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HIPPOCAMPUS: NUTRISI, NEUROTOKSIKA, DAN MEMORI
Ginus Partadiredja Bagian Ilmu Faal Fakultas Kedokteran Universitas Gadjah Mada [email protected] 081804287018
GARIS BESAR:
1. PENGANTAR: HIPPOCAMPUS
2. PENGARUH RESTRIKSI DIET SEBELUM DAN SESUDAH PENYAPIHAN
TERHADAP HIPPOCAMPUS
3. PENGARUH ALKOHOL DAN CURCUMIN TERHADAP HIPPOCAMPUS
4. PENGARUH ALKOHOL DAN RUMPUT TEKI (Cyperus rotundus)
TERHADAP HIPPOCAMPUS
5. PENGARUH MONOSODIUM GLUTAMAT DAN BLACK GARLIC (BAWANG
PUTIH/ Allium sativum TERFERMENTASI) TERHADAP HIPPOCAMPUS
6. DISKUSI
Figure 1.3.1. A micrograph of a 3 µm-thick horizontal section of hippocampal formation stained with toluidine blue. This shows the dentate gyrus with the granule cells and molecular layer, the hilus, the hippocampus proper (CA1, CA2, and CA3 regions), and subiculum. GL, granule cell layer; ML, molecular layer; H, hilus; S, subiculum
ML
GL H CA3
CA2 CA1
S
Perceptual memory Working memory
Cortical “association” areas Temporal Parietal Cingulate Olfactory Prefrontal
Parahippocampal region
Hippocampus
Amygdala Cerebellum Neostriatum
Brainstem & spinal motor outlets
Hypothalamus, autonomic & hormonal outputs
Procedural memory Habits neostriatum Skills
Sensorimotor adaptations (cerebellum)
Emotional memory Conditioned • preferences & aversions Memory modulation
Declarative memory Episodic & semantic Conscious recollection Flexible expression
Explicit memory: - associated with awareness - easily formed & easily forgotten - hippocampus & medial temporal lobes (entorhinal cortex, perirhinal cortex, parahippocampal cortex) - can be converted into implicit memory (e.g. athlete training)
Hippocampus • Connected to cerebral cortex and basal structures of limbic system (amygdala, hypothalamus, septum, mammilary bodies) • Almost any type of sensory experience activates hippocampus • Stimulation of areas of hippocampus pleasure, rage, passivity, sex drive • Can become hyperexcitable: weak stimuli focal epileptic seizures persisting after stimulation ends
• Connection of limbic system (closed circuit; circuit of Papez): Hippocampus Fornix Corpus mamillaris Nuclei thalamus anterior Cortex cingulatum Hippocampus
Role of Hippocampus in Learning • Bilateral removal of hippocampi (treatment of epilepsy) normal working memory; anterograde amnesia • Theoretical function:
Originally part of olfactory cortex (smell food, danger, or sex decision for life or death Critical decision making determining the importance of incoming sensory stimuli Provides the drive that causes the translation of short-term memory long-term memory (i.e. hippocampus transmit signals causing the rehearsal of new information permanent storage
1. PENGARUH RESTRIKSI DIET SEBELUM DAN
SESUDAH PENYAPIHAN TERHADAP HIPPOCAMPUS
The Effects of Diet Restriction on Ageing and Lifespan
• Western & eastern tradition on diet restriction
Kompas, 14 Maret 2014
H. Sanusi (83 tahun)
• Jalan tegak, tak ada
problem kolesterol,
hipertensi, asam urat, DM
• Hanya makan siang, pk
14.00 (mulai usia 15 tahun)
• Lapar biskuit 2 potong
• McCay et al (1935) seminal research on rats
Hypotheses Relating Diet Restriction and Ageing
• Retardation of Body Growth Hypothesis
• Reduction of Body Fat Hypothesis
• Cell Survival Hypothesis
• Attenuation of Insulin-Like Signaling Hypothesis
• Reduced Metabolic Rate Hypothesis
• Reduced Oxidative Damage Hypothesis
Experiments:
1. ROS Enzymes Experiments:
1. mRNA
2. Anti-Oxidant Activities
3. Oxidative Damage
2. The Number of Cells on CA2-CA3 Regions of Hippocampus
3. Behavioural Experiments:
1. Morris water maze
2. Revolving drum test
Materials and Methods
Dietary restriction schedule (75% of normal diet for QBS mice & 50% for rats)
1. G+ L+ W+
2. G- L- W+
3. G+ L+ W-
G0 G19 P21 P61
G0 G19 P21 P61 G0 G19 P21
G0 G19 P21 P61 P21 P61
Results
Figure 1.1.1. Examples of a control rat at post-natal day 23 (A) and a previously undernourished rat at post-natal day 22 (B)
Figure 1.1.2. Examples of a control rat at post-natal day 62 (C) and a previously undernourished rat at post-natal day 61 (D)
THE EFFECTS OF EARLY LIFE UNDERNUTRITION ON THE NUMBER OF PYRAMIDAL NEURONS IN THE CA2 –
CA3 REGION OF THE HIPPOCAMPUS OF WISTAR RATS
• Unbiased stereology physical disector & Cavalieri
principle
• The Volume of CA2 – CA3 Region of Hippocampus (V = Put
N/n)
• The Numerical Density of Pyramidal Neurons of CA2 – CA3
Region of Hippocampus (NV = Σ Q- / ah)
• The Total Number of Pyramidal Neurons of CA2 – CA3
Region of Hippocampus (Nv x V)
Results Table 1.3.1. Means ± SEM of the volumes (mm3), the numerical density (Nv [/mm3]), and the total number of pyramidal neurons of CA2 – CA3 regions of hippocampus in the left cerebral hemisphere of control and experimental rats.
Volume Numerical Density Total Number
Day 21
Control 0.898 ± 0.12 (n = 5) 210,989 ± 14,489 (n= 5) 183,879 ± 16,518 (n= 5)
Undernourished 0.661 ± 0.06 (n = 8) 227,392 ± 8,460 (n = 8) 151,952 ± 16,948 (n= 8)
Day 62
Control 1.283 ± 0.05 (n = 8) 165,813 ± 5,830 (n = 8) 212,481 ± 10,914 (n= 8)
Undernourished 1.293 ± 0.12 (n = 9) 163,963 ± 7,286 (n = 9) 206,380 ± 13,881 (n= 9)
Results of two-way ANOVA
Group (1, 26) F = 1.4 (1, 26) F = 0.7 (1, 26) F = 1.6
Age (1, 26) F = 28.1** (1, 26) F = 39.2** (1, 26) F = 7.6*
Group x Age (1, 26) F = 1.7 (1, 26) F = 1.1 (1, 26) F = 0.7
n = sample size; ANOVA – Analysis of variance; F = F value; degrees of freedom shown in brackets; * p < 0.05; ** p < 0.001
Morris Water Maze Test • Aims: Spatial learning and memory
• Mice to find a platform Trial 1 Trial 8
• 8 random starting points; fixed platform
• Escape latency times recorded
• 8 trials per day, 3 consecutive days; day 8, day 15
• Ages: 1 yr old
THE EFFECTS OF EARLY LIFE UNDERNUTRITION ON SPATIAL LEARNING TASKS IN QUACKENBUSH MICE
2. Spatial Learning and Memory Tests Figure 1.4.3. Means ± SEM of the escape latency of G-+L+W+, G--L-W+, and G-+L+W- mice during the three consecutive days of trials (8 trials per day) in the escape acquisition tests of the Morris water maze
Figure 1.4.4. Means ± SEM of the log10 of the escape latency of G-+L+W+, G--L-W+, and G-+L+W- mice during the three consecutive days of trials (8 trials per day) in the escape acquisition tests of the Morris water maze
Figure 1.4.5. Means ± SEM of the escape latency of G+L+W+, G-L-W+, and G+L+W- mice during trial 1 at day 3, and single trials at day 10 and day 17 of testing in the memory persistence tests of the Morris water maze.
Figure 1.4.6. Means ± SEM of the log10 of the escape latency of G+L+W+, G-L-W+, and G+L+W- mice during trial 1 at day 3, and single trials at day 10 and day 17 of testing in the memory persistence tests of the Morris water maze.
2. PENGARUH ALKOHOL DAN CURCUMIN TERHADAP
HIPPOCAMPUS
Materials and Methods
Groups Oral aquadest
IP 0.9% NaCl
IP 15% ethanol 1.5 g/ kgBW
Oral curcumin 50 mg/kgBW
Control group
+ + - -
Ethanol group
+ - + -
Ethanol-curcumin
group
- - + +
Figure 2.1. Means ± SEM log 10 latency data of control (A), ethanol (B), and ethanol-curcumin (C) groups, during the three-day session of escape acquistion phase of Morris water maze procedure
Source of Variation dF F p
Groups 2, 414 4,502 0,026
Days / Trials 23, 414 13,01 <0,001
Groups x Days / Trials 46, 414 0,929 0,607
Table 2.1. The results of two-way repeated measures ANOVA on log 10 latency data during escape acquisition phase of Morris water maze.
ANOVA, analysis of variance; dF, degree of freedom; F, F values; p, p values
4. PENGARUH ALKOHOL DAN RUMPUT TEKI (Cyperus
rotundus) TERHADAP HIPPOCAMPUS
Materials and Methods
Groups Treatments
G1 Oral and IP aquadest
G2 IP 2.5 gr/kgBW/day alcohol 25% + oral aquadest
G3 IP 2.5 gr/kgBW/day alcohol 25% + oral C. rotundus 0.325 gr/kgBW/day
G4 IP 2.5 gr/kgBW/day alcohol 25% + oral C. rotundus 0.75 gr/kgBW/day
G5 IP 2.5 gr/kgBW/day alcohol 25% + oral C. rotundus 1.5 gr/kgBW/day
Table 4.1. Means ± SEM of body weights (g), cerebral and cerebellar weights (mg) of control and experimental rats
Groups n Body weights Cerebral weights Cerebellar weights
G1 5 198 ± 5.1 1389.2 ± 42.1 316.9 ± 14.6 G2 5 194 ± 6.2 1334.6 ± 34.2 321.1 ± 34.7 G3 5 211 ± 3.3 1352.1 ± 57.8 398.8 ± 51.4 G4 5 208 ± 1.2 1392.8 ± 33.6 304.5 ± 16.9 G5 5 208 ± 5.1 1445.9 ± 44.1 311.5 ± 10.5
Results of one-way ANOVA
dF 4,20 4,20 4,20
F 2,61 0,99 1,68 p 0,67 0,44 0,20
n = number of rats; ANOVA – Analysis of variance; dF - degrees of freedom; F = F value; p = probability value
Table 4.2. Mean ± SEM of the log10 latency during escape acquisition phase of Morris water maze test
Groups n Log10 latency G1 5 1.824 ± 0.043 G2 5 1.997 ± 0.380 G3 5 1.965 ± 0.270 G4 5 1.830 ± 0.290 G5 5 1.961 ± 0.185
Two-way ANOVA repeated measures
dF F p
Groups 4,460 11.971 0.001 Day / trial 23,460 1.483 0.071 Groups x day / trial interaction
92,460 1.019 0.440
n = number of rats; ANOVA – Analysis of variance; dF - degrees of freedom; F = F value; p = probability value
Groups Significance
G2 versus G1 p = 0,0000471
G2 versus G4 p = 0,0000729
G3 versus G1 p = 0,000416
G5 versus G1 p = 0,000559
G3 versus G4 p = 0,000650
G5 versus G4 p = 0,000869
Tabel 4.3. Level of significance (unadjusted p) of comparisons between groups on log 10 data of escape latency test in Morris water maze procedure
Table 4.3. Mean ± SEM of the log10 escape latency in the memory persistence test of the Morris water maze
Groups n Day 3 Day 10 Day 17 G1 5 1.726 ± 0.340 1.635 ± 0.290 1.537 ± 0.419 G2 5 1.851 ± 0.285 1.911 ± 0.276 1.956 ± 0.378 G3 5 1.740 ± 0.270 1.655 ± 0.256 1.763 ± 0.326 G4 5 1.862 ± 0.350 1.620 ± 0.238 1.630 ± 0.286 G5 5 1.794 ± 0.010 1.858 ± 0.223 1.915 ± 0.226
Two-way ANOVA
dF F p
Groups 4,60 1.177 0.330 Day 2,60 3.388 0.040 Groups x Day interaction
8,60 2.594 0.017
n = number of rats; ANOVA – Analysis of variance; dF - degrees of freedom; F = F value; p = probability value
Table 4.4. Means ± SEM of the volume, numerical density, and the total number of pyramidal neurons in the CA1 region of the right hippocampus of control (G1) and experimental (G2 and G4) rats
Groups n Volume
(mm3) Numerical density
(x103/mm3) Total number
G1 5 0.658 ± 0.007 36056.9 ± 231,6 84713 ± 922.5 G2 5 0.582 ± 0.019 10077.2 ± 0,5 56900 ± 959.1 G4 5 0.620 ± 0.001 11277.7 ± 0,7 69936 ± 660.6
One-way ANOVA
dF 2,14 2,14 2,14
F 10.341 2.044 263.184 p 0.002 0.033 0.000
n = sample size; ANOVA = Analysis of variance; dF = degrees of freedom; F = F value; p = probability value
Table 4.5. Means ± SEM of the volume, numerical density, and the total number of pyramidal neurons in the CA2-CA3 regions of the right hippocampus of control (G1) and experimental (G2 and G4) rats
Groups n Volume
(mm3) Numerical density
(x103/mm3) Total number
G1 5 0.764 ± 0.007 127.3 ± 0.6 96801 ± 1000.6 G2 5 0.656 ± 0.005 100.3 ± 0.4 65869 ± 787.7 G4 5 0.736 ± 0.002 111.1 ± 0.97 81926 ± 958.9
One-way ANOVA dF 2,14 2,14 2,14 F 108.666 360.000 282.484 p 0.000 0.000 0.000
n = sample size; ANOVA = Analysis of variance; dF = degrees of freedom; F = F value; p = probability value
5. PENGARUH MONOSODIUM GLUTAMAT DAN BLACK
GARLIC (BAWANG PUTIH/ Allium sativum
TERFERMENTASI) TERHADAP HIPPOCAMPUS
Group MSG Black garlic NaCl 0.9 %
C1 - - 2 ml NaCl 0.9%, ip 2 ml NaCl 0.9 % oral
C2 2 mg/gr BW, ip - 2 ml NaCl 0.9 % oral T1 2 mg/gr BW, ip 2.5 mg/ 200 g BW -
T2 2 mg/gr BW, ip 5 mg/ 200 g BW - T3 2 mg/gr BW, ip 10 mg/ 200 g BW -
Materials and Methods
Groups‡ p* C1 C2 T1 T2 T3 Body weights before treatment (g)
117.4 +3.5 95.8+14.7 94+13.3 94.2+10.4 89+11.1 0.448
Body weights after treatment (g)
186.6+10.9 174.6+18 175.2+14.4 166+13.2 171.8+9.6 0.869
p† 0.001 0.000 0.001 0.002 0.000
Table 5.1. Means + SEM of body weights of rats prior and subsequent to treatments
‡ C1: NaCl 0.9% intra peritoneal/ ip + NaCl 0.9% per oral/ po; C2: MSG 2 mg/g bw (ip) + NaCl 0.9% (po); T1: MSG 2 mg/g bw (ip) + A.sativum 2.5 mg/200g bw (po), T2: MSG 2 mg/g bw (ip) + A.sativum 5 mg/200g bw (po); T3: MSG 2 mg/g bw (ip) + A.sativum 10 mg/200g bw (po) * p values of one way ANOVA (between groups) † p values of paired t test (within groups)
Morris water maze test: Escape acquisition test
Figure 5.2. Mean ± SEM of escape latency during 3 days test of Morris water maze test
Figure 5.3. Mean ± SEM of path length during 3 days test of Morris water maze test
Table 5.3. Post hoc test using LSD test of path length during 3 days test of Morris water maze test
Trials Groups Post hoc
Trial 2 C1 and C2 C2 and T2 C2 and T3 T1 and T2
p = 0.035 p = 0.003 p = 0.034 p = 0.017
Trial 13 C1 and T2 C1 and T3 C2 and T3 T1 and T3
p = 0.033 p = 0.001 p = 0.014 p = 0.027
Trial 14 C1 and C2 C1 and T2
p = 0.003 p = 0.006
Trial 17 C1 and T1 C2 and T1 T1and T2
p = 0.003 p = 0.036 p = 0.039
Trial 18 C1 and C2 C2 and T1 C2 and T2 C2 and T3
p = 0.020 p = 0.041 p = 0.004 p = 0.003
Memory persistence test
Table 5.4. Mean ± SEM of the log10 escape latency in the memory persistence test of the Morris Water Maze
Groups Day 3 Day 10 Day 17 C1 1.29 ± 0.22 1.25 ± 0.24 1.53 ± 0.12 C2 1.11 ± 0.18 0.93 ± 0.16 1.38 ± 0.30 T1 0.66 ± 0.09 1.05 ± 0.19 1.26 ± 0.09 T2 1.05 ± 0.16 1.30 ± 0.11 1.02 ± 0.15 T3 0.95 ± 0.16 1.18 ± 0.19 1.27 ± 0.24 One way ANOVA
p = 0.178 p = 0.653 p = 0.503
Table 5.5. Mean ± SEM of the log10 path length in the memory persistence test of the Morris Water Maze
Groups Day 3 Day 10 Day 17 C1 2.69 ± 0.19 2.55 ± 0.26 2.82 ± 0.13 C2 2.44 ± 0.15 2.17 ± 0.16 2.52 ± 0.27 T1 1.97 ± 0.08 2.30 ± 0.17 2.45 ± 0.10 T2 2.43 ± 0.14 2.57 ± 0.13 2.26 ± 0.17 T3 2.30 ± 0.14 2.41 ± 0.19 2.53 ± 0.22
p = 0.036* p = 0.416** p = 0.395*
* one-way ANOVA ** Kruskal-Wallis test
The estimated total number of hippocampal pyramidal cells
Figure 5.4. Mean of the estimated total number of pyramidal cell of hippocampus
Sensitif terhadap CA1 CA2-CA3 duroquinone +
colchicine +
ischemia +
alcohol + +
chronic mild stress +
paraquat +
malnutrition +
toluene +
dexamethasone +
kainate +
fluid percussion injury +
ferrous sulphate +
Sensitivitas regio CA1 dan CA2-CA3 terhadap berbagai kondisi
CA 3
CA 1
DG
Fisiologi Hippocampus
• Hippocampus regio dorsal/ septal fungsi kognitif
• Hippocampus regio ventral/ temporal fungsi emosi
• Dual inputs di CA3 (dari EC dan DG), CA1 (dari EC dan
CA3), subiculum (dari EC dan CA1) (Muller, 1996)
• Perbedaan peta gen: dorsal, intermediate, ventral (Fanselow &
Dong, 2010)
• Perforant path medial komponen spasial
• Perforant path lateral komponen non-spasial
• Place cells di seluruh hippocampus (Muller, 1996)
• Place cells berdekatan dapat punya place fields jauh
• Place fields hippocampus ventral 4-5x > hippocampus
dorsal (Ahmed & Mehta, 2009)
• Sel pyramidal tidak selalu beraksi sebagai place cells
(Muller, 1996)
• CA3: • Duplikasi input dari EC (perforant path/ PP) dan DG (mossy fibers/ MF) • Recurrent connections/ RC memori autoasosiasi (koneksi sinaptik yang merepresentasikan komponen2 berbeda dari memori diperkuat) (Gilbert & Brushfield, 2009) • MF mendorong penyimpanan representasi baru (Cerasti &
Treves, 2010) tapi tidak terlibat pada retrieval (Gilbert & Brushfield, 2009) • PP relay cue yang menginisiasi pengambilan kembali representasi yang sudah disimpan melalui dinamika atraktor (akibat RC) (retrieval) tapi tidak pada penyandian info baru
(Gilbert & Brushfield, 2009) • Akuisisi, bukan recall (?) (Florian & Roullet, 2004)
CA3: (Gilbert & Brushfield, 2009) • Asosiasi arbitrary spasial (e.g. Informasi lokasi dari lobus parietalis diasosiasikan dengan informasi identitas obyek dari lobus temporalis; asosiasi object-place dan odor-place) • Diperlukan untuk asosiasi baru; peran sedikit pada retrieval asosiasi yang sudah dipelajari sebelumnya • Spatial working memory • Spatial pattern completion (RC dapat melengkapi pola informasi selama retrieval, berdasarkan input tak lengkap) • Spatial pattern separation (mekanisme memisahkan pola-pola tumpang tindih sebagian, sehingga satu pola dapat diambil dari pola yang lain)
CA3a,b: (Kesner, 2007)
• Menyandi informasi spasial baru dalam memori jangka
pendek
• Menyandi informasi spasial dengan trials jamak,
termasuk akuisisi asosiasi arbitrary (dengan komponen
spasial) dan relasional
• Pengambilan kembali informasi memori jangka pendek
berdasarkan proses pattern completion spasial (karena
adanya RC)
• Pemrosesan geometri lingkungan
• CA1:
• Input dari CA3 (30.000 sinaps)
• Input dari EC (1800 sinaps)
• Inhibisi > eksitasi pada CA1
• 2/3 sel pyramidal CA1 tak mempunyai place fields
silent cells (Ahmed & Mehta, 2009)
• Berfungsi untuk retrieval pada Hebb-Williams maze
(Kesner, 2007)
• Asosiasi arbitrary dengan komponen temporal (Kesner, 2007)
• Detektor match-mismatch pembanding prediksi dari
CA3a,b dorsal dengan input langsung EC (Kesner 2007)
• Apakah fungsi sebenarnya dari hippocampus dorsal,
intermediate, ventral, CA1 dan CA2-CA3?
• Bagaimana pengaruh selective neuronal vulnerability
pada perilaku tikus maupun manusia?
• Dapatkah dideteksi dengan tes-tes perilaku?
• Hitung sel pyramidal terpisah antara segmen
hippocampus dorsal, intermediate, dan ventral?
• Morris water maze murni uji memori/ terpengaruh
fear/ anxiety?
Ucapan Terima Kasih
• Dr. Kuldip S Bedi
• Zul Izhar Mohd Ismail, MBBS, MPhil
• dr. Rina Susilowati, PhD, Bagian Histologi, FK UGM
• dr. Ch Tri Nuryana, MKes, Bagian Anatomi, FK UGM
• dr. Dwi Cahyani Ratna Sari, MKes, Bagian Anatomi, FK
UGM
• dr. Suwono, Bagian Ilmu Faal, FK UGM
• dr. Sutarman
• dr. Taufik Nur Yahya
• dr. Agung Prasetyo Wicaksono
• dr. Doddy Hendro Susilo
• dr. Ery Hermawati, MKes, Bagian Faal, FK Untan
• Aminuddin, SKep, MKes, Poltekkes Kaltim
• dr. Titis Nurmasitoh, MKes, Bagian Faal, FK UII
• Ronal Tolkhah, Skep, MKes, Poltekkes Semarang
• Tehnisi laboratorium Bagian Ilmu Faal, Histologi, dan
Patologi Anatomi, FK UGM
Referensi
• Ahmed OJ, Mehta MR. 2009. The hippocampal rate code:
anatomy, physiology, and theory. Trends Neurosci 32(6):
329-338
• Cerasti E, Treves A. 2010. How informative are spatial
CA3 representations estabkished by the dentate gyrus?
PLoS Comput Biol 6(4)
• Fanselow MS, Dong H-W. 2010. Are the dorsal and ventral
hippocampus functionally distinct structures? Neuron 65(1):
1-7
• Florian C, Roullet P. 2004. Hippocampal CA3-region is
crucial for acquisition and memory consolidation in Morris
water maze task in mice. Behav Brain Res
• Gilbert PE, Brushfield AM. 2009. The role of the CA3
hippocampal subregion in spatial memory: A process oriented
behavioral assessment. Prog Neuropsychopharmacol Biol
Psychiatry 33(5):774-781
• Kesner RP. 2007. Behavioral functions of the CA3 subregion
of the hippocampus. Learning & Memory 14:771-781
• Muller R. 1996. A quarter of a century of place cells. Neuron
17:813-822