Seizure Threshold
Materials and Methods
Pulse Generator, # 57800-001, UGO BASILE Biological Research Apparatus, Stoelting, USA (modified at TJL to generate a frequency of 299Hz and a pulse width of 9.9msec)
Stimulation Electrodes, # 7803, Stoelting, USA
Foot Pedal, 7805, Stoelting, USA
Tetracaine hydrochloride (99%), Sigma
Pentylenetetrazole, Sigma
Syringe, 0.5 ml, hypodermic needle
Clear plastic box
Timer
Datasheet
Working Solution
5% Tetracaine hydrochloride in saline, stored in light-protected bottle.
Primary Screen
The animals are tested for the occurrence of minimal clonic seizures. For that purpose the pulse generator is set at parameters representing the estimated critical current intensity for minimal seizure induction in 3% of the population: for 13 weeks old C57BL/6J mice the current is set to 7mA for female, and to 8.5mA for male mice. For 6-10 weeks old C57BL/6J mice the current is set to 6.5 mA for female, to 8.0 mA for male. In both cases the pulse frequency, width and duration are set to 299 Hz, 1.6 ms and 0.2 s.
Prior to electrode placement, one drop of Tetracaine-solution is applied to each eye. Following stimulation, the mouse is placed into a clear plastic box for observation and the behavior is classified according to seizure duration and degree of generalization (0= no observable symptoms, 3= rearing, forelimb clonus and jaw clonus, 5= tonic hind limb extension seizure). Intermediate values (1, 2, 4) are also noted for future reference. Animals exhibiting low seizure thresholds are further investigated by examining their susceptibility to seizure inducing and seizure preventing drugs, i.e. pentylenetetrazole, valproate, phenytoin, ethosuximide, lamotrigine.
Secondary Screens
Susceptibility to pentylenetetrazole (PTZ) induced seizures.
To test mice with a low electroconvulsive threshold for sensitivity to PTZ, mutant mice and the appropriate genetic controls are injected with a sub-threshold dose of PTZ (40 mg/kg, i.p.), and monitored for 30 minutes. To test mice with a high electroconvulsive threshold for sensitivity to PTZ, mice are injected with a super or peri-threshold dose of PTZ (80 mg/kg, i.p.). Latencies to the first three occurrences of the following seizure endpoints are recorded: twitch - stage 2; repeated clonic seizures or abortive generalized seizure - stage 3; fully generalized seizure - stage 4; tonic-hind limb extension seizure - stage 5. Average latencies to particular endpoints are analyzed parametrically by |t|-test; incidence of a particular endpoint is analyzed by the contingency Chi-square or Fisher's exact test, depending on the number of samples.
Determination of the ED50s for anti-epileptic drugs (AED) (for parental strains, and for mutant strains when 30 mice are available)
In order to obtain the ED50 (effective dose for 50% response) for a given AED, the current is fixed at the CC97 measured for the strain, i.e. the estimated critical current intensity for minimal seizure induction in 97% of the population, and the drug concentration is varied. The current response curve to determine the CC97 is determined by the staircase procedure, and typically requires at least 20 subjects to obtain reliable results. To determine the ED50 for an AED, the time to peak effect (TPE) of the drug is first determined, and 4-5 subjects are tested preliminary at 3-4 drug concentrations based on ED50 estimates obtained either by other investigators, or by using the drug in other strains, or by an educated guess. To complete the experiment, the remaining subjects are tested at doses estimated to flank the ED50. Typically, at least 40 subjects are required to obtain reliable results for AED response curves.
Estimating whether mutant mice have an abnormal response to AED’s (when smaller numbers of mutants are available).
To estimate whether mutants are more readily protected by an AED, at the CC97 (determined as above) for the respective strain, 8-10 mutants are tested at the control strain’s ED15 (predetermined as above) simultaneously with 8-10 controls. To estimate whether mutants are less readily protected by an AED, they are similarly tested at the control strain’s ED85, simultaneously with controls. Differences between groups are assessed using Fisher’s exact test.
Electroencephalogram Recording (EEG) *
Materials and Methods
* (This protocol was developed in consultation with Xiaoxi Qiao [Louisana State University] and Margaret Sutherland [George Washington University, Washington DC])
Epi-dural Electrodes (items purchased from Fisher Scientific, VWR [USA] or in-house Lab store unless noted otherwise)
Teflon coated silver wire (0.005” diameter), World Precision Instruments, USA
GF-4 or GF-6 ultra miniature connectors with GM-4 (GM-6) plugs (Microtech, Inc., USA)
Two-sided foam tape (Hardware store)
Duco Cement (Hardware store)
Soldering iron with fine tip, Pace Electronics, Inc., USA
Solder
Straight # 4 or finer forceps
Straight scissors
Bench top vice
Magnifying lamp
Surgery (use sterile material)
Standard dual stereotax with mouse adaptor, Stoelting, Co., USA
Minimite cordless drill, Dremel
Dremel drill bits, 1/64 inches
Electric shaver
Microspatula
Scalpel, blades (#10)
Q-tips
70% EtOH
Microspatula, rounded end 8”
Syringes, needles
Betadine
Tribromoethanol, Sigma, USA
Jet repair powder, clear +,
Jet acrylic liquid, Lang Dental Products, USA, or
Fleck’s Zinc cement liquid Type 1, class 2
and snow-white powder (Mizzy, Inc.)
Glass plate, toothpicks
Superglue Gel
GF-4 socket with silver wires and two-sided foam tape base attached (previously prepared)
Lamp with magnifying glass or access to dissecting scope
Preparation of epi-dural electrodes: Turn on the soldering iron, and test the fitting of the female socket (GF-4) and the male connector (GM-4), i.e. the connector should fit easily and smoothly into the socket; if necessary ‘widen’ the GF-4 socket carefully by inserting and turning a forceps; fix socket in the vice (prawns protruding out of the vice), cut approximately 5-7cm of silver wire, and clear the Teflon off the tip (about 0.5mm).
Heat the GF-4 with the soldering iron, and dab a small amount of solder on each prawn that protrudes from the bottom of the GF-4, preferably on the surface of prawn # 1 and 3 on one side, and of # 2 and 4 on the other side (Figure A). Hold the cut and stripped off end of the silver wire close to the attached solder while holding the heated soldering iron to the other side of the prawn; the wire will attach quickly. Repeat, so a piece of silver wire is attached to each of the 4 (or 6; we usually use GF-6 to allow for simultaneous EMG recordings) prawns. Firmly tug on each silver wire to make sure it is well attached, and carefully fold the wires up, 2 (or 3) on each side. Cut a small piece of two-sided foam tape, large enough to hold the GF-4; apply Duco cement, gently press the GF-4 onto it, prawns touching the cement; let settle and dry for 24 hours, before using in surgery. Arrange for the GM-4 to be attached to leads, and to be connected to the electrode board; these leads are made in-house by a very patient and skilled person!
Surgery and placement of epi-dural electrodes: Mice are anesthetized with tribromoethanol (Avertin - 400mg/kg; about 21-25ml/10g body weight), and the status of anesthesia is determined by checking the withdrawal reflex (i.e. the absence of it) and observing the rate of respiration (repeat throughout the procedure). Subsequently, the head of the mouse should be shaved, the skin cleaned with 70% EtOH and Betadine, and the animal should be placed into the mouse stereotax; confirm proper position of the incisor bars by gently pulling the tail of the mouse to stretch it’s body. Make a small incision starting just rostral to ears to just caudal to eyes, gently push the skin back, and clean the skull with Q-tips. Once the skull is completely cleaned and dried, drill four burr holes into the skull, two into each side, approximately 1 mm lateral to midline, and 1 to 2 mm anterior to and 2 to 4mm posterior to Bregma (Figure B).
Trim the double-sided foam strip under the GF-4 mount to size if necessary, carefully adjust the silver wires of the GF-4 so they are straight and horizontally oriented. Peel the protective paper off the foam strip, place 1-2 drops of Superglue on midline between the burr holes, and gently place and press the GF-4 socket mount on to the skull; release pressure, then gently press again.
If necessary, cut the silver wires to an appropriate length, so they can be guided comfortably into the burr holes and under the skull. Carefully bent the silver wires into position to insert each of the wires through the nearest burr hole, and, with the help of a magnifying lamp or dissection scope and fine, straight forceps, slide each wire under the skull so that it lies on top of the dura mater (Figure C and D). Make sure those parts of wire that remain outside the burr holes are bent in such a way that they remain close to the GF-4. Following insertion of the wires, place another drop of superglue at the rostral and caudal aspect of the socket mount to assure lasting adhesion.
Prepare dental cement of choice on a glass plate, and quickly (but carefully) spread it with a toothpick around the base of the GF-4, covering the contact area between skull and GF-4, including prawns and wires up to the base of the GF-4. Allow the dental cement to set for 2-3 minutes, then remove the mouse from the stereotax by placing it into a clean box (under a heating lamp if necessary). The animal should recover for 24-48 hours before EEG recordings are made.
EEG recording: Restrain the mouse and connect the GF-4 head mount to the GM-4-leads, making sure the orientation of the GM-4 leads is in agreement with the sequence of leads on the electrode board. The amplifier software should be set to differentially record between all four electrode wires. Recordings are taken on at least two different days for a minimum of two hours.
