HIPPOCAMPUS: NUTRISI, NEUROTOKSIKA, DAN MEMORIpsikoneurologi.psikologi.ugm.ac.id/.../Nutrisi_Neurotoksika_Memori.pdfHIPPOCAMPUS: NUTRISI, NEUROTOKSIKA, DAN MEMORI Ginus Partadiredja

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


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


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



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


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


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 -


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

Documents

HIPPOCAMPUS: NUTRISI, NEUROTOKSIKA, DAN MEMORIpsikoneurologi.psikologi.ugm.ac.id/.../Nutrisi_Neurotoksika_Memori.pdfHIPPOCAMPUS: NUTRISI, NEUROTOKSIKA, DAN MEMORI Ginus Partadiredja