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

Thalamus-Anterior cingulate cortex circuit Introduction Methods Results Summary Acknowledge ment 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. 1998. 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. 2007. 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. 1997. 2

PF & MD involve in nociceptive responses Introduction Methods Results Summary Acknowledge ment 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. 1990. 3

PF terminates in Layer I, V and VI Introduction Methods Results Summary Acknowledge ment 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. 1991. 4

MD terminates in Layer II/III of ACC Introduction Methods Results Summary Acknowledge ment Appendix BDA-labeled fibers from MD injection terminate in layer III (section C) and layer I (section D). CC Wang et al. Brain Res. 2004. 5

Aim Introduction Methods Results Summary Acknowledge ment Appendix 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

Male Sprague-Dawley rats (250~300g). Anesthesia Animal Preparation d) Craniotomy Introduction Methods Results Summary Acknowledge ment 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

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

Intracellular Recording & Analysis Introduction Methods Results Summary Acknowledge ment Appendix (mV) Voltage Time (ms)

Intracellular Recording & Analysis SNS & PFS & MDS Introduction Methods Results Summary Acknowledge ment Appendix (mV) Voltage EPSP (Excitatory Post-Synaptic Potential) Stimulation Time (ms)

Intracellular Recording & Analysis Intracellular Labeling & Immunohistochemical Methods Introduction Methods Results Summary Acknowledge ment 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.

SN stimulation Introduction Methods Results Summary Acknowledge ment -85mV (- withdraw) (mv) Introduction Methods Results Summary Acknowledge ment Appendix +0.8nA -85mv +0.6nA -85mv (mv) +0.4nA -85mv +0.2nA -85mv +0nA -85mv -0.2nA EPSP -85mv -85mv -0.4nA -85mv -0.6nA -85mv -0.8nA Time (ms) 100ms 200ms 300ms 400ms 500ms 600ms 700ms 800ms Time (ms) Sciatic nerve stimulation (0.5 ms 5mA) Sciatic nerve stimulation (0.5 ms 5mA) (mv) 0.8nA 0.6nA 0.4nA 0.2nA -0.0nA -0.2nA -0.4nA -0.6nA -0.8nA Sciatic nerve stimulation (0.5 ms 5mA) 100ms 200ms 300ms 400ms 500ms 600ms 700ms 800ms Time (ms)

PF stimulation Introduction Methods Results Summary Acknowledge ment -89mV (- withdraw) Introduction Methods Results Summary Acknowledge ment Appendix (mv) -89mv +0.8nA (mv) -89mv +0.6nA +0.4nA -89mv EPSP -89mv +0.2nA -89mv +0nA -0.2nA -89mv -0.4nA -89mv -0.6nA -89mv -89mv -0.8nA 100ms 200ms 300ms 400ms 500ms 600ms 700ms 800ms Time (ms) Time (ms) PF stimulate (0.5ms 150uA) PF stimulate (0.5ms 150uA) (mv) 0.8nA 0.6nA 0.4nA 0.2nA -0.0nA -0.2nA -0.4nA -0.6nA -0.8nA PF stimulation (0.5 ms 150uA) 100ms 200ms 300ms 400ms 500ms 600ms 700ms 800ms Time (ms)

Layer V pyramid neuron response to PF stim. 40x Introduction Methods Results Summary Acknowledge ment Appendix T1-1700 400x T1-1700

SN Stimulation Introduction Methods Results Summary Acknowledge ment Appendix -60mV (- withdraw) (mv) (mv) +0.6nA -60mv +0.4nA -60mv EPSP -60mv +0.2nA -60mv +0nA -60mv -0.2nA -60mv -0.4nA -60mv -0.8nA 100ms 200ms 300ms 400ms 500ms 600ms 700ms 800ms Time (ms) Time (ms) Sciatic nerve stimulation (0.5 ms 5mA) Sciatic nerve stimulation (0.5 ms 5mA) (mv) 0.6nA 0.4nA 0.2nA -0.0nA -0.2nA -0.4nA -0.6nA -0.8nA Sciatic nerve stimulation (0.5 ms 5mA) 100ms 200ms 300ms 400ms 500ms 600ms 700ms 800ms Time (ms)

MD Stimulation Introduction Methods Results Summary Acknowledge ment -67mV (- withdraw) Introduction Methods Results Summary Acknowledge ment Appendix (mv) +0.8nA (mv) -67mv +0.6nA EPSP -67mv +0.4nA Mem Potential -67mv +0.2nA +0nA IPSP -67mv -0.2nA -67mv -0.4nA -67mv -0.6nA -67mv -0.8nA IPSP (Inhibitory Post-Synaptic Potential) 100ms 200ms 300ms 400ms 500ms 600ms 700ms 800ms Time (ms) MD stimulate (0.5ms 200uA) Time (ms) MD stimulate (0.5ms 200uA) (mv) 0.8nA 0.6nA 0.4nA 0.2nA -0.0nA -0.2nA -0.4nA -0.6nA -0.8nA MD stimulation (0.5 ms 200uA) 100ms 200ms 300ms 400ms 500ms 600ms 700ms 800ms Time (ms)

Layer V pyramid neuron response to MD stim. 40x Introduction Methods Results Summary Acknowledge ment Appendix T1-1396 400x T1-1396

Atlas-Photo Merge (lesion site of PF) Introduction Methods Results Summary Acknowledge ment Appendix

Atlas-Photo Merge (lesion site of MD) Introduction Methods Results Summary Acknowledge ment Appendix

Data Analysis Layer_Data Analysis Introduction Methods Results Summary Acknowledge ment 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: -57.31 and PF: -71.22 (p=0.03<0.05); and in layer V neurons, MD: -62.94 and PF: -70.77 (p=0.03<0.05). In EPSP duration of layer II/III neurons, MD: 18.25 and PF: 15.11 (p=0.01<0.05); and in layer V neurons, MD: -62.94 and PF: 40.01 (p=0.01<0.05). In IPSP duration of layer II/III neurons, MD: 87.93 and PF: 0 (p=0.01<0.05); and in layer V neurons, MD: 87.14 and PF: 0 (p=0.00<0.05).

Data Analysis Layer_Data Analysis Introduction Methods Results Summary Acknowledge ment Appendix

Data Analysis Layer_Data Analysis Introduction Methods Results Summary Acknowledge ment Appendix

Data Analysis Layer_Data Analysis Introduction Methods Results Summary Acknowledge ment Appendix

Summary Introduction Methods Results Summary Acknowledge ment 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.

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

Thanks for your listening. Acknowledgement Introduction Methods Results Summary Acknowledge ment 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.

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