Weixing Shen

Prestin is the motor protein of cochlear outer hair cells.

The outer and inner hair cells of the mammalian cochlea perform different functions. In response to changes in membrane potential, the cylindrical outer hair cell rapidly alters its length and stiffness. These mechanical changes, driven by putative molecular motors, are assumed to produce amplification of vibrations in the cochlea that are transduced by inner hair cells. Here we have identified an abundant complementary DNA from a gene, designated Prestin, which is specifically expressed in outer hair cells. Regions of the encoded protein show moderate sequence similarity to pendrin and related sulphate/anion transport proteins. Voltage-induced shape changes can be elicited in cultured human kidney cells that express prestin. The mechanical response of outer hair cells to voltage change is accompanied by a ‘gating current’, which is manifested as nonlinear capacitance. We also demonstrate this nonlinear capacitance in transfected kidney cells. We conclude that prestin is the motor protein of the cochlear outer hair cell.

D1 and D2 dopamine-receptor modulation of striatal glutamatergic signaling in striatal medium spiny neurons

Dopamine shapes a wide variety of psychomotor functions. This is mainly accomplished by modulating cortical and thalamic glutamatergic signals impinging upon principal medium spiny neurons (MSNs) of the striatum. Several lines of evidence suggest that dopamine D1 receptor signaling enhances dendritic excitability and glutamatergic signaling in striatonigral MSNs, whereas D2 receptor signaling exerts the opposite effect in striatopallidal MSNs. The functional antagonism between these two major striatal dopamine receptors extends to the regulation of synaptic plasticity. Recent studies, using transgenic mice in which cells express D1 and D2 receptors, have uncovered unappreciated differences between MSNs that shape glutamatergic signaling and the influence of DA on synaptic plasticity. These studies have also shown that long-term alterations in dopamine signaling produce profound and cell-type-specific reshaping of corticostriatal connectivity and function.

Dichotomous dopaminergic control of striatal synaptic plasticity.

At synapses between cortical pyramidal neurons and principal striatal medium spiny neurons (MSNs), postsynaptic D1 and D2 dopamine (DA) receptors are postulated to be necessary for the induction of long-term potentiation and depression, respectively—forms of plasticity thought to underlie associative learning. Because these receptors are restricted to two distinct MSN populations, this postulate demands that synaptic plasticity be unidirectional in each cell type. Using brain slices from DA receptor transgenic mice, we show that this is not the case. Rather, DA plays complementary roles in these two types of MSN to ensure that synaptic plasticity is bidirectional and Hebbian. In models of Parkinsons disease, this system is thrown out of balance, leading to unidirectional changes in plasticity that could underlie network pathology and symptoms.

Distinct subclasses of medium spiny neurons differentially regulate striatal motor behaviors

The direct and indirect pathways of the basal ganglia have been proposed to oppositely regulate locomotion and differentially contribute to pathological behaviors. Analysis of the distinct contributions of each pathway to behavior has been a challenge, however, due to the difficulty of selectively investigating the neurons comprising the two pathways using conventional techniques. Here we present two mouse models in which the function of striatonigral or striatopallidal neurons is selectively disrupted due to cell type–specific deletion of the striatal signaling protein dopamine- and cAMP-regulated phosphoprotein Mr 32kDa (DARPP-32). Using these mice, we found that the loss of DARPP-32 in striatonigral neurons decreased basal and cocaine-induced locomotion and abolished dyskinetic behaviors in response to the Parkinsons disease drug L-DOPA. Conversely, the loss of DARPP-32 in striatopallidal neurons produced a robust increase in locomotor activity and a strongly reduced cataleptic response to the antipsychotic drug haloperidol. These findings provide insight into the selective contributions of the direct and indirect pathways to striatal motor behaviors.

Cholinergic modulation of Kir2 channels selectively elevates dendritic excitability in striatopallidal neurons

Dopamine-depleting lesions of the striatum that mimic Parkinsons disease induce a profound pruning of spines and glutamatergic synapses in striatopallidal medium spiny neurons, leaving striatonigral medium spiny neurons intact. The mechanisms that underlie this cell type–specific loss of connectivity are poorly understood. The Kir2 K+ channel is an important determinant of dendritic excitability in these cells. Here we show that opening of these channels is potently reduced by signaling through M1 muscarinic receptors in striatopallidal neurons, but not in striatonigral neurons. This asymmetry could be attributed to differences in the subunit composition of Kir2 channels. Dopamine depletion alters the subunit composition further, rendering Kir2 channels in striatopallidal neurons even more susceptible to modulation. Reduced opening of Kir2 channels enhances dendritic excitability and synaptic integration. This cell type–specific enhancement of dendritic excitability is an essential trigger for synaptic pruning after dopamine depletion, as pruning was prevented by genetic deletion of M1 muscarinic receptors.

Cholinergic Suppression of KCNQ Channel Currents Enhances Excitability of Striatal Medium Spiny Neurons

In response to glutamatergic synaptic drive, striatal medium spiny neurons in vivo transition to a depolarized “up state” near spike threshold. In the up state, medium spiny neurons either depolarize enough to spike or remain below spike threshold and are silent before returning to the hyperpolarized “down state.” Previous work has suggested that subthreshold K+ channel currents were responsible for this dichotomous behavior, but the channels giving rise to the current and the factors determining its engagement have been a mystery. To move toward resolution of these questions, perforated-patch recordings from medium spiny neurons in tissue slices were performed. K+ channels with pharmacological and kinetic features of KCNQ channels potently regulated spiking at up-state potentials. Single-cell reverse transcriptase-PCR confirmed the expression of KCNQ2, KCNQ3, and KCNQ5 mRNAs in medium spiny neurons. KCNQ channel currents in these cells were potently reduced by M1 muscarinic receptors, because the effects of carbachol were blocked by M1 receptor antagonists and lost in neurons lacking M1 receptors. Reversal of the modulation was blocked by a phosphoinositol 4-kinase inhibitor, indicating a requirement for phosphotidylinositol 4,5-bisphosphate resynthesis for recovery. Inhibition of protein kinase C reduced the efficacy of the muscarinic modulation. Finally, acceleration of cholinergic interneuron spiking with 4-aminopyridine mimicked the effects of exogenous agonist application. Together, these results show that KCNQ channels are potent regulators of the excitability of medium spiny neurons at up-state potentials, and they are modulated by intrastriatal cholinergic interneurons, providing a mechanistic explanation for variability in spiking during up states seen in vivo.

FGF acts as a co-transmitter through adenosine A2A receptor to regulate synaptic plasticity

Abnormalities of striatal function have been implicated in several major neurological and psychiatric disorders, including Parkinsons disease, schizophrenia and depression. Adenosine, via activation of A2A receptors, antagonizes dopamine signaling at D2 receptors and A2A receptor antagonists have been tested as therapeutic agents for Parkinsons disease. We found a direct physical interaction between the G protein–coupled A2A receptor (A2AR) and the receptor tyrosine kinase fibroblast growth factor receptor (FGFR). Concomitant activation of these two classes of receptors, but not individual activation of either one alone, caused a robust activation of the MAPK/ERK pathway, differentiation and neurite extension of PC12 cells, spine morphogenesis in primary neuronal cultures, and cortico-striatal plasticity that was induced by a previously unknown A2AR/FGFR-dependent mechanism. The discovery of a direct physical interaction between the A2A and FGF receptors and the robust physiological consequences of this association shed light on the mechanism underlying FGF functions as a co-transmitter and open new avenues for therapeutic interventions.

Dopamine and synaptic plasticity in dorsal striatal circuits controlling action selection

The striatum is thought to play a central role in learning how to choose acts that lead to reward and avoid punishment. Dopamine-dependent modification of striatal synapses in the action selection circuitry has long been thought to be a key step toward this type of learning. The development of new genetic and optical tools has pushed this field forward in the last couple of years, demanding a re-evaluation of models of how experience controls dopamine-dependent synaptic plasticity and how disease states like Parkinsons disease affect the striatal circuitry.

Prestin topology: localization of protein epitopes in relation to the plasma membrane.

Computer modeling of the outer hair cell (OHC) motor protein prestin produces ambiguous results regarding transmembrane regions and localization of its termini. To determine the location of prestins N- and C-termini, we created prestin constructs with synthetic epitopes located immediately upstream or downstream of prestin. The spatial distribution of these epitopes was studied in prestin-transfected cells using immunofluorescence. In permeabilized cells, antibodies label the plasma membrane of 30% of the cells, reflecting transfec- tion efficiency. Under non-permeabilizing conditions, the few labeled cells also displayed a lack of plasma membrane integrity. These data suggest that prestins N-and C-termini are cytoplasmic. Furthermore, prestin staining in OHCs was observed only under permeabilizing conditions. These results implicate prestins N- and C-termini as portions that may interact with other cytoplasmic proteins. A model of prestin membrane topology is also considered based on the results.

Effects of membrane potential and tension on prestin, the outer hair cell lateral membrane motor protein

1 Under whole‐cell voltage clamp, the effects of initial voltage conditions and membrane tension on gating charge and voltage‐dependent capacitance were studied in human embryonic kidney cells (TSA201 cell line) transiently transfected with the gene encoding the gerbil protein prestin. Conformational changes in this membrane‐bound protein probably provide the molecular basis of the outer hair cell (OHC) voltage‐driven mechanical activity, which spans the audio spectrum. 2 Boltzmann characteristics of the charge movement in transfected cells were similar to those reported for OHCs (amax= 0.99 ± 0.16 pC, z = 0.88 ± 0.02; n= 5, means ±s.e.m.). Unlike that of the adult OHC, the voltage at peak capacitance (apkcm) was very negative (‐74.7 ± 3.8 mV). Linear capacitance in transfected cells was 43.7 ± 13.8 pF and membrane resistance was 458 ± 123 MΩ. 3 Voltage steps from the holding potential preceding the measurement of capacitance‐voltage functions caused a time‐ and voltage‐dependent shift in Vpkcm. For a prepulse to ‐150 mV, from a holding potential of 0 mV, Vpkcm shifted 6.4 mV, and was fitted by a single exponential time constant of 45 ms. A higher resolution analysis of this time course was made by measuring the change in capacitance during a fixed voltage step and indicated a double exponential shift (τ0= 51.6 ms, τ1= 8.5 s) similar to that of the native gerbil OHC. 4 Membrane tension, delivered by increasing pipette pressure, caused a positive shift in Vpkcm. A maximal shift of 7.5 mV was obtained with 2 kPa of pressure. The effect was reversible. 5 Our results show that the sensitivity of prestin to initial voltage and membrane tension, though present, is less than that observed in adult OHCs. It remains possible that some other interacting molecular species within the lateral plasma membrane of the native OHC amplifies the effect of tension and prior voltage on prestins activity.