1. Effects of Mediodorsal and Parafascicular Nuclei Stimulation on Synaptic Responses of Cingulate Neurons: An Intracellular Study in Vivo
Student: Ting-Hsuan, Chang (張珽瑄) Life Science Department, NCKU
Advisors: Bai-Chuang, Shyu Ph.D (徐百川) IBMS, Academia Sinica
Hsiang-Chin, Lu (呂享晉) IBMS, Academia Sinica

2. Thalamus-Anterior cingulate cortex circuit
Introduction
Methods
Results
Summary
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Appendix
Anterior cingulate cortex (ACC) is involved in the integration of nociception and the anticipation of pain that recedes the avoidance of noxious stimuli.
Tetsuo K. et al. Neuroreport
The spinothalamic tract is the most important ascending pain system, especially in primates and humans. In medial thalamus, fibers terminate in the intralaminar nuclear group, especially the parafascicular (PF) nuclei, and in the medial dorsal nucleus (MD).
Shulamith Kreitler et al. The Handbook of Chronic Pain
Medial thalamus (MT) nuclei mediate different aspects of nociceptive information to specific ACC areas, and that nociceptive information in the MT is modulated reciprocally by activities from the ACC.
MM. Hsu et al. NeuroReport 2

3. PF & MD involve in nociceptive responses
Introduction
Methods
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Summary
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Appendix
The parafascicular nucleus (PF) receives spinothalamic afferents, contains nociceptive neurons, projects to ACC and transmits nociceptive information thereto.
Vogt BA. Cingulate Neurobiology and Disease.
Mediodorsal (MD) neurons discharges after somatosensory nociceptive stimuli.
JO Dostrovsky et al. Pain 3

4. PF terminates in Layer I, V and VI
Introduction
Methods
Results
Summary
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Appendix
The highest density of PHA-L-labeled fibers is present in the dorsal part of the prelimbic cortex, the anterior cingular cortex, and the caudal part of the medial agranular cortex.
In the latter areas, the label is restricted to layers I, V and VI. PF
HW Berendse et al. Neuroscience 4

5. MD terminates in Layer II/III of ACC
Introduction
Methods
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Appendix
BDA-labeled fibers from MD injection terminate in layer III (section C) and layer I (section D).
CC Wang et al. Brain Res 5

6. Aim
Introduction
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Because PF and MD nuclei project to different layers in ACC, thus the aim of the present study is to compare the synaptic response in cingulate neurons in response to PF and MD stimulation.
ACC PF MD 6

7. Male Sprague-Dawley rats (250~300g). Anesthesia
Animal Preparation
d) Craniotomy
Introduction
Methods
Results
Summary
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Appendix
Male Sprague-Dawley rats (250~300g).
Anesthesia
Animal were maintained under anesthesia with 1.5% isofluarane in 100% oxygen during the surgery.
Tracheostomy
Injection muscle relaxant Gallamine (50mg/kg) to avoid tiny movements of animal.
Craniotomy PF
ACC MD
Bregma

8. Intracellular Recording & Analysis
Introduction
Methods
Results
Summary
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Appendix
Intracellular Recording
SNS (Sciatic nerve Stimulation)
PFS & MDS (Parafascicular and mediodorsal Stimulation)
Intracellular Labeling & Immunohistochemical Methods

9. Intracellular Recording & Analysis
Introduction
Methods
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Summary
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Appendix
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10. Intracellular Recording & Analysis
SNS & PFS & MDS
Introduction
Methods
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Summary
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Appendix
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EPSP (Excitatory Post-Synaptic Potential)
Stimulation
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11. Intracellular Recording & Analysis
Intracellular Labeling & Immunohistochemical Methods
Introduction
Methods
Results
Summary
Acknowledge
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Appendix
Intracellular recording use Neurobiotin with 2 nA depolarizing pulses.
The Neurobiotin-filled cells were revealed using the ABC-kit, nickel, diaminobenzidine (DAB) reaction.
We will calculate the information of AP, EPSP, input resistance, and IV-curve to analysis our data.

12. SN stimulation Introduction Methods Results Summary Acknowledge ment
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Introduction
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Appendix
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13. PF stimulation Introduction Methods Results Summary Acknowledge ment
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Appendix
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14. Layer V pyramid neuron response to PF stim.
40x
Introduction
Methods
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Summary
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Appendix
T1-1700
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T1-1700

15. SN Stimulation Introduction Methods Results Summary Acknowledge ment
Appendix
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16. MD Stimulation Introduction Methods Results Summary Acknowledge ment
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Introduction
Methods
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Appendix
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17. Layer V pyramid neuron response to MD stim.
40x
Introduction
Methods
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Appendix
T1-1396
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T1-1396

18. Atlas-Photo Merge (lesion site of PF)
Introduction
Methods
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Summary
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Appendix

19. Atlas-Photo Merge (lesion site of MD)
Introduction
Methods
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Appendix

20. Data Analysis Layer_Data Analysis Introduction
Methods
Results
Summary
Acknowledge
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Appendix
In PF stimulation, we have recorded 18 cells, 33% cells are in layer II/III, and 67% cells are in layer V.
In MD stimulation, we have recorded 32 cells, 28% cells are in layer II/III, and 72% cells are in layer V.
There is significant difference between PF and MD stimulation in Mem potential, EPSP duration and IPSP duration.
In Mem Potential of layer II/III neurons, MD: and PF: (p=0.03<0.05); and in layer V neurons, MD: and PF: (p=0.03<0.05).
In EPSP duration of layer II/III neurons, MD: and PF: (p=0.01<0.05); and in layer V neurons, MD: and PF: (p=0.01<0.05).
In IPSP duration of layer II/III neurons, MD: and PF: 0 (p=0.01<0.05); and in layer V neurons, MD: and PF: 0 (p=0.00<0.05).

21. Data Analysis Layer_Data Analysis Introduction Methods Results Summary
Acknowledge
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Appendix

22. Data Analysis Layer_Data Analysis Introduction Methods Results Summary
Acknowledge
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Appendix

23. Data Analysis Layer_Data Analysis Introduction Methods Results Summary
Acknowledge
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Appendix

24. Summary
Introduction
Methods
Results
Summary
Acknowledge
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Appendix
We can find EPSP along with IPSP after the mediodorsal stimulation, but we can just find EPSP after the parafascicular stimulation.
There is significant difference in Mem potential, EPSP duration and IPSP duration.
Our result confirm the hypothesis, and it confirms that the differential projections of PF and MD to ACC will cause different responses to their post-synaptic targets.

25. Summary I II/III V VI Other PF MD Introduction Methods Results Summary
Acknowledge
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Appendix
Pyramidal Neuron
Inhibitory Neuron I
II/III V
EPSP+IPSP VI
EPSP
Other PF MD

26. Thanks for your listening.
Acknowledgement
Introduction
Methods
Results
Summary
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Appendix
To accomplish my studies, I want to thank all lab members. Thanks teacher Shyu to give me advices about my project, and thanks senior colleagues Hsiang- Chin and Wei-Jen to teach me how to perform the experiments. And I want to extend my gratitude to senior colleagues Wei-Pang, Jiun-Hsian, Yung-Hui and Hsi-Chien for answering my questions about basic knowledge. And I thank teacher Hwang to encourage me. At last, I want to thank my co-mates Ming-Che and Jing-Yun to accompany with me through two-month internship.
Thanks for your listening.

27. Intracellular Labeling & Immunohistochemical Methods
Appendix
Intracellular Labeling & Immunohistochemical Methods
Introduction
Methods
Results
Summary
Acknowledge
ment
Appendix
HRP
Biotinylated Enzyme
Triton
Avidin
ABC-kit
Nickel
DAB
NeuroBiotin
Interact with H2O2
NeuroBiotin-filled Neuron