This is a small wet-lab in which solutions are prepared, electrodes are constructed, etc.
This is the vibratome that is used for obtaining thick (300-400 um) slices of living tissue for in vitro recordings. The brain is kept in artificial cerebrospinal fluid during the sectioning, and the solution is kept cold to reduce metabolic demands on the brain.
This is a close-up of a slice being cut on the vibratome.
This is a horizontal electrode "puller" that is used to manufacture recording pipettes for intracellular recordings.
This is a close-up, showing a glass capillary tube in the process of being heated by a filament, and being pulled at the same time into two halves. You can see the filament glowing red around the capillary tube.
Two pipette recording electrodes are generated from each capillary tube. The tip of each of the electrodes is about 3 um in diameter. The recording pipettes are then filled with a solution that mimics the intracellular fluid of neurons.
A spacious antechamber leads into both the wet-lab, and into the room in which two in vitro recording set-ups are located. Not a bad place to analyse a little data.
This is one of the in vitro rigs for visually-guided whole-cell patch clamp recordings from individual neurons. The set-up allows for current- and voltage-clamp recordings from neurons, audio monitoring of recorded signals, bath-application of pharmacological agents to slices, "spritzing" of small quantities of drugs onto neurons, and ratiometric fluorescent imaging of intracellular calcium concentrations.
This is the recording chamber and electrode manipulators that are used for "submerged" recordings. The slice of tissue is submerged in oxygenated artificial cerebrospinal fluid that flows continuously in and out of the recording chamber. There is a manipulator holding a stimulating electrode at the left of the photo, and you can see the recording pipette in its holder at the right. The microscope allows the pipette to be guided towards an individual neuron that is also viewed on a video monitor.
This is Dr Said Kourrich, a post-doctoral fellow in the lab, in the midst of an experiment.
Here is a photograph of the video monitor which shows a nice cluster of living neurons in layer II of the entorhinal cortex.
This photograph shows the tip of a recording pipette (left) being applied to the soma of a neuron. After a very tight seal is formed between the tip of the electrode and the membrane of the cell, the membrane within the tip of the electrode is ruptured. This allows the solution inside the electrode to come in contact with the intracellular fluid so that the membrane potential can be recorded.
This is one type of data that can be recorded using the whole-cell patch clamp technique. Pulses of current of different sizes were injected into the cell through the recording electrode, and positive and negative voltage responses were observed in the membrane potential. Action potentials were evoked when positive current steps were large enough to cause the membrane potential to exceed the action potential threshold.
This is myself sitting in front of the interface recording rig.
This is Dr. Bassam Hamam recording from a slice.
This is a close-up of the "interface" recording chamber. The slice of tissue is placed on a nylon net (held by the illuminated ring) that sits at the interface between artificial cerebrospinal fluid that flows beneath the slice, and a humidified oxygenated atmosphere above the slice. Two recording pipettes have been placed in the slice in this photo (one for field potentials, and the other for intracellular recordings).
Here is a blurry photo of the screen of an oscilloscope showing an excitatory postsynaptic potential (an EPSP) in an entorhinal cortex neuron. The synaptic response peaks about 6 ms after stimulating the synaptic inputs to the neuron.
This graphs show how the mean amplitude of synaptic responses (field EPSPs) in the entorhinal cortex changed during the course of the experiment. Previously, we found that repeated "paired-pulse" (PP) stimulation causes a long-lasting reduction in synaptic strength. In this study, we found that the weakening of synapses is dependent on activation of the NMDA subtype of glutamate receptor.
This is me standing in front of a rack of equipment in the first testing room.
In the first stage of many experiments, electrodes are implanted under anesthesia into targeted sites in the rat brain using a "stereotaxic" apparatus that has arms which hold the electrodes. The permanent implantation of stimulating and recording electrodes allows a large amount of data to be collected after the rat recovers from the surgery. Each rat is tested for a period of weeks to months.
This is a figure that shows a number of electrode placements on representative sections taken from the atlas of G.Paxinos and C.Watson (The Rat Brain in Stereotaxic Coordinates, Fourth Edition, Academic Press, 1998). Structures in the brain are located at known distances from landmarks on the skull, and electrodes can therefore be precisely aimed at their targets. This figure shows histologically verified electrode placements for stimulating electrodes located in the piriform (olfactory) cortex, and for recording electrodes in the entorhinal cortex.
After recovery from surgery, electrical leads can be connected to the electrodes through a connector on top of the rat's head. The rat moves freely in the testing chamber, and the electrical leads are used to deliver very brief (0.1ms), painless stimulation pulses through the stimulating electrode. The resulting synaptic responses that are recorded from the other electrode are very small, and require a great deal of amplification. Much of the equipment contained in the rack has to do with controlling the small stimulation pulses, amplifying and filtering synaptic responses, and digitizing the recordings onto computer hard disk for later analysis.
This is Douglas Caruana, a Masters student in the lab, standing in front of the rack in the second in vivo recording room.
This is Douglas in his younger years. Even then, he was thinking forward to a career in neuroscience, and had consulted one of his idols at the time.
Synaptic responses are recorded and analysed using an automated computer software program. This screen shows responses recorded from the sensorimotor cortex following stimulation of the corpus callosum. The vertical line is a stimulation artifact, and the synaptic response peaks about 6 to 10 ms later.
Synaptic responses are also monitored using oscilloscopes. This screen shows a negative-going evoked synaptic potential that peaks about 5 ms following the stimulation pulse.
Here is an example of the type of data that can be collected from behaving animals. The graph shows the amplitude of field excitatory postsynaptic potentials (fEPSPs) in the entorhinal cortex evoked by stimulation of the piriform cortex. Following a baseline period, Òpaired-pulseÓ stimulation (PP) was delivered for 15 minutes, and this resulted in a long-lasting reduction in the size of the fEPSPs. The inset traces show superimposed synaptic responses recorded before, and after (arrow), the paired-pulse stimulation. The reduction in the size of the fEPSPs indicates that the synapses were weakened by the paired-pulse stimulation. Both reductions and increases in synaptic strength are thought to contribute to memory formation.
This is the CSBN's Technical Officer, Dave Munro. He is incredibly knowledgeable, and has designed and created many specialized devices that we use in the lab every day.
Many labs in the CSBN use part of this area for histological analysis of tissue.
Here is a student using a cryostat which is used to cut the frozen brain into very thin (40 um) slices. Sections of brain are then stained and viewed on slides in order to verify the positions of electrodes. As you see, this is often the last, happy task in many experiments.
This is a close-up showing the frozen brain on a pedestal, with the cryostat blade (beneath) holding a frozen section.