Controlling auditory excitability: the benefits of a cultured environment

Auditory brainstem neurons, such as those of the medial nucleus of the trapezoid body (MNTB) of rodents, fire action potentials at hundreds of hertz with high temporal precision. In response to physiological changes in the auditory environment, these neurons, and their counterparts in other species, rapidly adjust their potassium channels. In the seconds and minutes following the onset of a sound stimulus, changes in potassium current occur in response to activation of protein kinases/phosphatases (Song et al. 2005). Within 30 min of physiological auditory stimulation, changes in net levels of potassium channel proteins can be detected, most likely mediated by alterations of protein synthesis (Strumbos et al. 2010). Loss of auditory inputs also produces changes in levels of potassium channel subunits that are evident within 3 h (Lu et al. 2004). More prolonged lack of cochlear input produces changes in levels of ion channels that can differ substantially from those occurring in response to transient changes in activity (Leao et al. 2004; Lu et al. 2004; Steinert et al. 2008). Thus far most studies of changes in channel expression have focused on alterations that occur in the intact brain. Although such studies are absolutely essential for establishing the physiological relevance of changes in excitability, they suffer from the disadvantage that the cellular and molecular mechanisms of the observed changes are generally impossible to test rigorously using intact animals. In this issue of The Journal of Physiology, Tong et al. (2010) report results of experiments on MNTB principal neurons maintained in organotypic culture for periods of 7–28 days. In these cultures, the presynaptic calyx that provides the major excitatory synaptic input is lost. To stimulate these neurons, some cultures were maintained in a medium-containing a high level of potassium (25 mm, high-K), which tonically depolarizes the neurons. Other cultures were kept in a low-potassium medium (5 mm, low-K), which corresponds to a lack of neuronal stimulation. This allowed them to characterize activity-dependent changes in potassium currents and in channel mRNAs, and to test the roles of specific biochemical pathways using pharmacological agents. MNTB neurons express several classes of voltage-dependent potassium channels. Two in particular regulate MNTB neuron excitability. ‘Low-threshold’ Kv1-family channels contribute to the resting potential and limit the number of action potentials evoked by sustained depolarizing current pulses. ‘High-threshold’ Kv3-family channels repolarize action potentials and are required for high frequency firing. It was found that both types of currents were enhanced in the high-K cultures over those in low-K cultures, and that action potential responses in the high-K cultures resemble those recorded in acutely prepared slices. Cytoplasmic calcium is a key player in the observed changes. Neurons in high-K cultures have higher basal calcium levels and the increases in potassium currents were reduced by pretreatment with blockers of voltage-dependent calcium channels or by blockers of intracellular calcium release. The intracellular calcium store blockers had a preferential effect on the high-threshold potassium currents. Elevations of cytoplasmic calcium levels trigger many signalling pathways, including the transcription factor CREB (cyclic AMP response element-binding protein). CREB is activated by phosphorylation following elevations of calcium or cyclic AMP, and alters the transcription of specific genes whose DNA contains sequences termed cAMP response elements (CRE). CREB is widely recognized as a factor required for some forms of learning and memory (Carew, 1996), but was originally described as an activator of transcription of neuropeptide genes during secretion. Consistent with the idea that CREB plays some role in the alterations of excitability, high-K MNTB cultures were found to have higher levels of phosphorylated CREB than the low-K cultures. Treatment with a CREB antagonist reduced the high threshold K+ current in the high-K treated cultures, producing a broadening of action potentials. To test the hypothesis of a calcium-dependent change in the transcription of specific genes, the study examined levels of mRNAs for both low threshold (Kv1.1 and Kv1.2) and high threshold (Kv3.1 and Kv3.3) potassium channels, as well as those for the c-fos gene, whose transcription is often used as a marker for enhanced neuronal activity. An increase in mRNA for the Kv1.1 channel was found in high-K cultures, although this was not blocked by the CREB antagonist and, paradoxically, was increased by a blocker of calcium influx. More consistent with a role for the CREB pathway, however, levels of mRNA for Kv3.3 were increased in high-K cultures and reduced by the CREB antagonist. Interestingly, however, mRNAs for the two genes, c-fos and Kv3.1, that have been demonstrated experimentally to be bona fide targets of CREB, and for which biochemical experiments have established direct binding of CREB to their promoter DNA, were unchanged in high-K over low-K cultures, although levels of c-fos mRNA were reduced by pretreatment with the CREB antagonist. The finding that different RNAs are altered in different ways by prolonged depolarization should not be surprising. Activity-dependent neuronal plasticity that may underlie learning and memory, and which relies on the CREB pathway, occurs in short-term, intermediate-term and long-term forms, each of which involve different cellular signalling pathways (Stough et al. 2006). As suggested by studies with intact brains (Lu et al. 2004), a persistent change in neuronal activation of auditory neurons is likely to trigger multiple parallel pathways that control channel expression and that have different time courses. The use of cultured preparations will allow detailed pharmacological and molecular dissection of these pathways.