are you afraid FINAL
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The Effects of Post-training Timolol on Hippocampal Excitability Priscilla D. Saenz, Eric S. Lovitz, Lucien T. Thompson Aging and Memory Lab, School of Behavioral and Brain Sciences, The University of Texas at Dallas, Richardson, TX Methods Subjects: Experiments were performed using male Long-Evans rats (2-3 mo). Rats were locally bred, and maintained under conditions approved by the UT Dallas IACUC on a 12/12 hr light/dark schedule prior to testing. Rats were handled daily for 5 min for 5 days prior to all experimental use. Cannula Implantation: Rats were anesthetized with isoflurane and implanted bilaterally with cannula (15 mm, 23 ga) stereotaxically into the amygdala (-2.7 mm AP, ±5.1 mm ML, -6.8 mm DV) 5 days prior to training and subsequent in vitro recordings. 1 mg/kg of antibiotic (enrofloxacin) was injected I.M. post- surgery. IA Behavioral Training: In a single trial, each rat was placed into the brightly lit compartment of a rectangular Plexiglas shuttle box. The rat was allowed to cross to the dark compartment, and a guillotine door was shut to lock the animal in the dark compartment. When the rat reached the end of the dark compartment and turned around, a single high intensity (0.5 mA, 1 s) footshock was given. The rat remained in the dark compartment for an additional 15 s following the footshock. Immediately following the single IA behavioral training trial or control procedures, rats were infused with timolol (0.75 μg/0.5 μl, made in 0.1 M PBS) in one hemisphere and vehicle (0.1 M PBS) in the control hemisphere contralateral to the drug infusion. Infusion rates of 0.5 μl over 60 s were used. Slice Preparation: 24 hr after infusion, rats were anesthetized with isoflurane and decapitated. The brain was hemisected and immersed in cooled (1°C) oxygenated (95% O 2 : 5% CO 2 ) s-aCSF [in mM: 124 sucrose; 3 KCl; 1.3 MgSO 4 ; 1.24 NaH 2 PO 4 ; 2.4 CaCl 2 ; 26 NaHCO 3 ; 10 d-glucose; pH 7.4]. After chilling for 3-4 min, the brain was blocked and 400 μm ventral brain slices were cut using vibratomes. Slices were placed in room temperature (25°C) aCSF [in mM: 124 NaCl; 3 KCl; 1.3 MgSO 4 ; 1.24 NaH 2 PO 4 ; 2.4 CaCl 2 ; 26 NaHCO 3 ; 10 d-glucose; pH 7.4]. Slices were continuously oxygenated (95% O 2 : 5% CO 2 ). Neurophysiological recording: Neurons were recorded from both drug-treated and control hemisphere slices. Sharp electrodes were prepared from borosilicate glass (filled with 3 M KCl; 30-80 MΩ), and intracellular recordings were made (using AxoClamp 2b amplifiers and National Instruments LabView interfaces) from submerged slices (31°C). Electrophysiological data was analyzed using ANOVA. Post-hoc Scheffe’s tests were performed to identify individual differences between groups. Future Directions In a previous study, post-trial infusion of the β-adrenergic antagonist propranolol into the amygdala was shown to cause impairment in memory retention for the IA task. Propranolol is shown to have non-specific effects on voltage-gated K+ channels, and affecting Ca2+ channels. Propranolol could be tested to see if an optimal dose could block AHP plasticity after a fearful event. Adrenergic systems are importantly involved in memory storage processes. Acknowledgements We would like to thank others who helped on this project: Michelle Chavez, Devin Proch, David Beddow, & Hallie Cox. We would also like to thank the Undergraduate Research Scholar Award for helping to fund this project. Neurophysiological Recordings 24 hr IA Training and Drug Infusion Handling (5 d) Recovery (3 d) Cannulation of the Amygdala Handling (5 d) Fig. 3. Infographic on the timeline of behavioral training. Fig. 1. The inhibitory avoidance (IA) shuttlebox. Animals are placed into the light-side and once they entered the dark-side, they received a mild foot-shock. Fig. 4. Measures of AHPs following inhibitory avoidance task. (A) AHP waveforms from untrained and IA trained rats 24 h after post-trial unilateral infusions of timolol or vehicle (0 μg; IA trained neurons n = 7, untrained controls: n = 7; 0.75 μg; IA trained neurons n = 11, untrained controls: n = 10). AHPs were reduced in IA trained vehicle and timolol infusion. (B) Medium and slow components of AHPs. The amplitudes of mAHPs, measured 250 ms post-burst, and of sAHPs, measured 500 ms, 750 ms, and 1 s and 2 s post-burst, showed reductions in AHP. Neurons from IA trained timolol-infused hemispheres showed significant reductions in the sAHP at 2 s compared to neurons from untrained timolol-infused hemispheres (⁺=p<0.04). (C) Peak AHP amplitudes. Neurons from IA trained vehicle hemispheres showed reduced peak AHP amplitudes compared to neurons from untrained vehicle-infused hemispheres (ᵃ=p<0.02). Timolol infusion did not cause significant changes. Fig. 5: Intrinsic excitability as measured by accommodation. (A) Waveforms of accommodation was reduced in neurons from IA trained vehicle- and timolol-infused hemispheres, and also in neurons from untrained timolol-infused hemispheres. (B) Action potential firing during an 800 ms sustained depolarization was significantly increased in neurons from vehicle- and timolol-infused IA trained hemispheres, and from timolol untrained hemispheres. Action potential firing was decreased (i.e. accommodation was increased) in neurons from timolol-infused IA trained hemispheres compared to vehicle-infused IA trained hemispheres (*=p<0.001). 0.5 mA, 1 s Introduction Stress from fear plays a key role in the formation of memories. This fear releases a surge of Norepinephrine (NE) which activates the amygdala through β-adrenergic receptors, in turn enhancing memory formation mediated by the hippocampus. In a previous study, timolol (an inverse β-agonist, or functional antagonist) was shown to dose-dependently impair memory by interfering with consolidation after a stressful event in a single-trial inhibitory avoidance (IA) learning task, a measure of retention latency was decreased with bilateral infusion of timolol (0μg, 0.25μg, 0.75μg, 1.25μg) 24 hr after IA training. A dose of 0.75μg was shown to have the most effective reduction in retention. In this study, the mechanism behind the effect of timolol (0.75 μg) infused into the amygdala of male rats immediately after aversive (IA) training was examined. Neurophysiological recordings were taken from CA1 hippocampal neurons to determine the effects of unilaterally infused timolol on intrinsic excitability (AHPs, accommodation). This study determined intrinsic excitability decreased in neurons from IA-trained timolol infused hemispheres, while IA training combined with vehicle will enhance intrinsic excitability in hippocampal CA1 neurons when compared to neurons from untrained vehicle-infused hemispheres. Results Afterhyperpolarizations (AHPs) and accommodation were measured from CA1 neurons in timolol-infused (0.75 μg/0.5 μl) hemispheres, contralateral vehicle-infused IA trained hemispheres as well as to neurons from timolol- or vehicle-infused untrained control hemispheres. Following IA training, recordings showed reductions in both AHP and accommodation, yet showed intrinsic excitability was enhanced in those that were trained and vehicle-infused (evaluated 24 h post-training). (Figure 4) AHP plasticity effects were shown in analyses of both the medium AHP (mAHPs) and slow AHP (sAHPs) (Fig. 4B). CA1 Pyramidal neurons showed significantly reduced AHP peak amplitudes from IA trained, vehicle-infused hemispheres compared to neurons from untrained vehicle-infused hemispheres at various time intervals. (Figure 4C)(ANOVA: F(3) = 3.461, p =0.0181; Scheffe’s test: IA trained vehicle vs. untrained vehicle: p = 0.02; untrained timolol vs. untrained vehicle: p >0.26; IA trained timolol vs. IA trained vehicle: p >0.84; Fig. 4C). Effects of AHP were also measured as the spikes elicited in spike-frequency accommodation. Infusion of timolol blocked the effect of training in hippocampal excitability (Fig. 5). Are You Afraid of the Dark? Fig. 2. Timolol affects retention latency in the IA task. All rats showed increased latency to enter the dark compartment during retention compared to initial latency (*=p<0.007). The 0.75 μg and 1.25 μg doses significantly decreased latency (a=p<0.0150 μg: n=13, 0.25 μg: n=11, 0.75 μg: n=10, 1.25 μg: n=12). The 0.75 μg dose produced the most significant impairment in memory.
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Transcript of are you afraid FINAL
The Effects of Post-training Timolol on Hippocampal Excitabi l ityPriscilla D. Saenz, Eric S. Lovitz, Lucien T. Thompson
Aging and Memory Lab, School of Behavioral and Brain Sciences, The University of Texas at Dallas, Richardson, TX
MethodsSubjects: Experiments were performed using male Long-Evans rats (2-3 mo). Rats were locally bred, andmaintained under conditions approved by the UT Dallas IACUC on a 12/12 hr light/dark schedule prior totesting. Rats were handled daily for 5 min for 5 days prior to all experimental use.Cannula Implantation: Rats were anesthetized with isoflurane and implanted bilaterally with cannula (15mm, 23 ga) stereotaxically into the amygdala (-2.7 mm AP, ±5.1 mm ML, -6.8 mm DV) 5 days prior totraining and subsequent in vitro recordings. 1 mg/kg of antibiotic (enrofloxacin) was injected I.M. post-surgery.IA Behavioral Training: In a single trial, each rat was placed into the brightly lit compartment of arectangular Plexiglas shuttle box. The rat was allowed to cross to the dark compartment, and a guillotinedoor was shut to lock the animal in the dark compartment. When the rat reached the end of the darkcompartment and turned around, a single high intensity (0.5 mA, 1 s) footshock was given. The ratremained in the dark compartment for an additional 15 s following the footshock. Immediately followingthe single IA behavioral training trial or control procedures, rats were infused with timolol (0.75 μg/0.5μl, made in 0.1 M PBS) in one hemisphere and vehicle (0.1 M PBS) in the control hemispherecontralateral to the drug infusion. Infusion rates of 0.5 μl over 60 s were used.Slice Preparation: 24 hr after infusion, rats were anesthetized with isoflurane and decapitated. Thebrain was hemisected and immersed in cooled (1°C) oxygenated (95% O2: 5% CO2) s-aCSF [in mM: 124sucrose; 3 KCl; 1.3 MgSO4; 1.24 NaH2PO4; 2.4 CaCl2; 26 NaHCO3; 10 d-glucose; pH 7.4]. After chilling for3-4 min, the brain was blocked and 400 µm ventral brain slices were cut using vibratomes. Slices wereplaced in room temperature (25°C) aCSF [in mM: 124 NaCl; 3 KCl; 1.3 MgSO4; 1.24 NaH2PO4; 2.4 CaCl2;26 NaHCO3; 10 d-glucose; pH 7.4]. Slices were continuously oxygenated (95% O2: 5% CO2).Neurophysiological recording: Neurons were recorded from both drug-treated and control hemisphereslices. Sharp electrodes were prepared from borosilicate glass (filled with 3 M KCl; 30-80 MΩ), andintracellular recordings were made (using AxoClamp 2b amplifiers and National Instruments LabViewinterfaces) from submerged slices (31°C). Electrophysiological data was analyzed using ANOVA. Post-hocScheffe’s tests were performed to identify individual differences between groups.
Future DirectionsIn a previous study, post-trial infusion of the β-adrenergic antagonist propranolol into the amygdalawas shown to cause impairment in memory retention for the IA task. Propranolol is shown to havenon-specific effects on voltage-gated K+ channels, and affecting Ca2+ channels. Propranolol couldbe tested to see if an optimal dose could block AHP plasticity after a fearful event. Adrenergicsystems are importantly involved in memory storage processes.
Acknowledgements We would like to thank others who helped on this project: Michelle Chavez, Devin Proch, David Beddow, & HallieCox. We would also like to thank the Undergraduate Research Scholar Award for helping to fund this project.
Neurophysiological Recordings
24 hr
IA Training and Drug Infusion
Handling (5 d)
Recovery (3 d)
Cannulation of the Amygdala
Handling (5 d)
Fig. 3. Infographic on the timeline ofbehavioral training.
Fig. 1. The inhibitory avoidance (IA) shuttlebox. Animals are placed into the light-side and oncethey entered the dark-side, they received a mild foot-shock.
Fig. 4. Measures of AHPs following inhibitory avoidance task. (A) AHP waveforms from untrained and IA trained rats 24 h after
post-trial unilateral infusions of timolol or vehicle (0 µg; IA trained neurons n = 7, untrained controls: n = 7; 0.75 µg; IAtrained neurons n = 11, untrained controls: n = 10). AHPs were reduced in IA trained vehicle and timolol infusion. (B) Mediumand slow components of AHPs. The amplitudes of mAHPs, measured 250 ms post-burst, and of sAHPs, measured 500 ms, 750 ms,
and 1 s and 2 s post-burst, showed reductions in AHP. Neurons from IA trained timolol-infused hemispheres showedsignificant reductions in the sAHP at 2 s compared to neurons from untrained timolol-infused hemispheres (⁺=p<0.04). (C)
Peak AHP amplitudes. Neurons from IA trained vehicle hemispheres showed reduced peak AHP amplitudes compared toneurons from untrained vehicle-infused hemispheres (ᵃ=p<0.02). Timolol infusion did not cause significant changes.
Fig. 5: Intrinsic excitability as measured by accommodation. (A) Waveforms of accommodation was reduced in neuronsfrom IA trained vehicle- and timolol-infused hemispheres, and also in neurons from untrained timolol-infusedhemispheres. (B) Action potential firing during an 800 ms sustained depolarization was significantly increased in neuronsfrom vehicle- and timolol-infused IA trained hemispheres, and from timolol untrained hemispheres. Action potentialfiring was decreased (i.e. accommodation was increased) in neurons from timolol-infused IA trained hemispherescompared to vehicle-infused IA trained hemispheres (*=p<0.001).
0.5 mA, 1 s
IntroductionStress from fear plays a key role in the formation of memories. This fear
releases a surge of Norepinephrine (NE) which activates the amygdalathrough β-adrenergic receptors, in turn enhancing memory formationmediated by the hippocampus.
In a previous study, timolol (an inverse β-agonist, or functional antagonist) wasshown to dose-dependently impair memory by interfering with consolidation aftera stressful event in a single-trial inhibitory avoidance (IA) learning task, a measureof retention latency was decreased with bilateral infusion of timolol (0µg, 0.25µg,0.75µg, 1.25µg) 24 hr after IA training. A dose of 0.75µg was shown to have themost effective reduction in retention.In this study, the mechanism behind the effect of timolol (0.75 µg) infused into theamygdala of male rats immediately after aversive (IA) training was examined.Neurophysiological recordings were taken from CA1 hippocampal neurons todetermine the effects of unilaterally infused timolol on intrinsic excitability (AHPs,accommodation). This study determined intrinsic excitability decreased in neuronsfrom IA-trained timolol infused hemispheres, while IA training combined withvehicle will enhance intrinsic excitability in hippocampal CA1 neurons whencompared to neurons from untrained vehicle-infused hemispheres.
ResultsAfterhyperpolarizations (AHPs) and accommodation weremeasured from CA1 neurons in timolol-infused (0.75 μg/0.5 μl) hemispheres, contralateral vehicle-infused IA trained hemispheres as well as to neurons from timolol-or vehicle-infused untrained control hemispheres. Following IA training, recordings showed reductions in both AHP and accommodation, yetshowed intrinsic excitability was enhanced in those that were trained and vehicle-infused(evaluated 24 h post-training). (Figure 4) AHP plasticity effects were shown in analyses ofboth the medium AHP (mAHPs) and slow AHP (sAHPs) (Fig. 4B). CA1 Pyramidal neuronsshowed significantly reduced AHP peak amplitudes from IA trained, vehicle-infusedhemispheres compared to neurons from untrained vehicle-infused hemispheres at varioustime intervals. (Figure 4C)(ANOVA: F(3) = 3.461, p =0.0181; Scheffe’s test: IA trainedvehicle vs. untrained vehicle: p = 0.02; untrained timolol vs. untrained vehicle: p >0.26; IAtrained timolol vs. IA trained vehicle: p >0.84; Fig. 4C). Effects of AHP were also measuredas the spikes elicited in spike-frequency accommodation. Infusion of timolol blocked theeffect of training in hippocampal excitability (Fig. 5).
Are You Afraidof the Dark?
Fig. 2. Timolol affects retention latency in the IA task. All ratsshowed increased latency to enter the dark compartment duringretention compared to initial latency (*=p<0.007). The 0.75 μgand 1.25 μg doses significantly decreased latency (a=p<0.0150µg: n=13, 0.25 µg: n=11, 0.75 µg: n=10, 1.25 µg: n=12). The 0.75μg dose produced the most significant impairment in memory.
