Molecular and Electrophysiological Characterization of GFP-Expressing CA1 Interneurons in GAD65-GFP Mice

The use of transgenic mice in which subtypes of neurons are labeled with a fluorescent protein has greatly facilitated modern neuroscience research. GAD65-GFP mice, which have GABAergic interneurons labeled with GFP, are widely used in many research laboratories, although the properties of the labeled cells have not been studied in detail. Here we investigate these cells in the hippocampal area CA1 and show that they constitute ∼20% of interneurons in this area. The majority of them expresses either reelin (70±2%) or vasoactive intestinal peptide (VIP; 15±2%), while expression of parvalbumin and somatostatin is virtually absent. This strongly suggests they originate from the caudal, and not the medial, ganglionic eminence. GFP-labeled interneurons can be subdivided according to the (partially overlapping) expression of neuropeptide Y (42±3%), cholecystokinin (25±3%), calbindin (20±2%) or calretinin (20±2%). Most of these subtypes (with the exception of calretinin-expressing interneurons) target the dendrites of CA1 pyramidal cells. GFP-labeled interneurons mostly show delayed onset of firing around threshold, and regular firing with moderate frequency adaptation at more depolarized potentials

Precision of Inhibition : Dendritic Inhibition by Individual GABAergic Synapses on Hippocampal Pyramidal Cells Is Confined in Space and Time

Inhibition plays a fundamental role in controlling neuronal activity in the brain. While perisomatic inhibition has been studied in detail, the majority of inhibitory synapses are found on dendritic shafts and are less well characterized. Here, we combine paired patch-clamp recordings and two-photon Ca(2+) imaging to quantify inhibition exerted by individual GABAergic contacts on hippocampal pyramidal cell dendrites. We observed that Ca(2+) transients from back-propagating action potentials were significantly reduced during simultaneous activation of individual nearby inhibitory contacts. The inhibition of Ca(2+) transients depended on the precise spike-timing (time constant < 5 ms) and declined steeply in the proximal and distal direction (length constants 23-28 μm). Notably, Ca(2+) amplitudes in spines were inhibited to the same degree as in the shaft. Given the known anatomical distribution of inhibitory synapses, our data suggest that the collective inhibitory input to a pyramidal cell is sufficient to control Ca(2+) levels across the entire dendritic arbor with micrometer and millisecond precision

Improved Hidden Markov Models for Molecular Motors, Part 1: Basic Theory

Hidden Markov models (HMMs) provide an excellent analysis of recordings with very poor signal/noise ratio made from systems such as ion channels which switch among a few states. This method has also recently been used for modeling the kinetic rate constants of molecular motors, where the observable variable—the position—steadily accumulates as a result of the motor's reaction cycle. We present a new HMM implementation for obtaining the chemical-kinetic model of a molecular motor's reaction cycle called the variable-stepsize HMM in which the quantized position variable is represented by a large number of states of the Markov model. Unlike previous methods, the model allows for arbitrary distributions of step sizes, and allows these distributions to be estimated. The result is a robust algorithm that requires little or no user input for characterizing the stepping kinetics of molecular motors as recorded by optical techniques

Improved Hidden Markov Models for Molecular Motors, Part 2: Extensions and Application to Experimental Data

Unbiased interpretation of noisy single molecular motor recordings remains a challenging task. To address this issue, we have developed robust algorithms based on hidden Markov models (HMMs) of motor proteins. The basic algorithm, called variable-stepsize HMM (VS-HMM), was introduced in the previous article. It improves on currently available Markov-model based techniques by allowing for arbitrary distributions of step sizes, and shows excellent convergence properties for the characterization of staircase motor timecourses in the presence of large measurement noise. In this article, we extend the VS-HMM framework for better performance with experimental data. The extended algorithm, variable-stepsize integrating-detector HMM (VSI-HMM) better models the data-acquisition process, and accounts for random baseline drifts. Further, as an extension, maximum a posteriori estimation is provided. When used as a blind step detector, the VSI-HMM outperforms conventional step detectors. The fidelity of the VSI-HMM is tested with simulations and is applied to in vitro myosin V data where a small 10 nm population of steps is identified. It is also applied to an in vivo recording of melanosome motion, where strong evidence is found for repeated, bidirectional steps smaller than 8 nm in size, implying that multiple motors simultaneously carry the cargo

GFP-positive GABAergic interneurons.

<p>A: Maximal projection image illustrating the distribution of GFP-labeled profiles in the CA1 layers. Calbindin (red), labeling a fraction of pyramidal cells, is only shown to facilitate recognition of the layers. B: Distribution of GFP-labeled interneurons in the hippocampal CA1 area in GAD65-GFP mice. C: Percentage of GFP-labeled cells that expressed GABA (blue), GAD67 (red) or both (purple). Data from 268 GFP cells; 10 sections. D: Example of triple immunostaining for GFP (green), GABA (blue) and GAD67 (red). Scale bars are 30 µm. Abbreviations of CA1 layers: Or - oriens; Pyr – pyramidale; Rad – radiatum; LM – lacunosum moleculare.</p

Firing properties of GFP-positive interneurons.

<p>A: Representation of all recorded GFP-labeled interneurons, arranged according to characteristics of their firing patterns and their classification in 5 groups. Each segment represents a single interneuron. Inner ring: adapting (Ad; dark blue) and strongly adapting (S-Ad; dark red) cells. Second ring: cells showing delayed onset (DO; blue) and immediate onset (IO; red) firing. Third ring: cells displaying regular (reg; light blue) and irregular (irr; light red) firing. Interneurons were divided in five groups (1–5) as indicated with yellow and green colors. The letters correspond to example cells (a–g) in B and C. B: Examples of firing patterns of example cells a–g (as indicated in A). Upper traces show firing around threshold, middle traces show responses to hyperpolarizing steps (100 pA step size) and intermediate firing and lower traces show maximal firing. C: Morphology of example cells a–g (same as in B). Dendrites are shown in black, axons in red.</p

The majority of GFP-positive interneurons contain reelin or VIP.

<p>A: Immunohistochemistry for reelin (red) and vasoactive intestinal peptide (VIP; blue). Most GFP-positive interneurons (green) contained either reelin (yellow arrowheads) or VIP (blue arrowheads). Very few GFP-positive cells were lacking both (green arrowheads). B: Immunohistochemistry for parvalbumin (PV; red) and somatostatin (SOM; blue), showing minimal overlap with GFP-positive interneurons (green arrowheads). Red and blue arrowheads point to GFP-negative parvalbumin- and somatostatin-positive interneurons. C: Summary for all GFP-positive interneurons. D: Distribution of reelin- and VIP-containing GFP-labeled interneurons in the layers of the CA1 area. Overlap between both markers are indicated with purple. Scale bars are 30 µm.</p

<b>Table 2.</b> Morphological properties of GFP-labeled interneurons.

<p>*Tangential cells have an orientation close to 90 degrees; 0 degrees reflect radially oriented cells (similar to pyramidal cells).</p><p>No significant differences in morphological parameters between groups were detected (ANOVA). Values are given as mean ± standard error.</p