HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 97/1/892    most recent 00626.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (8) Citing Articles via Google Scholar Google Scholar Articles by Best, T. K. Articles by Galdzicki, Z. Search for Related Content PubMed PubMed Citation Articles by Best, T. K. Articles by Galdzicki, Z.

Ts65Dn, a Mouse Model of Down Syndrome, Exhibits Increased GABA_B-Induced Potassium Current

¹Neuroscience Graduate Program, ²Department of Anatomy, Physiology, and Genetics, Uniformed Services University of the Health Sciences School of Medicine, Bethesda, Maryland

Submitted 15 June 2006; accepted in final form 2 November 2006

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Down syndrome (DS) is the most common nonheritable cause of mental retardation. DS is the result of the presence of an extra chromosome 21 and its phenotype may be a consequence of overexpressed genes from that chromosome. One such gene is Kcnj6/Girk2, which encodes the G-protein-coupled inward rectifying potassium channel subunit 2 (GIRK2). We have recently shown that the DS mouse model, Ts65Dn, overexpresses GIRK2 throughout the brain and in particular the hippocampus. Here we report that this overexpression leads to a significant increase (2-fold) in GABA_B-mediated GIRK current in primary cultured hippocampal neurons. The dose response curves for peak and steady-state GIRK current density is significantly shifted left toward lower concentrations of baclofen in Ts65Dn neurons compared with diploid controls, consistent with increased functional expression of GIRK channels. Stationary fluctuation analysis of baclofen-induced GIRK current from Ts65Dn neurons indicated no significant change in single-channel conductance compared with diploid. However, significant increases in GIRK channel density was found in Ts65Dn neurons. In normalized baclofen-induced GIRK current and GIRK current kinetics no difference was found between diploid and Ts65Dn neurons, which suggests unimpaired mechanisms of interaction between GIRK channel and GABA_B receptor. These results indicate that increased expression of GIRK2 containing channels have functional consequences that likely affect the balance between excitatory and inhibitory neuronal transmission.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Mental retardation can be attributed to abnormal neural functioning caused by a spectrum of abnormalities that disrupt CNS function. The cognitive deficits associated with Down syndrome (DS) are the result of expression of genes from the third copy of chromosome (Chr.) 21 and their effects on CNS function. The major neurological DS phenotypes include mental retardation, muscle hypotonia, and appearance of Alzheimer disease neuropathology after age 35 (Antonarakis et al. 2004). Among the genes on Chr. 21 (Hattori et al. 2000) that might contribute to mental retardation in DS are genes located either within or in proximity to the DS critical region (DSCR) (Galdzicki et al. 2001; Toyoda et al. 2002). However, genes outside the DSCR also can be involved in the DS phenotype (Korenberg et al. 1994), and recently the concept and existence of the DSCR has been challenged (Olson et al. 2004). Although much investigation has focused on anatomic, chemical, and metabolic deficiencies in DS and DS mouse models, neurophysiological abnormalities in single neurons derived from segmental trisomy mouse models (Ts65Dn, Ts16Cje and Ts1Rhr) has not been widely investigated. In this study, we investigated electrophysiological properties of cultured Ts65Dn neurons to evaluate the impact that an extra gene copy of a potassium channel subunit (Kcnj6/Girk2) located within common segment of extra Chrs. in Ts65Dn, Ts1Cje, and Ts1Rhr mice has on intrinsic neuronal properties.

The distal segment of mouse Chr. 16 is homologous to nearly the entire long arm of human Chr. 21, thus trisomic mouse models have been generated that genetically model the human condition. Full trisomy 16 and segmental trisomy 16 (Ts65Dn, Ts1Cje, and Ts1Rhr) mice have been developed. Ts65Dn, and Ts1Cje mice mimic many of the behavioral, learning, and developmental deficits characteristics in DS individuals (Galdzicki and Siarey 2003; Galdzicki et al. 2001; Holtzman et al. 1996; Reeves et al. 1995; Sago et al. 1998). Protocols that induce long-term potentiation and depression (LTP and LTD) show abnormal synaptic plasticity in these mice. LTP is decreased, whereas LTD is elevated in Ts65Dn and Ts1Cje hippocampi (Costa and Grybko 2005; Kleschevnikov et al. 2004; Siarey et al. 1997, 1999, 2005). In Girk2 knockout mice, LTP and LTD are also abnormal (Adeniji-Adele 2004) but in the opposite direction, suggesting that the level of GIRK2 expression correlates with the level of potentiation or depression of hippocampal synapses.

Kcnj6, which encodes GIRK subunit 2 of the G-protein-coupled inward rectifying potassium channel (GIRK), is located within the DSCR on human Chr. 21 (Hattori et al. 2000) and likewise within triplicated segments of mouse Chr. 16 from DS mouse models. Mammalian GIRK channels are homo- and hetero-tetramers formed from GIRK1–GIRK4 subunits and are activated by neuromodulators acting on G-protein-coupled receptors (GPCRs). Within the CNS, GIRK1–GIRK3 subunits contribute to the formation of functional GIRK channels with GIRK3 playing a minor role in neuronal membrane potassium conductance (Koyrakh et al. 2005). In the hippocampus, GIRK1 and GIRK2 subunits are primarily localized to postsynaptic compartments and particularly found in peri- and extrasynaptic regions of dendritic spines of CA1 pyramidal neurons (Drake et al. 1997; Koyrakh et al. 2005). Furthermore, tonic GIRK open channel probability increases with dendritic distance from the soma (Chen and Johnston 2005). The postsynaptic location and function of GIRK containing channels signify that these channels play an important role in synaptic function and modulation and that the expression levels would impact intrinsic dendritic properties and the ability of neurons to properly integrate, modulate, and encode excitatory and inhibitory input. Through their contribution to the potassium conductance they can influence resting membrane potentials (Koyrakh et al. 2005; Luscher et al. 1997) and can impede neuronal excitability via shunting and slowing frequency of spike trains (Ehrengruber et al. 1997; Hille 2001).

High hippocampal GIRK expression indicates the importance that GIRK channels may have in modulating learning and memory. Indeed, reports have established that rodents lacking GIRK1 and GIRK4 subunits demonstrate defective learning and memory (Kourrich et al. 2003; Wickman et al. 2000). Our previous study (Harashima et al. 2006) showed that the presence of an extra GIRK2 gene copy in Ts65Dn hippocampus leads to elevation in GIRK2 mRNA. As a consequence, GIRK2 subunit protein was overexpressed in Ts65Dn neurons. Interestingly, GIRK1 subunit protein was also overexpressed, whereas GIRK1 mRNA was normal (Girk1 gene is not localized to the Ts65Dn chromosome). These data strongly suggest that GIRK current should be greater in Ts65Dn hippocampus in comparison to diploid neurons because heterotetramers of GIRK1 and GIRK2 are the most predominant form of GIRK channel in CNS neurons. The physiological impact of GIRK1-2 channel subunit overexpression in DS has not been demonstrated. In this study, we sought to reveal that GIRK2 (and consequential GIRK1) subunit overexpression leads to an increase in GIRK current that would likely impact tonic inhibitory tone. We demonstrate herein that the reported overexpression levels of GIRK channel subunits in Ts65Dn mice correlates with increases in GIRK currents and changes in intrinsic neuronal properties from hippocampal neurons cultured from the Ts65Dn neonatal mouse.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Cell culture

Mouse hippocampal neurons were cultured in a similar manner to that described previously (Galdzicki et al. 1998). Litters from Ts65Dn mothers and diploid fathers were taken at postnatal days one and two (P1-2). Pups were decapitated, and hippocampi dissected out, after which free hippocampi were cut into pieces and incubated in 0.05% trypsin (wt/vol) for 10 min at 37°C. Tissue was washed twice with plating media after which hippocampal cells were mechanically separated by trituration through a polished glass Pasteur pipette. Hippocampal cells were then plated on 35-mm Nunc brand dishes previously coated with 25 µg/ml poly-D-lysine and maintained at 37°C and 5% CO₂ in the presence of plating media. One day after plating, the media was replaced with maintenance media. Plating media was composed of Neurobasal-A media with B27 supplement (Invitrogen/Gibco BRL, Carlsbad, CA), fetal bovine serum (FBS, 10%), horse serum (HS, 5%), and glutamine (1%; Biosource, Camarillo, CA), whereas maintenance media was of the same composition but without FBS or HS. Throughout the dissection and plating procedure tissue from each pup was processed separately, and ploidy was determined afterward. Mice were karyotyped with blood as described previously (Harashima et al. 2006). All recordings and the initial analysis were done without prior knowledge of the genotype.

Electrophysiological recordings

Whole cell patch-clamp recordings of isolated hippocampal neurons were performed 10–20 days after plating. This age complies with functional GABA_B receptors and GIRK channels in cultured neurons (Correa et al. 2004). Pipettes were backfilled with a solution containing (in mM) 100 K-gluconate, 20 KCl, 10 HEPES, 10 EGTA, 1 CaCl₂, 4 Mg-ATP, 0.3 Na-GTP, and 7 Na-phosphocreatine, pH 7.35. In some instances, the 0.3 mM Na-GTP was replaced with 0.3 mM Li₃-GTPS, a nonhydrolyzable GTP analogue. At the time of recording, maintenance media was removed and replaced with room temperature bathing solution containing (in mM) 150 NaCl, 4 KCl, 2 CaCl₂, 2 MgCl₂, 10 Na-HEPES, and 10 glucose, pH 7.35. tetrodotoxin (1 µM) and MK-801 or D-2-amino-5-phosphonovaleric acid (D-APV; 50 µM) were added to block spontaneous firing, and N-methyl-D-aspartate (NMDA)-mediated currents, respectively. To elicit change in the potassium driving force, the bathing solution NaCl was replaced by equivalent KCl (high K⁺: 60 mM). The GABA_B receptor agonist baclofen was applied in both basal and high potassium solutions via a 9-barrel rapid solution changer (RSC-200, Biologic, Calix, France/Molecular Kinetics, Pullman, WA) positioned 200 µm from the patched neuron. Recordings were performed in voltage-clamp configuration at a holding potential of –70 mV, and data were acquired by way of an EPC-7 amplifier (HEKA), filtered at 5 kHz (8-pole Bessel filter, NPI, ALA Scientific Instruments, Westbury, NY), and recorded on a personal computer as per Klein et al. (2001). A 5 mV hyperpolarizing step from a holding potential of 8–70 mV was applied to estimate membrane capacitance and resistance at the initiation of whole cell access and at intervals throughout the recording. Similarly, resting membrane potential was measured at the beginning of each recording and throughout the experiment to assess and monitor cell viability.

Data analysis and statistics

High potassium (60 mM) currents were determined as steady-state values subtracted from basal currents measured under 4 mM extracellular potassium. When delivered under high potassium, baclofen-evoked peak and steady-state currents were determined by subtraction from the value of the high extracellular potassium steady-state currents. Under basal potassium, baclofen currents were subtracted from the steady-state basal current. Dose response relationships are the best least-squares fit to Eq. 1/(1+ EC₅₀/[baclofen]), and EC₅₀ is concentration of baclofen, which produces 50% of the maximum possible response.

To assess the conductance of individual channels from mice of each genotype, stationary noise analysis of current recordings were performed similar to previous reports (Sciancalepore et al. 1990; Takigawa and Alzheimer 1999; Traynelis and Jaramillo 1998). Current variance from 200 ms stretches of GIRK current were plotted as a function of the mean GIRK current value for that given stretch. Data points from individual neurons were fit to the equation: ² = iI –I²/N where ² is the current variance, i is the unitary current amplitude, I is the whole cell current amplitude, and N is the number of available channels. The value of i determined from the preceding equation was then related to single-channel conductance by the formula: = i/(V_h – E_K) where is the single-channel conductance, V_h is the holding potential (–83 mV after liquid junction potential correction), and E_K is the Nernst potential for potassium at high potassium recording conditions (–18 mV). Channel density was then determined for each neuron by taking the ratio of estimated N and membrane capacitance. Values of i, , N, and channel density from each neuron were averaged and analyzed for statistical significance based on ploidy.

To evaluate impact of ploidy on kinetics of baclofen-induced current, tau of current activation (_a) and deactivation (_d) were calculated by fitting to a single-exponential function. GIRK current desensitization was determined by subtraction of peak from steady-state current values (see Fig. 1B, right arrow and dashed line). Changes in current from peak to steady state are likely due to desensitization rather than cell dialysis. This is because only cells that showed a return to baseline on washout were used in the analysis, and long-term agonist application showed rates of desensitization comparable to short-term application.

View larger version (12K): [in this window] [in a new window]   FIG. 1. Baclofen induces an increase in G-protein-coupled inward rectifying potassium channel (GIRK) current density in Ts65Dn in comparison to diploid neurons. A: current-voltage relationships of diploid and Ts65Dn neurons show baclofen current is inwardly rectifying and the reversal potential approximates the K+ Nernst potential. I-V curves were determined by subtraction of ramps with and without baclofen at basal potassium concentrations. B: example of the increased inward current density induced by 25 µM baclofen in a Ts65Dn neuron compared with diploid.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Preliminary experiments with agonists, such as baclofen, serotonin, acetylcholine, and 2-chloroadenosine, to receptors coupled to GIRK channels regularly elicited inward rectifying potassium channel in diploid and Ts65Dn neurons. However, the GABA_B receptor agonist baclofen evoked the largest and most consistent current in both diploid and Ts65Dn neurons. Therefore we proceeded to focus on currents elicited by baclofen in our experiments. GABA_B specificity was determined through use of CGP-55845 (2 µM), which completely blocked baclofen induced current (n = 3). Voltage ramps indicate that the reversal potential of this current approximates the Nernst potential for potassium ions as well as showing inward rectification consistent with Kir channels (Fig. 1A). All passive membrane properties were similar between genotypes (see Table 1). Resting membrane potential under current clamp was not significantly hyperpolarized in Ts65Dn neurons (although, P = 0.08). These results are similar to those obtained in full trisomy 16 cultured hippocampal neurons (Galdzicki et al. 1993).

Baclofen was applied under both basal and high potassium solutions. Under basal potassium concentrations and at the holding potential (–70 mV, junction potential adjusted to –83 mV), the potassium driving force is outward. We measured this outward current after application of 25 µM baclofen and found current density responses was significantly increased in Ts65Dn (1.3 ± 0.3 pA/pF, n = 16) compared with diploid (0.6 ± 0.2 pA/pF, n = 11) neurons (P < 0.05). Under high potassium where the potassium driving force is inward, baclofen-induced GIRK current density of Ts65Dn neurons were significantly shifted left toward smaller concentrations of baclofen. Peak current density of baclofen-induced GIRK current shows dose dependence that is significantly different between diploid (n = 11–23) and Ts65Dn (n = 9–13) neurons [2-way repeated-measures (RM) ANOVA, P = 0.0002]. Peak EC₅₀ shifts from 4.2 µM in diploid to 0.9 µM in Ts65Dn (Fig. 2A). The steady-state current density was also significantly different (2-way RM ANOVA, P < 0.0001). The EC₅₀ shifted left from 3.0 µM in diploid to 1.0 µM in Ts65Dn (Fig. 2B). The increase in current density efficacy is consistent with an increase in channel number.

View larger version (32K): [in this window] [in a new window]   FIG. 2. Baclofen-induced normalized GIRK current in diploid and Ts65Dn neurons show similar dose dependence. A: normalized peak EC50 of 2.5 µM for diploid and 1.4 µM for Ts65Dn. B: normalized steady-state EC50 of 2.1 µM for diploid and 1.4 µM for Ts65Dn are not significantly different. Peak and steady-state current values were normalized to the maximum current in each cell. C: peak ( in Fig. 1B); D: steady state (- - - in Fig. 1B), current density of baclofen-induced GIRK current show dose dependence that is significantly different between diploid and Ts65Dn neurons (P < 0.001). Peak EC50 shifts from 3.2 µM in diploid to 1.4 µM in Ts65Dn and steady-state EC50 shifts 2.1 µM in diploid to 0.9 µM in Ts65Dn. Current density is determined by dividing current by capacitance value (pA/pF).

GIRK channel blockers were used to block the baclofen-induced currents. Tertiapin-Q (50 nM), a specific GIRK channel blocker inhibited baclofen (25 µM) induced current by 46 ± 8% in diploid (n = 7) and 56 ± 5% in Ts65Dn (n = 12) neurons (Fig. 3A). No significant difference in the percentage of block by tertiapin-Q were found between genotypes (P = 0.25). Tertiapin-Q produced a biphasic response as shown in Fig. 3A. This unique response may be indicative of the use dependency of tertiapin-Q block on GIRK channels (Huang et al. 2005; Kanjhan et al. 2005). Application of barium (Ba²⁺) (200 µM), which blocks inward rectifying potassium channels, also inhibited baclofen (25 µM)-induced currents in both Ts65Dn and diploid neurons Fig. 3B. Interestingly, the inhibitory effect of Ba²⁺ on diploid neurons was dichotic. In one subset of neurons (7 of 11), the Ba²⁺ blocked 60 ± 14% of baclofen-induced current. In the other subset of diploid neurons (4 of 11), Ba²⁺ blocked 540 ± 23% of the baclofen-induced current (i.e., 100% of baclofen current and 55 ± 5% of the high potassium current). This dichotic effect was not seen in Ts65Dn neurons. Ba²⁺ block in Ts65Dn neurons was similar to the first subset of diploid neurons in that block was 56 ± 9% (n = 11) of baclofen current. This suggests that the overexpression of GIRK leads to a decrease in endogenously active Ba²⁺-sensitive potassium channels in a subset of hippocampal cultured neurons.

View larger version (13K): [in this window] [in a new window]   FIG. 3. Block of baclofen-induced current by tertiapin-Q and Ba2+. A: example traces showing the action of tertiapin-Q (50 nM) on baclofen-induced (25 µM) current. Tertiapin-Q blocked 46 ± 8 and 56 ± 5% of the baclofen-induced current in diploid (n = 7) and Ts65Dn (n = 12) neurons, respectively. The biphasic block by tertiapin-Q in the Ts65Dn recording may be indicative of use dependence. B: traces demonstrating Ba2+ (200 µM) block of all baclofen (25 µM)-induced current. Diploid neurons responded in disparate ways to Ba2+. In 4 of 11 neurons Ba2+ blocked all of the baclofen-induced current and part (55 ± 5%) of the high K+ current. In the other 7 of 11 neurons, the response was similar to that in Ts65Dn neurons where block was 60 ± 14% compared with 56 ± 9% in Ts65Dn neurons (n = 11).

Because the use of GTPS was unable to substantially discriminate possible explanations for the increase in GIRK current density, we used stationary fluctuation analysis. An increase in channel number or density would likely be a principle cause for the increased GIRK current density and not changes in GABA_B receptor or G-protein properties. The values of the single-channel conductance () estimated for diploid (n = 12; 19.5 pS) and Ts65Dn neurons (n = 8; 25.1 pS) are within range of GIRK conductance obtained from acutely dissociated rat hippocampal neurons (Takigawa and Alzheimer 1999) but slightly smaller than that estimated from excised dendritic recordings (Chen and Johnston 2005). is not significantly different between diploid and Ts65Dn neurons (P = 0.46; Fig. 4, Table 2). However, when evaluating channel number per membrane capacitance, Ts65Dn neurons show a significant increase in channel density (80%, P < 0.05) that can explain the significant shift in EC₅₀ of GIRK currents and corroborate our previous findings that GIRK channel number is increased in Ts65Dn neurons compared with diploid (Harashima et al. 2006).

View larger version (35K): [in this window] [in a new window]   FIG. 4. Single-channel conductance () and channel number (N) are similar between diploid and Ts65Dn neurons. A: sample recordings from increasing doses of baclofen in diploid and Ts65Dn neurons. B: current variance from 200 ms stretches of GIRK current were plotted as a function of the mean GIRK current value for that given stretch. Single-channel conductance () and channel number (N) are not significantly different between genotypes (P = 0.46, P = 0.74, respectively); however, channel density is increased by 80% (P = 0.02; see Table 2).

View larger version (22K): [in this window] [in a new window]   FIG. 5. Normal kinetics of baclofen induced GIRK current. A: kinetics of GIRK current activation are dependent on baclofen dose (P < 0.001) but are not significantly different between diploid and Ts65Dn neurons. B: decay kinetics show no dose dependence and are similar between ploidy. C: short-term (15 s) desensitization of GIRK current density evaluated by the difference between peak and steady-state values show dependence on baclofen dose for diploid and Ts65Dn neurons (2-way RM ANOVA, P < 0.05). A significant interaction was found (P < 0.05) and Bonferroni posttests indicate significant differences at 5 and 50 µM baclofen (*P < 0.05, **P < 0/01).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The data presented in this report suggest that cultured hippocampal neurons from Ts65Dn neonates have elevated responses to GABA_B receptor activation as evidenced by a leftward shift in the dose-response curves of GIRK current density. Variance analysis of GIRK current fluctuations suggests that the leftward shift of the dose response curve can be explained by a significant increase in channel density but not by a significant change in single-channel conductance.

The GIRK currents seen in Ts65Dn neurons were consistently larger than those measured in diploid neurons for all tested doses. We see no evidence for a change in the activation and deactivation kinetics of GIRK current in Ts65Dn neurons suggesting that coupling of receptor and channel remain similar to diploid neurons. Disparate desensitization responses to baclofen between diploid and Ts65Dn neurons, however, may be indicative of more complex changes in GIRK current induced by changes in channel subunit ratios.

Because hippocampal neurons from GIRK2 knockout animals show significant depolarization (Koyrakh et al. 2005; Luscher et al. 1997), we evaluated the impact of the extra Ts65Dn segment, which contains GIRK2, on resting membrane properties. Resting membrane potential from each genotype was not significantly different. In full trisomy 16 hippocampal culture, we also did not find significant changes in membrane resting potential (Galdzicki et al. 1993). Membrane resting potentials in Ts65Dn neurons may not be affected by level of GIRK function alone because the overexpression of other genes from the Ts65Dn Chr. may contribute to neuronal resting membrane potentials.

Chen and Johnston (2005) suggested that the low GIRK conductance at the soma and low surface to volume ratio would contribute little to somatic membrane properties. Yet at the dendrites where GIRK conductance is high and surface to volume ratio is also high, GIRK expression would have profound influence on dendritic membrane properties. Within the CA1, it is at these distal synapses where the GABA_B-mediated potassium currents generated by perforant pathway stimulation within the stratum lacunosum-moleculare are greater than those within the more proximal stimulation of Schaffer collaterals within the stratum radiatum (Otmakhova and Lisman 2004). This poses the possibility that perforant path input at CA1 pyramidal synapse is more profoundly influenced in the Ts65Dn mouse than Schaeffer collateral input. A change in the integrative properties of pyramidal neurons is the likely result.

Plasma membrane associated GIRK1 and GIRK2 channel subunits have been identified primarily in extra- and perisynaptic regions of the postsynaptic membrane of mouse stratum radiatum. GIRK2 but not GIRK1 signal was also found within postsynaptic specializations (Koyrakh et al. 2005). More specifically, GIRK2 immunoreactivity colabeled with GABA_B receptors on dendritic spines of adult rat hippocampal pyramidal neurons. This coexpression, however, was not found in dendritic shafts where GABA_B receptor and GIRK2 protein were segregated (Kulik et al. 2006). Remarkably, in rat hippocampus, extrasynaptic GABA_B receptors show heterogenous potassium currents and may not couple Ba²⁺-sensitive inward rectifying potassium channels, whereas synaptic GABA_B receptors are homogenous and solely elicit characteristic GIRK currents (Pham et al. 1998). What an excess of GIRK1-2 expression would do to the balance between extrasynaptic and synaptic GABA_B-potassium channel coupling remains to be seen, but the extra GIRK1-2 would increase the overall inhibitory tone. (Note: because our protocol involves bath application of baclofen, both synaptic and extrasynaptic receptors were activated).

An increase in GIRK current through overexpression should have profound impact on the physiology of the neuron. It could perhaps account for the abnormalities in synaptic plasticity of Ts65Dn and Ts1Cje mice (Costa and Grybko 2005; Kleschevnikov et al. 2004; Siarey et al. 1997, 1999, 2005) through shifts in the dynamic range of these synapses and a disruption of the balance between excitation and inhibition. Intrinsic interneuron/GABAergic tone would impinge an elevated level of GABA_B receptor mediated inhibition in mice overexpressing GIRK1-2. In native systems, it is likely that lower concentrations of GABA would elicit larger GIRK currents. Thus minimal GABAergic neurotransmission should have greater shunting ability.

A heteromeric GABA_B receptor consisting of subunits 1 and 2 is considered necessary for metabotropic GABA signaling. GABA_B1 is responsible for binding GABA, whereas GABA_B2 mediates surface trafficking and G-protein coupling (Calver et al. 2001; Galvez et al. 2001; Kaupmann et al. 1998; Margeta-Mitrovic et al. 2000, 2001; Pagano et al. 2001; Robbins et al. 2001). In hippocampal primary cultures from E18 rats, GABA_B1 isoforms and GABA_B2 receptor expression matches the time-dependent increases in baclofen induced potassium currents. In contrast to diffuse GABA_B staining and minimal potassium current at 3 days in vitro (DIV), dendritic and punctate distribution of staining after 14 DIV paralleled the time at which maximum current density occurred. Interestingly, as the expression of the GABA_B subunits developed according to DIV, the levels of GIRK1 protein remained constant suggesting that GABA_B receptor expression was independent of GIRK (Correa et al. 2004). It is therefore unlikely that increases in baclofen-induced currents are mediated by changes in GABA_B receptor expression. Likewise, our data derived from variance analysis indicating increases in channel density alone can account for the shift in dose response and argue against a change in GABA_B receptor expression in these cultures.

For the most part any neurotransmitter that activates a G_i/o-coupled receptor can activate GIRK channels. Indeed, in native neurons and expression systems, acetylcholine, adenosine, endocannabinoids, dopamine, GABA, glutamate, histamine, melatonin, neuropeptide Y, norepinephrine, orexins (hypocretins), opioids, serotonin, and somatostatin, have all been shown to modulate GIRK channel activity (Bunemann et al. 2001; Hoang et al. 2003; Kobayashi et al. 1996; Kofuji et al. 1995; Kreienkamp et al. 1997; Kuzhikandathil et al. 1998; Luscher et al. 1997; McAllister et al. 1999; Nelson et al. 1996; Paredes et al. 2003; Saugstad et al. 1996; Spauschus et al. 1996; Takigawa and Alzheimer 1999; Ulens et al. 1999). Therefore it is likely that any of these systems with activity in the hippocampus and CNS that activate GIRK channels would result in abnormal DS function and may contribute to neurological and cognitive phenotypes found in DS individuals. In fact, Ts65Dn mice show an increase in GIRK2 mediated hypothermic responses to a serotonin (5-HT)1A/5-HT7 receptor agonist (Stasko et al. 2006), an effect that is most likely due to the presence of the extra Girk2 gene.

In summary, hippocampal neurons derived from Ts65Dn mice show increased sensitivity to GABA_B signaling with significant shift to the left in the dose-dependence relationships. The kinetics of GIRK current activation, deactivation, and acute desensitization were unchanged in Ts65Dn neurons, suggesting that the increased GIRK current does not affect coupling mechanisms among the GABA_B receptor, G protein, and GIRK channel. The increase in GIRK current can be attributed to a 80% increase in channel density and not significant changes in GIRK single-channel conductance.

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
This work was supported by National Institute of Child Health and Human Development Grant HD-38417, The Jerome Lejeune Foundation, and Uniformed Services University of the Health Sciences.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The authors thank M. Cho for assistance with the care and genotyping of the Ts65Dn mice. We are also grateful to A. Balbo for help with the neuronal cultures.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: Z. Galdzicki, Department of Anatomy, Physiology, and Genetics, USUHS, School of Medicine, 4301 Jones Bridge Rd., Bethesda, MD 20814 (E-mail: zgaldzicki{at}usuhs.mil)

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Adeniji-Adele A, Harashima C, Best TK, Stoffel M, Galdzicki Z, Siarey R. Long-term potentiation and depression in the GIRK2-/- mouse. Soc Neurosci Abstr 28: 56.16, 2004.

Antonarakis SE, Lyle R, Dermitzakis ET, Reymond A, Deutsch S. Chromosome 21 and down syndrome: from genomics to pathophysiology. Nat Rev Genet 5: 725–738, 2004.[CrossRef][Web of Science][Medline]

Bunemann M, Bucheler MM, Philipp M, Lohse MJ, Hein L. Activation and deactivation kinetics of alpha 2A- and alpha 2C-adrenergic receptor-activated G-protein-activated inwardly rectifying K⁺ channel currents. J Biol Chem 276: 47512–47517, 2001.[Abstract/Free Full Text]

Calver AR, Robbins MJ, Cosio C, Rice SQ, Babbs AJ, Hirst WD, Boyfield I, Wood MD, Russell RB, Price GW, Couve A, Moss SJ, Pangalos MN. The C-terminal domains of the GABA(b) receptor subunits mediate intracellular trafficking but are not required for receptor signaling. J Neurosci 21: 1203–1210, 2001.[Abstract/Free Full Text]

Chen X, Johnston D. Constitutively active G-protein-gated inwardly rectifying K⁺ channels in dendrites of hippocampal CA1 pyramidal neurons. J Neurosci 25: 3787–3792, 2005.[Abstract/Free Full Text]

Correa SA, Munton R, Nishimune A, Fitzjohn S, Henley JM. Development of GABAB subunits and functional GABA_B receptors in rat cultured hippocampal neurons. Neuropharmacology 47: 475–484, 2004.[CrossRef][Web of Science][Medline]

Costa AC, Grybko MJ. Deficits in hippocampal CA1 LTP induced by TBS but not HFS in the Ts65Dn mouse: a model of Down syndrome. Neurosci Lett 382: 317–322, 2005.[CrossRef][Web of Science][Medline]

Drake CT, Bausch SB, Milner TA, Chavkin C. GIRK1 immunoreactivity is present predominantly in dendrites, dendritic spines, and somata in the CA1 region of the hippocampus. Proc Natl Acad Sci USA 94: 1007–1012, 1997.[Abstract/Free Full Text]

Ehrengruber MU, Doupnik CA, Xu Y, Garvey J, Jasek MC, Lester HA, Davidson N. Activation of heteromeric G protein-gated inward rectifier K⁺ channels overexpressed by adenovirus gene transfer inhibits the excitability of hippocampal neurons. Proc Natl Acad Sci USA 94: 7070–7075, 1997.[Abstract/Free Full Text]

Galdzicki Z, Coan E, Rapoport SI. Cultured hippocampal neurons from trisomy 16 mouse, a model for Down's syndrome, have an abnormal action potential due to a reduced inward sodium current. Brain Res 604: 69–78, 1993.[CrossRef][Web of Science][Medline]

Galdzicki Z, Coan EJ, Rapoport SI, Stoll J. Increased expression of voltage-activated calcium channels in cultured hippocampal neurons from mouse trisomy 16, a model for Down syndrome. Brain Res Mol Brain Res 56: 200–206, 1998.[Medline]

Galdzicki Z, Siarey RJ. Understanding mental retardation in Down's syndrome using trisomy 16 mouse models. Genes Brain Behav 2: 167–178, 2003.[CrossRef][Web of Science][Medline]

Galdzicki Z, Siarey R, Pearce R, Stoll J, Rapoport SI. On the cause of mental retardation in Down syndrome: extrapolation from full and segmental trisomy 16 mouse models. Brain Res Brain Res Rev 35: 115–145, 2001.[CrossRef][Medline]

Galvez T, Duthey B, Kniazeff J, Blahos J, Rovelli G, Bettler B, Prezeau L, Pin JP. Allosteric interactions between GB1 and GB2 subunits are required for optimal GABA(B) receptor function. Embo J 20: 2152–2159, 2001.[CrossRef][Web of Science][Medline]

Harashima C, Jacobowitz DM, Witta J, Borke RC, Best TK, Siarey RJ, Galdzicki Z. Abnormal expression of the G-protein-activated inwardly rectifying potassium channel 2 (GIRK2) in hippocampus, frontal cortex, and substantia nigra of Ts65Dn mouse: A model of Down syndrome. J Comp Neurol 494: 815–833, 2006.[CrossRef][Web of Science][Medline]

Hattori M, Fujiyama A, Taylor TD, Watanabe H, Yada T, Park HS, Toyoda A, Ishii K, Totoki Y, Choi DK, Groner Y, Soeda E, Ohki M, Takagi T, Sakaki Y, Taudien S, Blechschmidt K, Polley A, Menzel U, Delabar J, Kumpf K, Lehmann R, Patterson D, Reichwald K, Rump A, Schillhabel M, Schudy A, Zimmermann W, Rosenthal A, Kudoh J, Schibuya K, Kawasaki K, Asakawa S, Shintani A, Sasaki T, Nagamine K, Mitsuyama S, Antonarakis SE, Minoshima S, Shimizu N, Nordsiek G, Hornischer K, Brant P, Scharfe M, Schon O, Desario A, Reichelt J, Kauer G, Blocker H, Ramser J, Beck A, Klages S, Hennig S, Riesselmann L, Dagand E, Haaf T, Wehrmeyer S, Borzym K, Gardiner K, Nizetic D, Francis F, Lehrach H, Reinhardt R, Yaspo ML. The DNA sequence of human chromosome 21. Nature 405: 311–319, 2000.[CrossRef][Medline]

Hille B. Ion Channels of Excitable Membranes. Sunderland, MA: Sinauer, 2001.

Hoang QV, Bajic D, Yanagisawa M, Nakajima S, Nakajima Y. Effects of orexin (hypocretin) on GIRK channels. J Neurophysiol 90: 693–702, 2003.[Abstract/Free Full Text]

Holtzman DM, Santucci D, Kilbridge J, Chua-Couzens J, Fontana DJ, Daniels SE, Johnson RM, Chen K, Sun Y, Carlson E, Alleva E, Epstein CJ, Mobley WC. Developmental abnormalities and age-related neurodegeneration in a mouse model of Down syndrome. Proc Natl Acad Sci USA 93: 13333–13338, 1996.[Abstract/Free Full Text]

Huang CS, Shi SH, Ule J, Ruggiu M, Barker LA, Darnell RB, Jan YN, Jan LY. Common molecular pathways mediate long-term potentiation of synaptic excitation and slow synaptic inhibition. Cell 123: 105–118, 2005.[CrossRef][Web of Science][Medline]

Kanjhan R, Coulson EJ, Adams DJ, Bellingham MC. Tertiapin-q blocks recombinant and native large conductance k+ channels in a use-dependent manner. J Pharmacol Exp Ther 314: 1353–1361, 2005.[Abstract/Free Full Text]

Kaupmann K, Schuler V, Mosbacher J, Bischoff S, Bittiger H, Heid J, Froestl W, Leonhard S, Pfaff T, Karschin A, Bettler B. Human gamma-aminobutyric acid type B receptors are differentially expressed and regulate inwardly rectifying K⁺ channels. Proc Natl Acad Sci USA 95: 14991–14996, 1998.[Abstract/Free Full Text]

Klein RC, Siarey RJ, Caruso A, Rapoport SI, Castellino FJ, Galdzicki Z. Increased expression of NR2A subunit does not alter NMDA-evoked responses in cultured fetal trisomy 16 mouse hippocampal neurons. J Neurochem 76: 1663–1669, 2001.[CrossRef][Web of Science][Medline]

Kleschevnikov AM, Belichenko PV, Villar AJ, Epstein CJ, Malenka RC, Mobley WC. Hippocampal long-term potentiation suppressed by increased inhibition in the Ts65Dn mouse, a genetic model of Down syndrome. J Neurosci 24: 8153–8160, 2004.[Abstract/Free Full Text]

Kobayashi T, Ikeda K, Ichikawa T, Togashi S, Kumanishi T. Effects of sigma ligands on the cloned mu-, delta- and kappa-opioid receptors co-expressed with G-protein-activated K⁺ (GIRK) channel in Xenopus oocytes. Br J Pharmacol 119: 73–80, 1996.[Web of Science][Medline]

Kofuji P, Davidson N, Lester HA. Evidence that neuronal G-protein-gated inwardly rectifying K⁺ channels are activated by G beta gamma subunits and function as heteromultimers. Proc Natl Acad Sci USA 92: 6542–6546, 1995.[Abstract/Free Full Text]

Korenberg JR, Chen XN, Schipper R, Sun Z, Gonsky R, Gerwehr S, Carpenter N, Daumer C, Dignan P, Disteche C, Graham JM Jr, Hudgins L, McGillivray B, Miyazaki K, Ogasawara N, Park JP, Pagon R, Pueschel S, Sack G, Say B, Schuffenhauer S, Soukup S, Yamanaka T. Down syndrome phenotypes: the consequences of chromosomal imbalance. Proc Natl Acad Sci USA 91: 4997–5001, 1994.[Abstract/Free Full Text]

Kourrich S, Masmejean F, Martin-Eauclaire MF, Soumireu-Mourat B, Mourre C. Inwardly rectifying Kir3.1 subunit knockdown impairs learning and memory in an olfactory associative task in rat. Brain Res Mol Brain Res 113: 97–106, 2003.[Medline]

Koyrakh L, Lujan R, Colon J, Karschin C, Kurachi Y, Karschin A, Wickman K. Molecular and cellular diversity of neuronal G-protein-gated potassium channels. J Neurosci 25: 11468–11478, 2005.[Abstract/Free Full Text]

Kreienkamp HJ, Honck HH, Richter D. Coupling of rat somatostatin receptor subtypes to a G-protein gated inwardly rectifying potassium channel (GIRK1). FEBS Lett 419: 92–94, 1997.[CrossRef][Web of Science][Medline]

Kulik A, Vida I, Fukazawa Y, Guetg N, Kasugai Y, Marker CL, Rigato F, Bettler B, Wickman K, Frotscher M, Shigemoto R. Compartment-dependent colocalization of Kir3.2-containing K⁺ channels and GABAB receptors in hippocampal pyramidal cells. J Neurosci 26: 4289–4297, 2006.[Abstract/Free Full Text]

Kuzhikandathil EV, Yu W, Oxford GS. Human dopamine D3 and D2L receptors couple to inward rectifier potassium channels in mammalian cell lines. Mol Cell Neurosci 12: 390–402, 1998.[CrossRef][Web of Science][Medline]

Leaney JL. Contribution of Kir3.1, Kir3.2A and Kir3.2C subunits to native G protein-gated inwardly rectifying potassium currents in cultured hippocampal neurons. Eur J Neurosci 18: 2110–2118, 2003.[CrossRef][Web of Science][Medline]

Luscher C, Jan LY, Stoffel M, Malenka RC, Nicoll RA. G protein-coupled inwardly rectifying K⁺ channels (GIRKs) mediate postsynaptic but not presynaptic transmitter actions in hippocampal neurons. Neuron 19: 687–695, 1997.[CrossRef][Web of Science][Medline]

Margeta-Mitrovic M, Jan YN, Jan LY. A trafficking checkpoint controls GABA(B) receptor heterodimerization. Neuron 27: 97–106, 2000.[CrossRef][Web of Science][Medline]

Margeta-Mitrovic M, Jan YN, Jan LY. Function of GB1 and GB2 subunits in G protein coupling of GABA(B) receptors. Proc Natl Acad Sci USA 98: 14649–14654, 2001.[Abstract/Free Full Text]

McAllister SD, Griffin G, Satin LS, Abood ME. Cannabinoid receptors can activate and inhibit G-protein-coupled inwardly rectifying potassium channels in a Xenopus oocyte expression system. J Pharmacol Exp Ther 291: 618–626, 1999.[Abstract/Free Full Text]

Nelson CS, Marino JL, Allen CN. Melatonin receptors activate heteromeric G-protein coupled Kir3 channels. Neuroreport 7: 717–720, 1996.[Web of Science][Medline]

Nieminen K, Suarez-Isla BA, Rapoport SI. Electrical properties of cultured dorsal root ganglion neurons from normal and trisomy 21 human fetal tissue. Brain Res 474: 246–254, 1988.[CrossRef][Web of Science][Medline]

Olson LE, Richtsmeier JT, Leszl J, Reeves RH. A chromosome 21 critical region does not cause specific Down syndrome phenotypes. Science 306: 687–690, 2004.[Abstract/Free Full Text]

Otmakhova NA, Lisman JE. Contribution of Ih and GABAB to synaptically induced afterhyperpolarizations in CA1: a brake on the NMDA response. J Neurophysiol 92: 2027–2039, 2004.[Abstract/Free Full Text]

Pagano A, Rovelli G, Mosbacher J, Lohmann T, Duthey B, Stauffer D, Ristig D, Schuler V, Meigel I, Lampert C, Stein T, Prezeau L, Blahos J, Pin J, Froestl W, Kuhn R, Heid J, Kaupmann K, Bettler B. C-terminal interaction is essential for surface trafficking but not for heteromeric assembly of GABA(b) receptors. J Neurosci 21: 1189–1202, 2001.[Abstract/Free Full Text]

Paredes MF, Greenwood J, Baraban SC. Neuropeptide Y modulates a G-protein-coupled inwardly rectifying potassium current in the mouse hippocampus. Neurosci Lett 340: 9–12, 2003.[CrossRef][Web of Science][Medline]

Pham TM, Nurse S, Lacaille JC. Distinct GABAB actions via synaptic and extrasynaptic receptors in rat hippocampus in vitro. J Neurophysiol 80: 297–308, 1998.[Abstract/Free Full Text]

Reeves RH, Irving NG, Moran TH, Wohn A, Kitt C, Sisodia SS, Schmidt C, Bronson RT, Davisson MT. A mouse model for Down syndrome exhibits learning and behavior deficits. Nat Genet 11: 177–184, 1995.[CrossRef][Web of Science][Medline]

Robbins MJ, Calver AR, Filippov AK, Hirst WD, Russell RB, Wood MD, Nasir S, Couve A, Brown DA, Moss SJ, Pangalos MN. GABA(B2) is essential for G-protein coupling of the GABA(B) receptor heterodimer. J Neurosci 21: 8043–8052, 2001.[Abstract/Free Full Text]

Sago H, Carlson EJ, Smith DJ, Kilbridge J, Rubin EM, Mobley WC, Epstein CJ, Huang TT. Ts1Cje, a partial trisomy 16 mouse model for Down syndrome, exhibits learning and behavioral abnormalities. Proc Natl Acad Sci USA 95: 6256–6261, 1998.[Abstract/Free Full Text]

Sanchez JA, Gonoi T, Inagaki N, Katada T, Seino S. Modulation of reconstituted ATP-sensitive K(+)-channels by GTP-binding proteins in a mammalian cell line. J Physiol 507: 315–324, 1998.[Abstract/Free Full Text]

Saugstad JA, Segerson TP, Westbrook GL. Metabotropic glutamate receptors activate G-protein-coupled inwardly rectifying potassium channels in Xenopus oocytes. J Neurosci 16: 5979–5985, 1996.[Abstract/Free Full Text]

Sciancalepore M, Galdzicki Z, Zheng X, Moran O. Kainate activated single channel currents as revealed by domoic acid. Eur Biophys J 19: 63–68, 1990.[Web of Science][Medline]

Siarey RJ, Carlson EJ, Epstein CJ, Balbo A, Rapoport SI, Galdzicki Z. Increased synaptic depression in the Ts65Dn mouse, a model for mental retardation in Down syndrome. Neuropharmacology 38: 1917–1920, 1999.[CrossRef][Web of Science][Medline]

Siarey RJ, Stoll J, Rapoport SI, Galdzicki Z. Altered long-term potentiation in the young and old Ts65Dn mouse, a model for Down Syndrome. Neuropharmacology 36: 1549–1554, 1997.[CrossRef][Web of Science][Medline]

Siarey RJ, Villar AJ, Epstein CJ, Galdzicki Z. Abnormal synaptic plasticity in the Ts1Cje segmental trisomy 16 mouse model of Down syndrome. Neuropharmacology 49: 122–128, 2005.[CrossRef][Web of Science][Medline]

Sodickson DL, Bean BP. GABAB receptor-activated inwardly rectifying potassium current in dissociated hippocampal CA3 neurons. J Neurosci 16: 6374–6385, 1996.[Abstract/Free Full Text]

Sodickson DL, Bean BP. Neurotransmitter activation of inwardly rectifying potassium current in dissociated hippocampal CA3 neurons: interactions among multiple receptors. J Neurosci 18: 8153–8162, 1998.[Abstract/Free Full Text]

Spauschus A, Lentes KU, Wischmeyer E, Dissmann E, Karschin C, Karschin A. A G-protein-activated inwardly rectifying K⁺ channel (GIRK4) from human hippocampus associates with other GIRK channels. J Neurosci 16: 930–938, 1996.[Abstract/Free Full Text]

Stasko MR, Scott-McKean JJ, Costa AC. Hypothermic responses to 8-OH-DPAT in the Ts65Dn mouse model of Down syndrome. Neuroreport 17: 837–841, 2006.[CrossRef][Web of Science][Medline]

Takigawa T, Alzheimer C. Variance analysis of current fluctuations of adenosine- and baclofen-activated GIRK channels in dissociated neocortical pyramidal cells. J Neurophysiol 82: 1647–1650, 1999.[Abstract/Free Full Text]

Toyoda A, Noguchi H, Taylor TD, Ito T, Pletcher MT, Sakaki Y, Reeves RH, Hattori M. Comparative genomic sequence analysis of the human chromosome 21 Down syndrome critical region. Genome Res 12: 1323–1332, 2002.[Abstract/Free Full Text]

Trapp S, Tucker SJ, Ashcroft FM. Activation and inhibition of K-ATP currents by guanine nucleotides is mediated by different channel subunits. Proc Natl Acad Sci USA 94: 8872–8877, 1997.[Abstract/Free Full Text]

Traynelis SF, Jaramillo F. Getting the most out of noise in the central nervous system. Trends Neurosci 21: 137–145, 1998.[CrossRef][Web of Science][Medline]

Ulens C, Daenens P, Tytgat J. The dual modulation of GIRK1/GIRK2 channels by opioid receptor ligands. Eur J Pharmacol 385: 239–245, 1999.[CrossRef][Web of Science][Medline]

Wickman K, Karschin C, Karschin A, Picciotto MR, Clapham DE. Brain localization and behavioral impact of the G-protein-gated K⁺ channel subunit GIRK4. J Neurosci 20: 5608–5615, 2000.[Abstract/Free Full Text]

This article has been cited by other articles:

				M. Dierssen, Y. Herault, and X. Estivill Aneuploidy: From a Physiological Mechanism of Variance to Down Syndrome Physiol Rev, July 1, 2009; 89(3): 887 - 920. [Abstract] [Full Text] [PDF]

				D. K. Slonim, K. Koide, K. L. Johnson, U. Tantravahi, J. M. Cowan, Z. Jarrah, and D. W. Bianchi Functional genomic analysis of amniotic fluid cell-free mRNA suggests that oxidative stress is significant in Down syndrome fetuses PNAS, June 9, 2009; 106(23): 9425 - 9429. [Abstract] [Full Text] [PDF]

				F. K. Wiseman, K. A. Alford, V. L.J. Tybulewicz, and E. M.C. Fisher Down syndrome--recent progress and future prospects Hum. Mol. Genet., April 15, 2009; 18(R1): R75 - R83. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 97/1/892    most recent 00626.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (8) Citing Articles via Google Scholar Google Scholar Articles by Best, T. K. Articles by Galdzicki, Z. Search for Related Content PubMed PubMed Citation Articles by Best, T. K. Articles by Galdzicki, Z.