The overall goal of this procedure is to characterize GABA B receptor mediated effects in identified hippocampal parvalbumin positive inter neurons. This is accomplished by first producing high quality acute hippocampal slices. The second step is to use a video microscope to select neurons based on venous YFP expression and known characteristics of paral albumin positive neurons.
Next whole cell patch clamp recordings are obtained and either extracellular stimulation or paired recordings are used to examine GABA B receptor mediated effects. The final step is to visualize the neurons and to confirm that the recorded neurons are in fact par albumin positive, and are either para somatic or dendritic inhibitory neurons. Ultimately, this method enables the characterization of GABA B receptor mediated inhibitory effects and allows unequivocal classification of the recorded inter neurons.
This method can answer key questions in the inter neuron field. For example, how inter neurons communicate with each other, how their excitability, their synaptic output is controlled by neuromodulatory systems. Though this method provides insight into GABAergic, control of hippocampal into neurons, it can also be applied to other brain regions, neuromodulatory systems, or cell types.
Take the brain from a 17 to 24 day old transgenic rat expressing fluorescent venous YFP kept in semi frozen carbogen sucrose, A CSF using a scalpel, remove the frontal third of the cortex, the cerebellum, and separate the hemispheres. Remove the dorsal surface of the cortex to provide a flat surface to glue the brain for sectioning. Glue the hemispheres onto the viome stage with the viome bath filled with rozen carbogen native sucrose.
A CSF cut 300 micrometer transverse slices of the hippocampal formation. Transfer each slice to a submerged holding chamber containing carbogen sucrose, A CSF at 35 degrees Celsius. 30 minutes After placing the last slice in the warmed sucrose, A CSF transfer the slices to room temperature for storage.
Pull patch pipettes from glass capillaries so that a pipette resistance of two to four mega ohms is achieved. When filled, transfer a hippocampal slice to the recording chamber, holding it in place with a platinum ring strung with single fibers of silk. Place the chamber into the recording setup and begin perfusion with carbogen warmed recording A CSF at a flow rate of five to 10 milliliters per minute.
Use a video camera to assess the slice quality under I-R-D-I-C optics at 40 x objective magnification. Assume the slice is of good quality. If a large number of round moderately contrasted ca one parametal cells can be seen in the stratum parameter at depths of 20 to 30 micrometers below a smooth and lightly cratered surface.
Using epi fluorescent illumination, identify putative fast spiking inter neurons as those expressing venous YFP with large multipolar somatic in or near to the stratum parama. Do next, fill the patch pipettes with a solution containing a low and physiologically relevant chloride concentration for identification of post-synaptic currents and place it in the pipette holder on the head stage. Then apply a low positive pressure through the tube line.
Lower the pipette to the surface of the slice, slightly offset to the center of the selected neuron. Increase the pressure to 70 to 80 millibars and rapidly lower the pipette through the slice to just above the soma of the selected cell. Press the pipette against the cell membrane to produce a dimple on it.
Perform this step swiftly. In order to prevent biotin labeling of neighboring cells, create a giga ohm seal by releasing the pressure and simultaneously applying a negative 20 millivolt voltage command to the pipette. Once sealing begins, reduce the voltage command to the expected resting membrane potential typically between negative 70 and negative 60 millivolts.
Now apply a short pulse of negative pressure to rupture the membrane patch, thereby achieving the whole cell configuration. Using current clamp mode, identify fast spiking inter neurons by their response to a family of hyperpolarizing to depolarizing current pulses. To observe synaptically evoked responses, position an extracellular stimulation electrode in the slice at the border of the stratum radium and stratum laun oum molecular recording.
In voltage clamp mode, use an isolated constant voltage stimulator to deliver either single or 200 hertz trains of five 50 volt electrical stimuli to the pre-synaptic axons every 20 seconds. Next, apply ionotropic glutamate receptor antagonists to the bath in order to reveal the isolated monos. Synaptic IPSC.
Further isolate the GABA B receptor mediated IPSC with application of a GABA a receptor antagonist. To begin paired recordings. First, establish a whole cell recording of a pre-synaptic inter neuron as before and confirm the fast spiking phenotype.
Next, patch a CA one parametal cell within a 20 to 100 micrometer distance of the inter neuron holding the presynaptic inter neuron. In current clamp mode. Apply brief SRA threshold depolarizing current pulses to elicit action potentials in the inter neuron.
If a synaptic connection is present, action potentials in the inter neuron result in IPCs in the voltage clamped ca one parametal cell. Once a connection is established, elicit pairs of action potentials in the pre-synaptic fast spiking inter neuron with a typical paired pulse protocol of two depolarizing stimuli with a 50 millisecond interval. After collecting the control traces, apply the selective GABA B receptor agonist baclofen to the perfusing A CSF, thus activating GABA B receptors.
Next, apply the antagonist CGP 55 8 45 to fully block the receptor mediated effects. Once the recording is complete, slowly withdraw the pipette from the cell body in voltage clamp. As the series measured resistance increases, reduce the membrane voltage to negative 40 millivolts to seal the somatic membrane by forming an outside out patch.
Following the recordings, fix the slices by immersion in 4%para formaldehyde with 0.1 molar phosphate buffer overnight at four degrees Celsius washing phosphate buffer, then in phosphate buffered saline and block with 10%normal goat serum 0.03%TRITTON X 100 and 0.05%Sodium azide for one hour at room temperature to label for par albumin expression, use an anti parvalbumin monoclonal mouse antibody diluted in a solution containing 5%normal goat serum 0.3%TRITTON X 100 and 0.05%Sodium azide in PBS incubate the primary antibodies for two to three days at four degrees Celsius. Following incubation, rinse the slices thoroughly in PBS apply fluorescent anti-US secondary antibodies along with a biotin binding protein strippin conjugated to a fluorochrome and incubate in a solution containing 3%normal goat serum 0.1%tritton X 100 and 0.05%Sodium diluted in PBS incubate overnight at four degrees Celsius. The next day liberally rinse the slices two to three times with PBS followed by two to three rinses in phosphate buffer.
Mount the slices on glass slides and use a 300 micrometer agar spacer to prevent the slice from collapsing. Then cover slip the slices with a fluorescent mounting medium and seal with nail varnish. Assess the par albumin immunoreactivity of the inter neuron with a high magnification and numerical aperture.
Objective lens cells are deemed immunoreactive for par albumin. If immuno labeling is seen to align with the biotin labeled structures, take a Z axis series of images with a laser scanning confocal microscope, imaging fluorescent Aden for morphological identification and three dimensional reconstruction. Then reconstruct the image into neuron in three dimensions, incorporating dendritic and axonal arborization.
The compound synaptic response of a fast spiking inter neuron in response to a single extracellular stimulus is shown here. Application of the ionotropic glutamate receptor antagonists, DNQX and A PV isolates, the monos synaptic inhibitory post-synaptic current or IPSC application of the gabaa receptor antagonist. Gazin abolished the fast component of the IPSC.
A train of five stimuli revealed a slow GABA B receptor mediated IPSC, which was blocked by subsequent application of CGP. These traces show action potentials in the pre-synaptic, fast spiking inter neuron and short latency unitary I PSCs in CA one parametal cells under control conditions after bath, application of baclofen and subsequent application of CGP. Note that baclofen reduces the IPSC amplitude by approximately 50%whereas the GABA B receptor antagonist resulted in an almost full recovery of the IPSC amplitude.
A time course plot of the IPSC amplitude shows the effect of baclofen and CGPA fast spiking inter neuron shown at 20 x magnification had a somata located at the border of the stratum radium and parama doll of the ca one area with dendrites running vertically and spanning all layers. Whilst the majority of the axon is found in and around the cell body layer as typical for basket cells, A 60 x magnification image shows typical baskets of axons forming around putative ca one parametal salata, which are marked by the orange asterisk. A three dimensional reconstruction of the same inter neuron is shown here with the soma and dendrites in black and the axon in red.
The layers of the ca one are delineated in blue Once mastered. This technique can be performed in one day with postdoc morphological analysis performed on several slices at once After its development. This combined approach paved the way for researchers of hippocampal and cortical micro circuits to explore the morphological physiological diversity as well as the structure function interrelationship of inter neuron types.
