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
CA1 CA2
S ML
CA3
H
GL
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
Perceptual memory
Temporal
Cerebellum
Working memory
Cortical “association” areas Parietal Cingulate Olfactory
Neostriatum
Brainstem & spinal motor outlets Procedural memory Habits neostriatum Skills Sensorimotor adaptations (cerebellum)
Amygdala
Prefrontal
Parahippocampal region
Hypothalamus, autonomic & hormonal outputs Emotional memory Conditioned • preferences & aversions Memory modulation
Hippocampus
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
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
Kompas, 14 Maret 2014
• 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+ G0
G19
P21
P61
G0 G19 G0 G19 3. G+ L+ W-
P21 P21
P61
P21 P21
P61 P61
2. G- L- W+
G0
G19
Results Figure 1.1.1. Examples of a control rat at post-natal day 23 (A) and a previously undernourished rat at postnatal day 22 (B)
Figure 1.1.2. Examples of a control rat at post-natal day 62 (C) and a previously undernourished rat at postnatal 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)
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)
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
Day 62
Results of two-way ANOVA
n = sample size; ANOVA – Analysis of variance; F = F value; degrees of freedom shown in brackets; * p < 0.05; ** p < 0.001
THE EFFECTS OF EARLY LIFE UNDERNUTRITION ON SPATIAL LEARNING TASKS IN QUACKENBUSH MICE
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
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+Wmice 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+Wmice 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% Oral ethanol 1.5 g/ curcumin 50 kgBW mg/kgBW
Control group
+
+
-
-
Ethanol group
+
-
+
-
Ethanolcurcumin 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
Table 2.1. The results of two-way repeated measures ANOVA on log 10 latency data during escape acquisition phase of Morris water maze. 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
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 n
Body weights Cerebral weights Cerebellar weights
Groups 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 oneway 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
dF
F
p
Groups
4,460
11.971
0.001
Day / trial
23,460
1.483
0.071
Groups x day / trial 92,460 interaction
1.019
0.440
Two-way ANOVA repeated measures
n = number of rats; ANOVA – Analysis of variance; dF - degrees of freedom; F = F value; p = probability value
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 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
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
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
Two-way ANOVA
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 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
Groups
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 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
2,14
2,14
2,14
F
108.666
360.000
282.484
p
0.000
0.000
0.000
Groups
One-way ANOVA dF
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
Materials and Methods
Group
MSG
Black garlic
C1
-
-
2 ml NaCl 0.9%, ip 2 ml NaCl 0.9 % oral 2 ml NaCl 0.9 % oral -
C2 T1
2 mg/gr BW, ip 2 mg/gr BW, ip
- 2.5 mg/ 200 g BW
T2 T3
2 mg/gr BW, ip 2 mg/gr BW, ip
5 mg/ 200 g BW 10 mg/ 200 g BW
NaCl 0.9 %
- -
Table 5.1. Means + SEM of body weights of rats prior and subsequent to treatments
C1
C2
Groups‡ T1
T2
Body weights 117.4 +3.5 95.8+14.7 94+13.3 94.2+10.4 before treatment (g) Body weights after treatment 186.6+10.9 174.6+18 175.2+14.4 166+13.2 (g) p† 0.001 0.000 0.001 0.002
T3
p*
89+11.1
0.448
171.8+9.6
0.869
0.000
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* * one-way ANOVA ** Kruskal-Wallis test
p = 0.416**
p = 0.395*
The estimated total number of hippocampal pyramidal cells
Figure 5.4. Mean of the estimated total number of pyramidal cell of hippocampus
Sensitivitas regio CA1 dan CA2-CA3 terhadap berbagai kondisi
Sensitif terhadap
CA1
duroquinone
+
colchicine
+
ischemia
+
alcohol
+
chronic mild stress
+
paraquat
+
malnutrition
+
CA2-CA3
+
toluene
+
dexamethasone
+
kainate
+
fluid percussion injury
+
ferrous sulphate
+
CA 1
DG CA 3
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
