NOTE: All procedures were approved and carried out in accordance with the policies set forth by the East Carolina University IACUC. Long-Evans rats were generated from females mated with male breeders. Pups from all three treatment groups (see 1.1) were generated within a litter from the same dam. Five litters were produced and each litter was culled to 8 pups on postnatal day (PD) 3. The remaining two pups from each litter were assigned to separate experiments. Both male and female offspring (one per exposure group) were included in the study. A total of N = 27 adult offspring were examined in this study; 3 rats were excluded due to broken 3T wire leads (see 3.1.1) which were irreparable on Day 1 of ECC training.
1. Preparation of Groups, Materials, and Solutions
2. Neonatal Alcohol Exposure (Postnatal Days 4-9)
3. Fabrication and Modification of Electrodes
Figure 2: Electromyographic (EMG) recording electrodes and bipolar electrode. The finished EMG headstage (right, orange) is constructed from three male contact pins, two size 3T PTFE-coated wires, one size 10T PTFE-coated wire, and a modified micro strip. The three wires are approximately 5 cm each and are crimped to the contact pins. The finished bipolar electrode (right, white) is untwisted, re-straightened, and molded in a V-shape (5 mm split). Shielding is removed from the tips of the two prongs. Please click here to view a larger version of this figure.
4. Eyelid Surgery Procedure (Postnatal Day 90)
5. Trace Eyeblink Classical Conditioning Procedure
Figure 3: Modified operant conditioning box for eyeblink conditioning. Rats are freely-moving mammals, and therefore a rotating commutator is used for maintaining electrical signal contact from the EMG and bipolar plugs that are attached to the head. The commutator is attached to the arm of the stanchion, which is counter-weighted for alleviating pressure on the rat. A piezo tweeter (speaker) delivers a 2.8 kHz tone at 85 dB and these values are calibrated regularly. Acoustical foam assists with attenuating environmental noise. Please click here to view a larger version of this figure.
Figure 4: Eyeblink conditioning system. This custom-built system consists of an EMG Integrator unit that filters and amplifies incoming signals from the rats, a Stimulus Control unit that delivers various stimuli in addition to tones and shocks, a pre-amplifier for each operant box to increase EMG signal gain, and a stimulus isolator for each operant box; it provides varying shock levels (in mA). A digital oscilloscope (not part of the stock eyeblink system) is used for diagnostic purposes during habituation and acquisition. Please click here to view a larger version of this figure.
The eyeblink software is capable of providing a large and comprehensive set of data for many types of measurements. For brevity, we report in this study, representative results for learning and performance measures that include adaptive CR percentage, adaptive CR amplitude, UR percentage and UR amplitude. The adaptive CR period was chosen as it represents the acquisition of well-timed eyeblink responses over repeated training, as a result of enhanced synaptic plasticity in the hippocampus during trace ECC50,51,52. The UR measures were chosen to elucidate whether the neonatal alcohol-induced learning deficits in trace ECC were due to disruptions in associative learning or disruptions in responding to the shock US – which may indicate motivational or motor differences, rather than learning differences among treatment groups. Data for each measure were analyzed using 2 (Sex) x 3 (Neonatal Group) x 6 (Session) mixed ANOVAs, with Session as the repeated-measures factor. Significant main effects for neonatal treatment were analyzed using Tukey's post hoc tests and significant interactions were analyzed using simple effects tests. All statistical analyses were conducted using a minimum alpha level of 0.05 and results in graphs are mean ± SEM.
Beginning with the adaptive CR percentage measure, the ANOVA indicated a significant main effect of neonatal group, F(2,21) = 11.69, p <0.001, but no significant main effect of sex (p = 0.71) or significant interaction between these factors (p = 0.20). As expected, adaptive CR percentage increased over the six sessions of training, F(5, 105) = 81.15, p <0.001, and the differences among neonatal groups were dependent on some level of session, F(10, 105) = 4.58, p <0.001. There were no other significant interactions involving the session factor. Likewise for adaptive CR amplitude, there was again a significant main effect of neonatal group, F(2,21) = 22.32, p <0.001, but no significant main effect of sex (p = 0.21) or significant interaction between these factors (p = 0.48). CR amplitude also increased significantly over the six sessions of training, F(5, 105) = 59.27, p <0.001, and the differences among neonatal groups were dependent on some level of session, F(10, 105) = 4.31, p <0.001. Overall, both CR measures showed significant differences among the group means and these means separated significantly at different sessions of training. To confirm which groups differed significantly, Tukey's post hoc tests showed that the alcohol-intubated (AI) rats performed significantly worse on both CR measures than the unintubated-control (UC) and sham-intubated (SI) rats (p <0.01 for CR percentage; p <0.001 for CR amplitude), which did not differ from each other (p's >0.05). Simple effects tests performed on the significant Neonatal Group x Session interactions for both CR measures, confirmed that the AI rats were more significantly impaired in acquiring CRs beginning at Session 2 and carrying on through Session 6 compared to both UC and SI rats (all p's <0.05), which did not differ from each other throughout six sessions. The only exception was adaptive CR amplitude for SI rats did not begin to differ significantly from AI rats until Session 3. These results are shown in Figure 5A, 5B.
There were no significant differences in the UR measures due to sex, neonatal group, or interactions of these factors with the session factor. These negative findings indicated that each group was able to emit eyeblink responses to the shock US equally, and that the learning deficits observed in the AI rats were not influenced by motivational or motor differences in blinking (Figure 6A, 6B).
Figure 5: Acquisition of trace conditioned responding (mean ± SEM). Early alcohol exposure (group AI) significantly affected acquisition of adaptive conditioned response (CR) percentage (A) and amplitude (B). Trace ECC is inherently difficult to acquire, therefore the measures are relatively lower for all groups – with delay ECC, percentages may reach 80-85% in rodent models of FASD21,53. Nonetheless, the trace ECC procedure is more taxing on the hippocampus, which is susceptible to alcohol effects during early brain development. * = p <0.05, ** = p <0.01, *** = p <0.001 between UC and AI rats; sample sizes are provided in parentheses. Please click here to view a larger version of this figure.
Figure 6: Acquisition of unconditioned responses (mean ± SEM). Eyeblink performance (UR percentage and UR amplitude) was not significantly different among groups. The lack of differences indicate that the shock intensity used during acquisition training did not differentially alter motivation in the AI rats or their ability to produce defensive blink responses to the shock, compared to both control groups (UC and SI). Sample sizes are provided in parentheses. Please click here to view a larger version of this figure.
Neonatal Alcohol Exposure | |||
190 Proof Ethyl Alcohol (USP) | Pharmco-AAPER | 225-36000 [ECU Medical Storeroom] | Can be substituted; should be USP; avoid using 200 proof ethyl alcohol |
Container/Basket for Pups | Any | ||
Corn Oil | Any | Food grade | |
Heated Water Therapy Pump w/ Pads | Gaymar | TP-500 | To keep pups warm; can be substituted |
Hypodermic Needles 22G x 1 in, Sterile | Any | ||
Hypodermic Needles 30G x 1/2 in, Sterile | Any | ||
Isopropyl Alcohol 70% | EMD Millipore | PX1840-4 [Fisher Scientific] | Can be substituted; reagent grade www.fishersci.com |
Long-Evans Rats (Female and Male Breeders) | Charles River Laboratories | N/A [ECU Dept. of Comparative Medicine] | Age and weight need to be specified; pricing varies by these factors www.criver.com |
Micro Dissecting Scissors, 3.5 in, 23 mm Blades | Biomedical Research Instruments | 11-2200 | For cutting PE tubing brisurgical.com |
Polyethylene 10 Tubing (0.011 in. I.D.; 0.024 in. O.D.) | BD Diagnostic Systems | 22-204008 [Fisher Scientific] | Can be substituted www.fishersci.com |
Polyethylene 50 Tubing (0.023 in. I.D.; 0.038 in. O.D.) | BD Diagnostic Systems | 22270835 [Fisher Scientific] | Can be substituted www.fishersci.com |
Regulated water heater or baby milk bottle warmer | Any | Optional; helps with warming up cold milk solutions | |
Tuberculin Syringes, Sterile, 1.0 ml | Any | ||
Tuberculin Syringes, Sterile, 10 ml | Any | Can be used to draw out ethyl alcohol or use appropriate size micropipet | |
Weigh Scale | Any | Should have good resolution (in gram units) | |
Name | Company | Catalog Number | Comments |
EMG Headstage Fabrication and Bipolar Electrode Modification | |||
Bipolar Electrode, 2 Channel SS Twisted | Plastics One, Inc. | MS303/2-B/SPC ELECT SS 2C TW .008" | Must specify custom length of 20 mm below pedestal www.plastics1.com |
Centi-Loc Strip Socket Insulator (aka, Micro Strip) | ITT Cannon / ITT Interconnect Solutions | CTA4-IS-60* or CTA4-1S-60* | *Depends on vendor; see www.onlinecomponents.com or www.avnetexpress.avnet.com |
Dental Pliers, Serrated | CMF Medicon | 390.20.05 | Can be substituted; use to crimp wires to male contact pins www.medicon.de |
Micro Dissecting Scissors, 3.5 in, 23 mm Blades | Biomedical Research Instruments | 11-2200 | Only use to cut 3T wires; cutting 10T wires will damage the blade – use the blade of the wire stripper instead brisurgical.com |
PTFE-Coated Stainless Steel Wire, 10T (Bare Diameter .010 in) | Sigmund Cohn-Medwire | 316SS10T | www.sigmundcohn.com |
PTFE-Coated Stainless Steel Wire, 3T (Bare Diameter 0.003 in) | Sigmund Cohn-Medwire | 316SS3T | www.sigmundcohn.com |
Razor Blade | Any | To strip 1 mm from prongs of bipolar electrode | |
Relia-Tac Socket Contact Pin, Male | Cooper Interconnect | 220-P02-100 | See Allied Electronics Cat # 70144761 www.alliedelec.com |
Tweezers, High Precision, Serrated, 4 3/4 in | Electron Microscopy Sciences | 78314-00D | To grasp 10T wire firmly while stripping PTFE with smooth tweezers www.emsdiasum.com |
Tweezers, High Precision, Smooth, 4 3/4 in | Electron Microscopy Sciences | 78313-00B | www.emsdiasum.com |
Tweezers, Ultra Fine Tips, 4 3/4 in | Electron Microscopy Sciences | 78510-0 | To strip 1 mm of PTFE from one end of 3T wire; grasp shielded portion with smooth tweezers www.emsdiasum.com |
Wire Stripper, 16-26 AWG | Any | Use the blade end to cut micro strips | |
Name | Company | Catalog Number | Comments |
Eyelid Surgery | |||
Surgical Instruments (High Quality Stainless Steel) | |||
2 x Dressing Forceps, 4 in Serrated | Biomedical Research Instruments | 30-1205 | Can be substituted; extra forceps for grasping electrodes/screws outside of surgery tray brisurgical.com |
Dressing Forceps, 3 in Serrated | Biomedical Research Instruments | 30-1200 | Can be substituted brisurgical.com |
Instrument Tray | Biomedical Research Instruments | 24-1355 | Can be substituted brisurgical.com |
Knife Handle No. 3, 5 in | Biomedical Research Instruments | 26-1000 | Can be substituted brisurgical.com |
Micro Dissecting Forceps, 3.5 in, Fine Points | Biomedical Research Instruments | 10-1630 | Can be substituted brisurgical.com |
Micro Dissecting Forceps, 3.5 in, Smooth Platform (0.3 x 5 mm) | Biomedical Research Instruments | 10-1720 | brisurgical.com |
Micro Dissecting Scissors, 3.5 in, Extremely Delicate, 15 mm Blades | Biomedical Research Instruments | 11-2000 | Can be substituted brisurgical.com |
Plain Splinter Forceps, 3.5 in | Biomedical Research Instruments | 30-1600 | Can be substituted brisurgical.com |
#10 Stainless Steel Surgical Blade for #3 Handle, Sterile | Any | Can be substituted | |
0-80 x 0.125 in Stainless Steel Screws | Plastics One, Inc. | 0-80 x 0.125 | Can be substituted www.plastics1.com |
Alcohol Prep Pads, Sterile | Fisher Scientific | 22-363-750 [Fisher Scientific | Can be substituted www.fishersci.com |
Betadine Povidone-Iodine | Purdue Frederick Co. | 6761815101 [Fisher Scientific] | Can be substituted www.fishersci.com |
Betadine Povidone-Iodine Prep Pads | Moore Medical | 19-898-946 [Fisher Scientific] | Can be substituted www.fishersci.com |
Cotton-Tipped Swabs, Autoclavable | Any | Typically 7.6 cm or 15.2 cm length | |
Drill Bit for Pin Vise, #55 (0.052 in) | Any | Metal should resist rusting and corrosion | |
Gauze Pads, 2 in x 2 in | Fisher Scientific | 22-362-178 [Fisher Scientific] | Can be substituted www.fishersci.com |
General Purpose Latex/Nitrile/Vinyl Gloves | Any | ||
Glass Bead Sterilizer | Any | Sterilize instruments between surgeries | |
Heated Water Therapy Pump w/ Pads x 2 | Gaymar | TP-500 | Can be substituted; separate pumps are recommended – 1 for surgery, 1 for recovery |
Hypodermic Needles 26G x 3/8 in, Sterile | Any | ||
Isoflurane | Vedco | NDC 50989-150-12 | Manfacturer can be substituted; veterinary approval may be required |
Isoflurane Vaporizer System, Tabletop, Non-Rebreathing | Parkland Scientific | V3000PK | Can be substituted www.parklandscientific.com |
Jewelers Screwdriver w/ 1.8-2 mm Blade | Any | Metal should resist rusting and corrosion | |
Ortho-Jet BCA Package (Dental Cement) | Lang Dental | B1334 | Contains powder (1 lb) and liquid www.langdental.com |
Oxygen Tank with Pressure Regulator, Large | Local supplier | ||
Porcelain Crucible, High-Form, Glazed, 10 ml | CoorsTek, Inc. | 07-965C [Fisher Scientific] | Can be substituted with Fisher FB-965-I Wide-Form Crucible www.fishersci.com |
Puralube Veterinary Ophthalmic Ointment, Sterile | Henry Schein Company | NC0144682 [Fisher Scientific] | Can be substituted www.fishersci.com |
Quatricide PV-15 | Pharmacal | PV-15 | Antimicrobial disinfectant; can be substituted www.pharmacal.com |
Rat Gas Anesthesia Masks for Stereotaxic Surgery | Stoelting Company | 51610 | www.stoeltingco.com |
Rat Stereotaxic Apparatus w/ Ear Bars (45 Degree) | Any | 45 degree bars are recommended to prevent damaging eardrums | |
Roboz Surgical Instrument Milk | Roboz Surgical | NC9358575 [Fisher Scientific] | Can be substituted; for lubricating instruments during autoclaving www.fishersci.com |
Rodent Hair Trimmer | Any | ||
Sodium Chloride | Fisher Scientific | S641-500 [Fisher Scientific] | To make 0.9% saline; reagent grade; USP www.fishersci.com |
Stainless Steel Microspatula (Blade: 0.75 L x 0.18 in. W) | Fisher Scientific | 21-401-15 [Fisher Scientific] | Can be substituted www.fishersci.com |
Starrett Pin Vise, 0.000 in – 0.055 in | Any | Nickel-plated or equivalent recommended to resist rusting and corrosion | |
Sterile Surgical Gloves | Any | ||
Sterilization Wraps, 20 in x 20 in, Autoclavable | Propper Manufacturing | 11-890-8C [Fisher Scientific] | Useful for wrapping autoclavable supplies and on sterile field during surgery www.fishersci.com |
Surgical Drape, Sterile/Autoclavable | Any | May need to cut to size for rats | |
Surgical Gown* | Any | *If required by IACUC | |
Surgical Mask | Any | ||
Tuberculin Syringes, Sterile, 1.0 ml | Any | ||
Weigh Scale | Any | Should have good resolution (in gram units) | |
Name | Company | Catalog Number | Comments |
Eyeblink System and Components (assuming 4-rodent system) | |||
5 Channel Commutator x 4 | Plastics One, Inc. | SL2 + 3C | www.plastics1.com |
Bipolar Electrode Cable, Dual 305 x 4 | Plastics One, Inc. | 305-305 80CM TT2 (C) | Provides plug end to bipolar electrode on rat and to commutator; must be modified www.plastics1.com |
Cable, 5 Channel, Shielded, 26 AWG x 4 | Any | To fabricate commutator cable; this must be made from scratch | |
Chamber for Operant Test Box (Inside: 24 H x 23 W x 14 D in) x 4 | Med-Associates | Can be substituted; inner dimensions should fit operant test box comfortably, with room for acoustical foam; fit with fan – 55-60 dB www.med-associates.com |
|
Eyeblink System and Software | JSA Designs | N/A | Proprietary and customized for research lab |
Heat Shrink Tubing (3/16 in, 1/4 in, 3/8 in, 1/2 in Diameters) | Any | To protect modified commutator cable soldered ends and splices | |
Melamine Triple Peak Acoustical Foam w/Black Hypalon (24 x 48 in) | McMaster-Carr | 9162T5 | Can be substituted; cut to fit 4 housing chambers www.mcmaster.com |
Operant Test Box (Exterior 12.5 L x 10 W x 13.5 in H), Complete x 4 | Med-Associates | ENV-007 Custom Package | With stainless steel grid floor and custom top (3 in hole in center for commutator cable) www.med-associates.com |
Oscilloscope (Optional) | Any | Recommended minimum specs: 200 MHz analog bandwidth, 1 GS/s real-time sampling, 4 channels; see www.picotech.com /td> |
|
Piezo Tweeters (Speakers) x 4 (7 x 3 in) | MCM Electronics | 53-805 | Must match frequency range specifications for eyeblink system (2500 Hz – 25 KHz) www.mcmelectronics.com |
Soldering Station, Solder, Flux, Tinner | Any | For soldering 26 AWG cables to female sockets (that fit male relia-tac contact pins) and bipolar plugs | |
Stimulus Isolators x 4 | WPI International | A365 | These units run on 16-9V alkaline batteries; a suitable rechargeable version (A365R) is available www.wpiinc.com |
Tripolar Electrode Cable for SL3C Commutator x 4 | Plastics One, Inc. | 335-335 80cm TT3 C | Provides plug end to EMG headstage on rat and to commutator; must be modified www.plastics1.com |
USB LED Lights x 4 | Any | USB-based lights do not cause electrical "noise" with the EMG signals from the rats www.plastics1.com |
|
Webcams x 4, Surveillance Software | Any | ||
PC Computer Running MS Windows OS | Any |
Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.
Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.
Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.