Comparative Effects of Event Detection Methods on the Analysis and Interpretation of Ca\u3csup\u3e2+\u3c/sup\u3e Imaging Data

Calcium imaging has gained substantial popularity as a tool to profile the activity of multiple simultaneously active cells at high spatiotemporal resolution. Among the diverse approaches to processing of Ca2+ imaging data is an often subjective decision of how to quantify baseline fluorescence or F0. We examine the effect of popular F0 determination methods on the interpretation of neuronal and astrocyte activity in a single dataset of rats trained to self-administer intravenous infusions of cocaine and compare them with an F0-independent wavelet ridgewalking event detection approach. We find that the choice of the processing method has a profound impact on the interpretation of widefield imaging results. All of the dF/F0 thresholding methods tended to introduce spurious events and fragment individual transients, leading to smaller calculated event durations and larger event frequencies. Analysis of simulated datasets confirmed these observations and indicated substantial intermethod variability as to the events classified as significant. Additionally, most dF/F0 methods on their own were unable to adequately account for bleaching of fluorescence, although the F0 smooth approach and the wavelet ridgewalking algorithm both did so. In general, the choice of the processing method led to dramatically different quantitative and sometimes opposing qualitative interpretations of the effects of cocaine self-administration both at the level of individual cells and at the level of cell networks. Significantly different distributions of event duration, amplitude, frequency, and network measures were found across the majority of dF/F0 approaches. The wavelet ridgewalking algorithm broadly outperformed dF/F0-based methods for both neuron and astrocyte recordings. These results indicate the need for heightened awareness of the limitations and tendencies associated with decisions to use particular Ca2+ image processing pipelines. Both quantification and interpretation of the effects of experimental manipulations are strongly sensitive to such decisions

Altered Gating of K\u3csub\u3ev\u3c/sub\u3e1.4 in the Nucleus Accumbens Suppresses Motivation for Reward

Deficient motivation contributes to numerous psychiatric disorders, including withdrawal from drug use, depression, schizophrenia, and others. Nucleus accumbens (NAc) has been implicated in motivated behavior, but it remains unclear whether motivational drive is linked to discrete neurobiological mechanisms within the NAc. To examine this, we profiled cohorts of Sprague-Dawley rats in a test of motivation to consume sucrose. We found that substantial variability in willingness to exert effort for reward was not associated with operant responding under low-effort conditions or stress levels. Instead, effort-based motivation was mirrored by a divergent NAc shell transcriptome with differential regulation at potassium and dopamine signaling genes. Functionally, motivation was inversely related to excitability of NAc principal neurons. Furthermore, neuronal and behavioral outputs associated with low motivation were linked to faster inactivation of a voltage-gated potassium channel, Kv1.4. These results raise the prospect of targeting Kv1.4 gating in psychiatric conditions associated with motivational dysfunction

Methamphetamine induces Shati/Nat8L expression in the mouse nucleus accumbens via CREB- and dopamine D1 receptor-dependent mechanism

Shati/Nat8L significantly increased in the nucleus accumbens (NAc) of mice after repeated methamphetamine (METH) treatment. We reported that Shati/Nat8L overexpression in mouse NAc attenuated METH-induced hyperlocomotion, locomotor sensitization, and conditioned place preference. We recently found that Shati/Nat8L overexpression in NAc regulates the dopaminergic neuronal system via the activation of group II mGluRs by elevated Nacetylaspartylglutamate following N-acetylaspartate increase due to the overexpression. These findings suggest that Shati/Nat8L suppresses METH-induced responses. However, the mechanism by which METH increases the Shati/Nat8L mRNA expression in NAc is unclear. To investigate the regulatory mechanism of Shati/Nat8L mRNA expression, we performed a mouse Shati/Nat8L luciferase assay using PC12 cells. Next, we investigated the response of METH to Shati/Nat8L expression and CREB activity using mouse brain slices of NAc, METH administration to mice, and western blotting for CREB activity of specific dopamine receptor signals in vivo and ex vivo. We found that METH activates CREB binding to the Shati/Nat8L promoter to induce the Shati/Nat8L mRNA expression. Furthermore, the dopamine D1 receptor antagonist SCH23390, but not the dopamine D2 receptor antagonist sulpiride, inhibited the upregulation of Shati/Nat8L and CREB activities in the mouse NAc slices. Thus, the administration of the dopamine D1 receptor agonist SKF38393 increased the Shati/Nat8L mRNA expression in mouse NAc. These results showed that the Shati/ Nat8L mRNA was increased by METH-induced CREB pathway via dopamine D1 receptor signaling in mouse NAc. These findings may contribute to development of a clinical tool for METH addiction

Behavioral History of Withdrawal Influences Regulation of Cocaine Seeking by Glutamate Re-Uptake.

Withdrawal from cocaine regulates expression of distinct glutamate re-uptake transporters in the nucleus accumbens (NAc). In this study, we examined the cumulative effect of glutamate re-uptake by multiple excitatory amino acid transporters (EAATs) on drug-seeking at two different stages of withdrawal from self-administered cocaine. Rats were trained on fixed ratio 1 (FR1), progressing to FR5 schedule of reinforcement. After one day of withdrawal, microinfusion of a broad non-transportable EAAT antagonist, DL-threo-beta-benzyloxyaspartate (DL-TBOA), into the NAc shell dose-dependently attenuated self-administration of cocaine. Sucrose self-administration was not affected by DL-TBOA, indicating an effect specific to reinforcing properties of cocaine. The attenuating effect on cocaine seeking was not due to suppression of locomotor response, as DL-TBOA was found to transiently increase spontaneous locomotor activity. Previous studies have established a role for EAAT2-mediated re-uptake on reinstatement of cocaine seeking following extended withdrawal and extinction training. We found that blockade of NAc shell EAATs did not affect cocaine-primed reinstatement of cocaine seeking. These results indicate that behavioral history of withdrawal influences the effect of re-uptake mediated glutamate clearance on cocaine seeking. Dynamic regulation of glutamate availability by re-uptake mechanisms may impact other glutamate signaling pathways to account for such differences

Comparison between α1β2γ2 and α3β2γ2 GABA-channel currents elicited by long-lasting GABA application

<p><b>Copyright information:</b></p><p>Taken from "Desensitization and binding properties determine distinct α1β2γ2 and α3β2γ2 GABA receptor-channel kinetic behavior"</p><p></p><p>The European Journal of Neuroscience 2007;25(9):2726-2740.</p><p>Published online 01 May 2007</p><p>PMCID:PMC1950087.</p><p>© The Authors (2007). Journal Compilation © Federation of European Neuroscience Societies and Blackwell Publishing Ltd</p> (A) Channel activity evoked by long-lasting applications of GABA to outside-out patches excised from human embryonic kidney (HEK) cells expressing α1β2γ2 (top, 100 µ GABA) and α3β2γ2 (bottom, 600 µ GABA) receptors (holding potential, −100 mV). Inset: a fraction of the single-channel current activity at an expanded time scale. Corresponding calibration bars are shown in the lower right corner of traces. (B) Distribution of open time (top) and burst durations (bottom) for the patches in A. Distributions are fitted with a double exponential function. (C) Summary of chord conductance and open times characterizing the main conductance state of single-channel current in at least 10 patches excised from HEK cells expressing α1β2γ2 and α3β2γ2 receptors. (D) A comparison of average burst length of α1β2γ2 and α3β2γ2 channels in these patches and the parameters (A, τ and τ) from double exponential fitting of burst duration distributions as in B. * < 0.05 indicates a significant difference between subunits

Comparison between α1β2γ2 and α3β2γ2 GABA-channel currents elicited by brief GABA applications

<p><b>Copyright information:</b></p><p>Taken from "Desensitization and binding properties determine distinct α1β2γ2 and α3β2γ2 GABA receptor-channel kinetic behavior"</p><p></p><p>The European Journal of Neuroscience 2007;25(9):2726-2740.</p><p>Published online 01 May 2007</p><p>PMCID:PMC1950087.</p><p>© The Authors (2007). Journal Compilation © Federation of European Neuroscience Societies and Blackwell Publishing Ltd</p> (A) Multiple records of 1 s illustrating channel activity evoked by 2 ms applications of GABA to outside-out patches in two patches excised from human embryonic kidney (HEK) cells expressing single α1β2γ2 (left, 10 m GABA) and α3β2γ2 (right, 50 m GABA) channels (holding potential, −100 mV). The mean current resulting from the average of the single-channel records is shown in the bottom trace (right and left panel). (B) Two records of 2 s illustrating channel activity evoked by 2 ms applications of GABA to outside-out patches in two patches excised from HEK cells expressing many α1β2γ2 (top, 10 m GABA) and α3β2γ2 (bottom, 50 m GABA) channels (holding potential, −100 mV). Individual channel activity is visible in the tail of these currents. (C) Comparison of mean burst length for α3β2γ2 and α3β2γ2 channels measured during the entire recording period (4 s). (D) Comparison of mean burst lengths in three epochs: 0–500 ms (Epoch 1), 500–1000 ms (Epoch 2) and >1000 ms (Epoch 3). Note that burst durations tend to decrease with time after applications. (E) Comparison of frequencies of late openings (in Epoch 3) for α1β2γ2 and α3β2γ2 receptors. Note that frequency of late openings is several times larger in α3β2γ2 receptors. * < 0.05 indicates a significant difference between subunits

Recovery process in the paired-pulse experiments differs between α1β2γ2 and α3β2γ2 channels

<p><b>Copyright information:</b></p><p>Taken from "Desensitization and binding properties determine distinct α1β2γ2 and α3β2γ2 GABA receptor-channel kinetic behavior"</p><p></p><p>The European Journal of Neuroscience 2007;25(9):2726-2740.</p><p>Published online 01 May 2007</p><p>PMCID:PMC1950087.</p><p>© The Authors (2007). Journal Compilation © Federation of European Neuroscience Societies and Blackwell Publishing Ltd</p> (A) Examples of normalized averages of five traces evoked by two successive applications of 2 ms GABA (10 and 50 m) pulses separated by 30 ms intervals in a patch excised from human embryonic kidney (HEK) cells expressing α1β2γ2 (left) and α3β2γ2 (right) receptors. (B) Comparison of the recovery time course of the second response from desensitization in at least 15 patches excised from HEK cells expressing α1β2γ2 (◊) and α3β2γ2 (•) receptors. The percent recovery at each designated separation of two brief GABA pulses is calculated as described in Materials and methods, and plotted against the interpulse interval. Each data point represents the mean ± SEM of eight patches studied. (C) The data in B are shown at an expanded time scale to better illustrate the components of the double exponential fitting of the recovery

Comparison of rise time for currents elicited by non-saturating GABA in α1β2γ2 and α3β2γ2 channels

<p><b>Copyright information:</b></p><p>Taken from "Desensitization and binding properties determine distinct α1β2γ2 and α3β2γ2 GABA receptor-channel kinetic behavior"</p><p></p><p>The European Journal of Neuroscience 2007;25(9):2726-2740.</p><p>Published online 01 May 2007</p><p>PMCID:PMC1950087.</p><p>© The Authors (2007). Journal Compilation © Federation of European Neuroscience Societies and Blackwell Publishing Ltd</p> (A) Typical examples of normalized α1β2γ2- and α3β2γ2-mediated current responses to a non-saturating (300 µ) GABA concentration. (B) Summary of data derived for the assessment of the onset rate of current elicited at four increasing non-saturating GABA concentrations in at least five patches excised from human embryonic kidney cells expressing α1β2γ2 and α3β2γ2 receptors. * < 0.05 indicates a significant difference. Note that the difference in the onset rate is so large between the two combinations that the vertical axis is shown in logarithmic scale