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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
• 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.
Results
Summary
Acknowledge
ment
Appendix
• 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.
PF & MD involve in nociceptive responses
Introduction
Methods
• The parafascicular nucleus (PF) receives spinothalamic
afferents, contains nociceptive neurons, projects to ACC and
transmits nociceptive information thereto.
Vogt BA. Cingulate Neurobiology and Disease.
Results
• Mediodorsal (MD) neurons discharges after somatosensory
nociceptive stimuli.
Summary
Acknowledge
ment
Appendix
JO Dostrovsky et al. Pain. 1990.
PF terminates in Layer I, V and VI
Introduction
Methods
• 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.
Results
Summary
Acknowledge
ment
Appendix
PF
HW Berendse et al. Neuroscience. 1991.
MD terminates in Layer II/III of ACC
Introduction
• BDA-labeled fibers from MD injection terminate in layer III
(section C) and layer I (section D).
Methods
Results
Summary
Acknowledge
ment
Appendix
CC Wang et al. Brain Res. 2004.
Aim
Introduction
Methods
• 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.
Results
Summary
Acknowledge
ment
Appendix
ACC
PF
MD
Animal Preparation
Introduction
Methods
d) Craniotomy
a) Male Sprague-Dawley rats (250~300g).
b) Anesthesia
Results
Summary
Acknowledge
ment
Animal were maintained under anesthesia with 1.5% isofluarane in
100% oxygen during the surgery.
c) Tracheostomy
ACC
Injection muscle relaxant Gallamine (50mg/kg) to avoid tiny
movements of animal.
d) Craniotomy
Appendix
PF
Bregma
MD
Intracellular Recording & Analysis
Introduction
Methods
Results
Summary
Acknowledge
ment
Appendix
a) Intracellular Recording
b) SNS (Sciatic nerve Stimulation)
c) PFS & MDS (Parafascicular and mediodorsal Stimulation)
d) Intracellular Labeling & Immunohistochemical Methods
Intracellular Recording & Analysis
Intracellular Recording
(mV)
Introduction
Methods
Results
Voltage
Summary
Acknowledge
ment
Appendix
Time
(ms)
Intracellular Recording & Analysis
SNS & PFS & MDS
(mV)
Introduction
Methods
Results
Acknowledge
ment
Voltage
Summary
EPSP (Excitatory Post-Synaptic Potential)
Appendix
Stimulation
Time
(ms)
Intracellular Recording & Analysis
Introduction
Methods
Intracellular Labeling & Immunohistochemical Methods
• Intracellular recording use Neurobiotin with 2 nA
depolarizing pulses.
Results
Summary
• The Neurobiotin-filled cells were revealed using the
ABC-kit, nickel, diaminobenzidine (DAB) reaction.
Acknowledge
ment
• We will calculate the information of AP, EPSP, input
resistance, and IV-curve to analysis our data.
Appendix
SN stimulation
(mv)
Introduction
Methods
-85mV (- withdraw)
+0.8nA
-85mv
-85mv
+0.6nA
-85mv
+0.4nA
(mv)
-85mv
+0.2nA
+0nA
-0.2nA
-85mv
-0.4nA
-85mv
-0.6nA
-85mv
-0.8nA
-85mv
Results
-85mv
EPSP
Summary
Time (ms)
Acknowledge
ment
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
Appendix
-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
(mv)
(mv)
Methods
EPSP
-89mV (- withdraw)
-89mv
+0.8nA
-89mv
+0.6nA
-89mv
+0.4nA
-89mv
-89mv
+0.2nA
+0nA
-0.2nA
-0.4nA
-89mv
-0.6nA
-89mv
Results
-89mv
Summary
-89mv
-0.8nA
100ms 200ms 300ms 400ms 500ms 600ms 700ms 800ms
Time (ms)
Acknowledge
ment
Time (ms)
PF stimulate (0.5ms 150uA)
PF stimulate (0.5ms 150uA)
(mv)
0.8nA
0.6nA
0.4nA
0.2nA
Appendix
-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
T1-1700
Methods
Results
400x
Summary
Acknowledge
ment
T1-1700
Appendix
SN Stimulation
Introduction
(mv)
(mv)
Methods
EPSP
Results
Summary
-60mV (- withdraw)
-60mv
+0.6nA
-60mv
+0.4nA
-60mv
+0.2nA
-60mv
+0nA
-60mv
-0.2nA
-60mv
-0.4nA
-60mv
-0.8nA
100ms 200ms 300ms 400ms 500ms 600ms 700ms 800ms
Time (ms)
Acknowledge
ment
Time (ms)
Sciatic nerve stimulation (0.5 ms 5mA)
Sciatic nerve stimulation (0.5 ms 5mA)
(mv)
0.6nA
0.4nA
0.2nA
Appendix
-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
(mv)
Introduction
-67mV (- withdraw)
+0.8nA
-67mv
(mv)
+0.6nA
Methods
-67mv
EPSP
+0.4nA
Mem Potential
-67mv
Results
Summary
+0.2nA
+0nA
-0.2nA
-0.4nA
-67mv
IPSP
-67mv
-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)
Acknowledge
ment
MD stimulate (0.5ms 200uA)
(mv)
0.8nA
0.6nA
0.4nA
0.2nA
-0.0nA
-0.2nA
Appendix
-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
T1-1396
Methods
Results
400x
Summary
Acknowledge
ment
Appendix
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
Introduction
Layer_Data Analysis
• In PF stimulation, we have recorded 18 cells, 33% cells are in layer
II/III, and 67% cells are in layer V.
Methods
• In MD stimulation, we have recorded 32 cells, 28% cells are in layer
II/III, and 72% cells are in layer V.
Results
• There is significant difference between PF and MD stimulation in Mem
potential, EPSP duration and IPSP duration.
Summary
Acknowledge
ment
Appendix
• 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
Mem Potential
Methods
(-mV)
Results
Summary
80
70
60
MD-Mem Potential
50
PF-Mem Potential
40
30
Acknowledge
ment
20
10
PF-Mem Potential
0
MD-Mem Potential
Layer II/III
Layer V
Appendix
Data Analysis
Layer_Data Analysis
Introduction
Methods
EPSP Duration
ms
Results
40.01
45
35.18
40
35
Summary
MD-EPSP Duration
30
PF-EPSP Duration
25
20
Acknowledge
ment
15
10
PF-EPSP Duration
5
0
MD-EPSP Duration
Layer II/III
Appendix
Layer V
Data Analysis
Layer_Data Analysis
Introduction
IPSP Duration
Methods
ms
90
80
Results
Summary
70
60
MD-IPSP Duration
50
PF-IPSP Duration
40
30
Acknowledge
ment
20
PF-IPSP Duration
10
0
MD-IPSP Duration
Layer II/III
Layer V
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
Introduction
Pyramidal Neuron
Inhibitory Neuron
I
Methods
II/III
Results
Summary
V
Acknowledge
ment
Appendix
EPSP+IPSP
VI
EPSP
Other
PF
MD
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.
Appendix
Introduction
Intracellular Labeling & Immunohistochemical Methods
HRP
Methods
Biotinylated Enzyme
Triton
Avidin
Results
ABC-kit
Summary
Acknowledge
ment
Appendix
Nickel
DAB
NeuroBiotin
Interact with H2O2
NeuroBiotin-filled Neuron