Prepulse Experiments
Effects of visual prestimulus on the acoustic startle reflex in hamsters.

ハムスタ−における視覚刺激を用いたprepulse の効果　

キーワード: 　驚愕反応, 　聴覚刺激、視覚機能、prepulse

序論
切断した視神経の断端に末梢神経を移植し、視神経の標的部位である上丘に架橋することにより、従来、成熟哺乳動物においては再生しないと考えられてきた網膜神経節細胞の軸索が再生し、網膜-上丘路を再形成することが知られている（So and Aguayo, 1985）。私たちは、末梢神経を架橋移植することにより網膜-上丘路を再形成させたハムスタ−を用いて、視覚機能の回復に関し、一連の実験を行ってきた（Sasaki et al. 1993, 1996, 佐々木, 井上 1995, 福田ら 1996）。 一方、突然の強い聴覚刺激の提示はヒトおよびラット、マウスの驚愕反応を引き起こすことが知られている。この聴覚刺激に先行して驚愕反応を引き起こさない強度の聴覚刺激を提示すると、驚愕反応が抑制される現象はそれぞれprepulse inhibition と呼ばれている(Hoffman and Ison, 1980)。また、この先行刺激は聴覚刺激のみならず、視覚刺激によっても同様の効果が引き起こされることが報告されているが（Campeau and Davis,1995）、ハムスタ−を用いた実験は見当たらない。 したがって、本実験では、ハムスタ−の聴覚性驚愕反応におよぼす視覚先行刺激の効果ついて調べ、ハムスタ−の視覚機能の簡便な評価法としての、本行動課題の有用性について検討した。

方法
正常、成熟ハムスター（80-100g）を用い、側面の２方が穴の開いたアルミ板からなる透明のアクリル製の実験箱（70 x 70 x 180 mm）に動物を入れた。実験箱はインシュレーターの上に置いた。聴覚刺激は110 dB (騒音計、SPL) の白色雑音（SANEI, 3G13）を増幅器（ROLAND）を介して、実験箱の側方30 cm の距離に設置したスピーカーから50 msec の持続時間で提示した。実験箱の反対側に別のスピーカーを置き、0-20 KHz の雑音（NF, WG-721,68dB）を常時提示することにより背景雑音レベルを一定に維持した。視覚刺激として、赤色の高輝度発光ダイオード（STANLEY, JS3102）を20 msecの持続時間で提示した。実験箱およびスピーカーは、防音室内に入れ、外部と隔離した。背景光のレベルは0.1 Lux, 視覚刺激強度は115Luxであった。動物の驚愕反応は実験箱に取り付けた加速度センサー（KEYENCE, GA-245 SO）を用いて検出した実験箱の振動を 60Hz のlow pass フィルター（NF, FV-624）を介し、サンプリング速度 1 msec でA/D変換後(MacLab), コンピューター(Macintosh LCII) で記録した。驚愕反応としては、聴覚刺激提示後200 msec間における振動波形の最大値と最小値の差を計測した。動物の行動は、赤外線ビデオカメラを用いて常時モニターした。動物を実験箱に入れ、5-10min の装置へのHabituation の後、24試行の驚愕刺激単独提示試行に続き、1)驚愕刺激のみ, 2)視覚刺激を驚愕刺激に50 msec 先行, 3)視覚刺激を驚愕刺激に100msec 先行させた３条件のいずれかの試行を平均試行間隔 25 secのランダムな順で1日合計120試行をおこなった。

結果
　白色雑音に対する正常ハムスターの驚愕反応は、全身反応（飛び上がり、重心移動）、頭部、頚部の動き、あるいは耳介の動きから成り、潜時は約40 msecであった。刺激の反復提示により反応は漸減し、最初の２４試行以内に著明に減少したが、その後はおよそ一定の大きさの反応を示した( Fig. 1 )。さらに、驚愕反応の振幅は刺激提示時の動物の行動状態に依存し、探索行動、毛づくろい行動時、および覚醒レベルの低下時に著明に抑制された。3匹のハムスターで、調べたprepulse の効果は、先行時間が 50 msec の条件では驚愕刺激単独提示条件に比べ、平均振幅に差は認められなかった。一方、先行時間が100 msec の条件では、驚愕刺激単独提示条件に比べ、平均振幅の増大が認められた（Fig.2, t (52)=4.116, p<.001)。

論議
正常ハムスタ−の聴覚性驚愕反応におよぼす視覚先行刺激の効果ついて調べた。ラットでは聴覚性prepulseの刺激強度が比較的強い場合には（85dB, 背景雑音レベル79dB）抑制が生ずるが、弱い場合（81dB）には逆に促進が生ずる（Reijmers and Peeters, 1994）。また、ｹﾞｯ歯類の視感度は緑色の波長 (500 nm）で閾値が低く、赤色の波長（600 nm）では高いことが知られている。本実験では高輝度のLEDとして入手可能な市販の赤色LEDを用いたが、この視覚刺激はハムスタ−にとって比較的弱い刺激であったと考えられる。したがって、本実験条件では 視覚刺激を100 msec 先行させた場合抑制ではなく、促進が認められたものと解釈される。今後、さらに 視覚刺激の強度および先行時間を変数としてprepulse の効果を調べることが必要である。

REFERENCE

So, K. -F. and Aguayo, A.J., Lengthy regrowth of cut axons from ganglion cells after peripheral nerve transplantation into the retina of adult rats. Brain Res., 328 (1985) 349-354.

Sasaki, H., Inoue, T., Fukuda, Y., Iso, H. and Hayashi, Y., Light-dark discrimination after sciatic nerve transplantation to the sectioned optic nerve in adult hamsters. Vision Res., 33 (1993) 877-880.

佐々木 仁、井上 徹, 再生視神経の行動的機能回復-視覚機能修復の行動学的解析, 神経眼科、Vol. 12, No.3 , 293-299, 1995.

H. Sasaki, P. Coffey, M.P. Villegas-Perez, M. Vidal-Sanz, M.J. Young, R.D. Lund and Y. Fukuda, Light induced EEG desynchronization and behavioral arousal in rats with restored retinocollicular projection by peripheral nerve graft. Neurosci. Lett. 218: 45-48, 1996.

福田　淳、渡部眞三、澤井元、佐々木 仁、井上　徹、三好　智満, 末梢神経移植による視神経の再生と視覚路の機能再建, 神経の再生と機能再建, 第７章、志水義房他（編集）西村書店, Pp. 273-287, 1997.

Hoffman HS, Ison JR (1980) Reflex modification in the domain of startle: 1. Some empirical findings and their implications for how the nervous system processes sensory input. Psychol Review 87:175-189.

Campeau, S. and Davis, M. Prepulse inhibition of the acoustic startle reflex using visual and auditory prepulses: disruption by apomorphine. Psychopharmacology, Suppl. 3, 117, 267-74, 1995.

Reijmers L.G. J. E., Peeters, B. W. M. M.　Acoustic prepulses can facilitate the startle reflex in rats: Discrepancy between rat and human data resolved.　Brain Research Bulletin, 35(4) 337-338,1994

Abstract : A weak visual prepulse was applied just before a burst of white noise with a various lead intervals from 50 to 400 ms in hamsters. After habituation each hamster received 96 trials of the auditory noise without or with the prepulse. Amplitude of startle response increased significantly at 100 and 200 ms (n=6). This facilitatory effect disappeared in blind control (n=6). Amplitude of startle response decreased during exploratory behavior, grooming, sniffing or other movements. However, the facilitatory effects of the visual prepulse were remained consistent, regardless of the animal's behavioral states. These results show that the visual prepulse task is a simple and repeatable task to evaluate visual perception in hamsters.

Keywords: Acoustic Startle Response; Prepulse Inhibition; Visual Perception; Hamsters

Introduction: It have been shown that the axons of retinal ganglion cells can regrow if peripheral nerve was grafted to their cut end in adults mammals (So and Aguayo 1985). In our previous studies, we have shown some functional recovery of the retino-tectal pathway after the peripheral nerve graft bridging from the cut end of the optic nerve and the superior colliculus in hamsters and rats (Sasaki et al. 1993, 1996). In this study, we will try to establish a new behavioral method to estimate recovery of the visual functions in the grafted hamsters. It is well known that a sudden intense noise cause a startle response in many kinds of animals, including fish and human (Davis 1984, Landis and Hunt 1939). A brief stimulus, which itself dose not cause the startle response, preceding the startle stimulus inhibit the startle response. This effect is called as prepulse inhibition ( ) and it has been found using different modality of prepulse, acoustic (Hoffman and Searle 1965), cutaneous (Pinckney 1976) or visual (Buckland et al. 1969). The purpose of the current experiment is to decide the effects of visual prepulse on auditory startle responses in hamsters. If the task is available for test visual functions in hamsters, it will be more effective, because the test can be applied repeatedly, which is impossible in the case of conditioning procedures such as shuttle box avoidance (Sasaki et al. ).

Methods: Eight adult golden hamsters (8-9 weeks old of age, weighting 80-100g ) were used in the experiment. Animals were put in a translucent acrylic chamber (70 x 70 x 170 mm), with two aluminum mesh at both sides. The chamber was put on four insulator. A piezoelectric accelerometer (GH313A, GA-245 SO, KEYENCE) was attached at the center of the bottom of chamber to detect movements of the animals. The signals were digitized at 1 KHz (MacLab, A/D Instruments), via a low-pass filter (60 Hz, FV-624, NF Corp.) then recorded on a computer (Macintosh Quadra-650, Apple). A white noise (3G13, SAN-EI,) with a duration of 50 ms at 110-120-dB (SPL, Type 2209, Bruel & Kjaer) was used as an acoustic startle stimulus. The white noise was amplified (PA-60, ROLAND) and presented by a speaker located at 50 cm from the chamber. Another acoustic noise （0-20KHz, WG-721, NF Corp.）was continuously presented by another speaker to maintain the background noise level. Arrays of red LED （115 lx, JS3102, STANLEY）was set 10 cm above the chamber and was used as a visual prepulse with a duration of 20 ms. Background illumination level was 0.1 lx. These apparatus was set in a sound-attenuating room (Rion). Animal's behavior was monitored by an infra-red video camera system. Difference between maximal and minimal values within 100 ms from the onset of the startle stimulus was measured using a software (IgorPro, WaveMetrics) and was used as startle amplitude. After 10 min of a habituation period in the chamber, animals received total of 120 trials; startle stimulus alone was presented in the first 24 trials, then 12 trials of startle stimulus alone, 12 trials in each of prepulse conditions with a random order. Two prepulse conditions with different lead time of 50 and 100 ms (n=3), three prepulse conditions with 50, 100 and 200 ms (n=12) or three prepulse conditions with 100, 200 and 400 ms (n=6). Six hamsters in the second prepulse condition, were tested after bilateral optic nerve section as a blind control group. After one week of recovery, these blind animal received the same procedures as normal group. Mean intertrial interval was 25 s (15 -45 sec). All stimulus presentation was controlled by a sequencer (Sysmac, OMRON).

Results: The amplitude of startle responses in hamsters decreased rapidly within the first 24 trials and reached at a relatively constant values. Three experiments were done using different lead times from 50 to 400 ms. The amplitude of the startle response was consistently increased at 100 ms in every experiment. Figure 1 shows the change of the startle amplitude in the different lead time conditions to the amplitude in the startle stimulus alone trial. There was no change at the lead time of 50 ms. The amplitude increased maximally at 100 ms condition, then decreased at 200 ms and the increment disappeared at 400 ms. On the other hand, there was no change in the blind hamsters. These results shows the increment was due to visual effects. The amplitude of startle response was dependent on the state of animals. The amplitude was decreased when the startle stimulus was presented during movements of animals such as grooming, exploratory behavior, sniffing or other movements. However, the effect of the visual prepulse was not dependent on these behavioral states (Fig. 2). The facilitatory effects at 100 ms lead interval was consistently observed even during these movements.

Discussions The most of the prepulse effect to the startle response are inhibitory in rats, mouse, rabbit, and humans ( ). Indeed, we have conducted the same experiment in rats and inhibitory effect can be obtained at 50 and 100 ms lead intervals. The startle amplitude was 50, 75 and 98 % corresponding to the lead intervals of 50, 100 and 200 ms, respectively (data not shown). These findings confirmed priviously reported findings and show that the facilitatory effects in hamsters are not due to the procedures we adopted but due to factors specific to the spices. It has been shown that facilitation can be induced to visual stimulus after fear conditioning in rats (Davis and Astrachan, 1978, Davis et al. 1989). Similar effects were reported in humans that amplitude of eyeblink component of airpuff-eliciting startle response increased to visual stimulus which was paired with electrical shocks (Spence and Runquist 1958, Ross 1961). Also, the amplitude of eyeblink increased to a visual stimulus under shock anticipating condition (Grillon et al. 1990, 1995). Thus, the facilitatory effects in hamster might be due to fear state in the current experimental conditions. However, in our preliminary experiment using auditory prepulse (80 dB, 20 ms) in 4 normal and 4 blind hamsters, every hamster showed not facilitation but inhibition (data not shown) . These data seems to not compatible to this hypothesis. An alternative explanation is that the off response to the visual stimulus has more dominant effects in hamsters than to the on response. It has been shown in rats that a dark prepulse elicits facilitation while light pulse elicits inhibition (Ison et al. 1991). If this is true, only inhibitory effect can be observed if we use a longer light duration which will terminate after onset of the startle stimulus. In such condition, only on response will occur and there is no off response. In present, this is still an open question. Although the mechanisms of the facilitation is not clear, the effect of visual prepulse to the auditory startle response in hamster was significant and consistent. Thus we can conclude that the visual prepulse task is effective to evaluate visual ability in the peripheral nerve grafted hamsters.


Fig. 2 Visual prepulse effect and behavioral states

WIDTH="100" HEIGHT="100">
  • Fig. 1 Visual prepulse effect in hamsters.
  • WIDTH="100" HEIGHT="300">