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Techniques

Combined patch clamp recording
and mRNA expression profiling in individual neurons

Patch Clamp Recording & Expression Profiling. Combined patch clamp recording and expression profiling in individual neurons. This is a powerful technique to explore the causative relationship between gene expression alterations and functional ion channel changes in individual neurons. The function of receptors expressed in neurons can be characterized, and then the relative levels of expression of multiple mRNA species can be determined in the same neuron. In the left panel, the experimental paradigm is schematized. Neurons are recorded, the cytoplasm collected in the electrode, reverse transcribed, and then amplified. This probe is then used to hybridize with cDNA arrays, and the relative levels of expression of various mRNA species determined. In the right panel, actual profile data from an individual dentate granule neuron from an animal 1 week post pilocarpine-induced Status Epilepticus is depicted, with a nylon phosphorimaged cDNA slot-blot and its quantitation for mRNAs encoding major neurotransmitter receptor subunits as well as mRNAs encoding transporters and housekeeping genes.

Hierarchical cluster analysis of ~ 1.3 K genes in the rat model of temporal lobe epilepsy. The microarray cluster analysis allows to identify drug targets for new therapeutical approaches and therefore find a better help or even cure for epileptic patients. Status Epilepticus (SE) induced in rats by pilocarpine injection. Total RNA isolated from dentate gyrus (DG) at 2 h, 24 h, 7 days post SE and controls. Targets hybridized to Rat Neurobiology U34 Arrays (Affymetrix). Data analysis performed using GeneSpring software. The cluster analysis of 25 DG samples yielded 2 clusters that correlate well with sample distribution between control (red branches) and SE (yellow branches) groups. The cluster analysis demonstrates that SE induced significant changes in gene expression in post-SE animals, and suggests that at least some of these gene alterations may be a part of epileptogenesis. The cluster analysis also revealed a number of clusters for up- and downregulated genes, primarily at 2 h post SE suggesting correlated function of those genes.

Imaging

 
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A dentate granule neuron was recorded under Dodt contrast two-photon laser microscope. The fluorescent image of the cell could be observed using two-photon excitation (Fig 1). The dendritic spines of the neuron can be seen under high magnifications (Fig 2). The laser can be tuned to a different wave-length and target the dendritic spines to uncage caged neurotransmitter, such as caged GABA or caged glutamate, to study the receptor properties at individual spines.

Acutely isolated neuron:

  1. The general procedure to acutely isolate hippocampal neurons including dentate granule cells and CA1 neurons using the combination of enzymatic and mechanical approaches.
  2. Fast drug application and change system was accomplished by computer-triggered fast step perfusion device and the technique of lifting up of recorded cells from the Petri-dish bottom.
  3. Parameters calculated and solution change time. (A) Using GABAR-gated current trace as an example to introduce the measured parameters. Current density was calculated as the maximum current amplitude (Ampmax) divided by the cell capacitance. Rise time was measured as the time needed for the current rose from 10-90% of Ampmax. Percentage of current desensitization was calculated as the amount of current desensitized during 2-second agonist application (Ampdes) divided by the Ampmax then timed 100%. Both current desensitization time constant (?des) and deactivation time constant (?deact) were measured by fitting curves with first-order exponential function using Marquardt-Levenberg nonlinear least-squares algorithm (Clampfit 8.01). Total charge transfer density was calculated by the integral area under the current trace then normalized to cell capacitance. (B) The current trace depicting an open tip electrode switched from different concentration of saline solution for 2 seconds. A rectangular shape of current trace indicated that there was no concentration fluctuation during the 2-second compound application. The current rise time from 0-100% was 2.6 ms which made valid the kinetic data in the current study. (C) Multiple ligand-gated neurotransmitter receptor functions can be examined on the same acutely isolated cells.

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Determining the role neurotransmitter recycling plays in regulating inhibitory synaptic strength

Regulation of synaptic vesicle neurotransmitter content is complex, dynamic and multifactorial. Activity-dependent alterations in vesicular content play a significant role in regulating synaptic strength, particularly at inhibitory synapses. One major laboratory direction is to study factors regulating synaptic vesicle GABA content in normal and pathological brain.

Schematic depicting the sources of g-aminobutyric acid (GABA) contributing to synaptic vesicles refilling in neurons. In synaptic vesicles of inhibitory neurons, GABA is locally synthesized mainly by glutamic acid decarboxylase (GAD) and is packaged into vesicles by the vesicular GABA transporter, VGAT. In addition, GABA is recycled from synaptic cleft through membrane GABA transporter, GAT. Glutamate is transported either by EAAC1 (excitatory amino acid carrier-1; EAAT-3) located on GABA neurons or by GLT-1 (glutamate transporter-1; EAAT-2) located on astrocytes. The latter pathway provides an efficient way to recycle glutamate via the glutamate-glutamine cycle. Glutamate, taken up by astrocytes, is converted by glutamine synthetase into glutamine, which is then released and taken up by neuronal system A (SA-1) transporters into GABAergic neurons. Neuronal glutamine then can be converted into glutamate via phosphate activated glutaminase (PAG) in the mitochondria. It is our hypothesis that the astrocytic glutamate-glutamine cycle contributes dynamically to GABA synaptic vesicle refilling.

What is the role of birth of new neurons in the development of epilepsy?

There is a tremendous increase in the birth of new neurons in the dentate gyrus as a result of brain injuries that cause epilepsy. We have been GFP labeling and recording from these newly born neurons in the adult brain (termed adult born neurons), and characterizing their properties and role in hippocampal seizure generation.

Figure 1. GFP+ adult born neurons co-label with NeuN (marker for mature neurons) and can appear ectopically in the hilus (blue arrow). GFP+ cell with glial morphology does not co-label with NeuN (gray arrow). Image taken 4 wks following viral infection in an epileptic animal. Red, NeuN; Green, GFP; Yellow, GFP and NeuN; GCL= granule cell layer, scale bar=10 µm.

Figure 2. Adult born neuron recurrent collaterals.. In epileptic animals, 8 wks following viral infection, GFP+ adult born born neurons commonly (6/6 rats) extend recurrent axons (arrow) into the molecular layer. Recurrent axons begin to sprout within 4 wks of viral infection (3/5 rats.) Scale bar, 10 µm.

Figure 3. Adult born neurons can innervate the opposite blade of the dentate gyrus potentially generating excitatory synapses between granule cells in the buried and exposed blades. Image taken 4 wks after viral infection in epileptic animal. Arrows follow axon across the hilus. Red, NeuN; Green, GFP; scale bar, 10 µm.