increased with maturation. In older neurons (greater than or equal to 12 d in vitro) with well- developed dendritic structure, a burst event, the complex spike, preceded the repetitive simple spike firing. Magnesium (10 mM) and cadmium (50–100 microM), calcium channel blockers, antagonized the repetitive simple spike firing in both young and old neurons. The complex spike of the older neurons was also antagonized by magnesium (10 mM) but was resistant to cadmium (50–100 microM), suggesting that a pharmacologically distinct calcium conductance mediated this spike event. Whole-cell voltage-clamp recordings showed that the older Purkinje neurons expressed two calcium currents, a low-threshold rapidly inactivating calcium current resistant to cadmium (50–100 microM) and a high-threshold slowly inactivating calcium current antagonized by cadmium (50–100 microM). In young Purkinje neurons without dendritic structure (6–9 d in vitro), only the high-threshold calcium current was evident. The amplitude of this current increased approximately 50% during development. These results indicate that the developmental expression of calcium conductances plays a prominent role in the physiological maturation of the cultured Purkinje neurons, which closely simulate the physiologic cells they model. The high-threshold calcium conductance is expressed early in development and contributes to repetitive simple spike firing of both the young and old neurons. The low-threshold calcium conductance appears later in development, coincident with dendritic expression, and plays a major role in the generation of the complex spike.

The cerebellar Purkinje neuron cultured from 20 d rat embryos is electrically inexcitable when immature, and acquires excitable membrane properties according to a programmed developmental sequence, thus providing a useful model for investigating mechanisms of CNS neuronal development. Using conventional patch-clamp techniques, we have characterized the the predominant classes of active K+-selective channels at a range of ages encompassing the entire developmental process from 5 to 29 d in vitro (DIV), and have shown pharmacologically that these channels are important contributors to the patterns of spontaneous activity generated by the Purkinje neurons. The 4 predominant classes of K+ channels that are active during steady-state depolarizing voltage commands are identified by unit conductances as the 27, 44, 70, and 100 pS channels, and show differences in several properties, including voltage dependence, sensitivity to tetraethylammonium chloride (TEA), mean open time, and time of appearance during development. Intracellular current-clamp recordings show that physiological maturation of the Purkinje neuron entails increases in the firing rate, the diversity of spike events that comprise spontaneous activity, and the sensitivity of spontaneous activity to disruption by the K+ channel blocker TEA. This increase in sensitivity to TEA correlates with the new expression of activity of the larger-conductance TEA-sensitive classes of K+ channel (70 and 100 pS types). These data show that developmental regulation of the activity of K+-selective channels contributes significantly to the ionic mechanisms that underlie the developmental transitions in spontaneous activity patterns in the Purkinje neuron.