The medial agranular cortex mediates attentional enhancement of prepulse inhibition of the startle reflex

Journal Pre-proof The Medial Agranular Cortex Mediates Attentional Enhancement of Prepulse Inhibition of the Startle Reflex Qingxin Meng, Yu Ding, Liangjie Chen, Liang Li

PII:

S0166-4328(19)31440-8

DOI:

https://doi.org/10.1016/j.bbr.2020.112511

Reference:

BBR 112511

To appear in:

Behavioural Brain Research

Received Date:

22 September 2019

Revised Date:

15 January 2020

Accepted Date:

24 January 2020

Please cite this article as: Meng Q, Ding Y, Chen L, Li L, The Medial Agranular Cortex Mediates Attentional Enhancement of Prepulse Inhibition of the Startle Reflex, Behavioural Brain Research (2020), doi: https://doi.org/10.1016/j.bbr.2020.112511

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The Medial Agranular Cortex Mediates Attentional Enhancement of Prepulse Inhibition of the Startle Reflex Qingxin Menga, Yu Dingb, Liangjie Chenb, Liang Lia,b,* a

Center for Brain Disorders Research, Capital Medical University and Beijing Institute of Brain

Disorders, Beijing, China b

School of Psychological and Cognitive Sciences and Beijing Key Laboratory of Behavior and Mental

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Health, Speech and Hearing Research Center, Key Laboratory on Machine Perception (Ministry of Education), Peking University, Beijing 100080, China

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*Correspondence:

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Liang Li, Ph.D.

E-mail: [email protected]

Type 1 of attentional enhancement of prepulse inhibition of startle (PPI) in rats: Fear conditioning

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Highlights

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School of Psychological and Cognitive Sciences, Peking University, Beijing 100080, China

of the prepulse enhances PPI induced by the conditioned prepulse. Type 2 of attentional enhancement of PPI: Under the noise-masking condition, the perceived

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spatial separation between the fear-conditioned prepulse and the noise masker further enhances PPI.

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Blocking excitatory glutamate neurotransmissions in the medial agranular cortex (AGm) abolish the two types of attentional enhancements of PPI without affecting baseline PPI.

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The AGm, which is a subdivision of the median prefrontal cortex (PFC), is critical for attentional enhancements of PPI.

ABSTRACT

The startle reflex, which interferes with on-going cognitive/behavioral activities, is of important

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protective function for humans and animals. Prepulse inhibition (PPI), as an operational measure of sensorimotor gating, is the suppression of the startle reflex in response to an intense startling stimulus (pulse) when this startling stimulus is shortly preceded by a weaker non-startling stimulus (prepulse).

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In both humans and laboratory animals, PPI can be enhanced by facilitating selective attention to the

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prepulse, suggesting that higher-order cognitive/perceptual processes modulate PPI. It has been well

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known that both the cholinergic system located in the basal forebrain and the deep layers of the superior colliculus in the PPI-mediating circuit are top-down modulated by the medial agranular cortex (AGm),

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which is a subdivision of the medial prefrontal cortex (PFC) and has wide axonal connections with both cortical regions (including the posterior parietal cortex) and subcortical structures critical for

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attention/orientation processes. This study investigated whether the AGm is involved in attentional modulation of PPI. The results showed that PPI was enhanced by fear conditioning of the prepulse,

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and then further enhanced by perceived spatial separation between the conditioned prepulse and a back-ground masking noise based on the auditory precedence effect. Bilateral injection of 2-mM kynurenic acid, a broad spectrum antagonist of glutamate receptors, into the AGm, but not the primary somatosensory cortex, eliminated these two types of attentional enhancement of PPI. Thus, the AGm plays a role in facilitating attention to the prepulse and is involved in the top-down modulation of PPI.

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Keywords: Startle reflex; Prepulse inhibition; Attentional modulation; Fear conditioning; Precedence effect; Perceptual separation.

1.

INTRODUCTION

The startle reflex is a type of systemic and rapid whole-body reflective response in mammals. It can

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be triggered by a sudden burst of intense sensory stimulus, and serves as an important protective mechanism for survival with a wide range of inter-species commonalities [1–4]. The neural circuitry mediating the startle reflex includes the cochlear nucleus, vestibular, trigeminal nuclei, the caudal

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pontine reticular nucleus, and the spinal motor neurons [4–7], and is able to summate signals from

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different sensory modalities.

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The startle reflex is the strongest whole-body reflexive response and may interfere with current ongoing cognitive/behavioral activities [8–10]. For example, the acoustic startle reflex can disrupt

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perception/motor tasks in humans [8,10] and learned lever-pressing behaviors in rats [9]. Prepulse inhibition (PPI) is the suppression of the startle reflex in response to an intense startling stimulus (pulse)

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when this startling stimulus is shortly preceded by a weaker, non-startling sensory stimulus (prepulse) [11,12]. Graham (1975) has assumed that a weaker prepulse stimulus preceding the intense stimulus

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(pulse) can generate both the central processing of the prepulse signals and a gating mechanism reducing the processing of the intense disruptive inputs of the pulse (the “Protection-of-Processing Theory” for explaining the function of PPI) [13]. Thus, PPI protects the early processing of the prepulse signals from interference by startling pulse stimuli and has been generally recognized as a simple operational measure of sensorimotor gating [14].This prepulse-induced inhibitory effect on the startle

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reflex is common across mammal species [14–16]. Although the basic auditory PPI-mediating circuitry includes some brainstem structures such as the inferior colliculus (IC) and the pedunculopontine tegmental nucleus (PPTg) [17–22], the neural circuitry mediating top-down cognitive/perceptual modulation of PPI is not completely clear [23]. In rats, PPI can be enhanced by fear conditioning of the prepulse stimulus with synchronized (paired) combination of the foot-shock and the prepulse [24-28]. The fearing-conditioning-induced

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enhancement is further increased by the perceived spatial separation between the conditioned prepulse and a background noise based on the auditory precedence effect [23,26-28](for the concept of the precedence effect see [73]), due to the facilitation of selective attention to the prepulse signal [22–28].

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In rats the PPI enhancement induced by fear conditioning and that by perceived spatial separation can

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be eliminated by blocking the neural activity of the lateral nucleus of the amygdala (LA) and posterior parietal cortex (PPC), respectively [26]. Moreover, blocking the auditory cortex (AC), which provides

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auditory inputs to both LA and PPC, completely abolishes PPI enhancements, suggesting that the AC,

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LA, and PPC are involved in the top-down attentional modulation circuitry of PPI. In our previous studies, blocking these brain structures was made by bilateral injection of kynurenic acid (KYNA, 2

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mM in Locke’s solution), which is a broad spectrum endogenous antagonist of excitatory amino acid receptors, into these structures (AC, 2.0 µL on each side; LA , 1.0 µL on each side; PPC, 2.0 µL on

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each side)[26]. The PPI enhancements also exhibits a remarkable specificity to the conditioned prepulse signal [26,27,106,107]. A recent study by Ding et al. (2019) has shown that the two types of PPI enhancement are mediated by the deeper (DpSC) but not superficial (superSC) layers of the superior colliculus [28]. More importantly, these brain areas mentioned above connect with the medial agranular cortex

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(AGm) either directly or indirectly [29–35]. Also, the AGm and the PPC have very similar neurological response patterns and spatial processing functions of cognitive tasks [36]. Therefore, the functional and anatomical relationship between the AGm and PPC may constitute the frontal-parietal cortical circuit of spatial attention [36–38]. Meanwhile, the DpSC receives direct descending axonal projections from the AGm for top-down modulation by higher-order cognitions [37,39]. Up to date, however, it is unclear whether the AGm is involved in the modulation of PPI driven by perception of

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the prepulse signal. This present study, for the first time, aimed to explore the role of the AGm in the attentional modulation of PPI.

In rodents, the AGm is a subdivision of the medial prefrontal cortex, called either the dorsomedial

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prefrontal cortex or the dorsally situated precentral medial area, and the AGm has also been recognized

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as the secondary motor cortex (M2), frontal orienting field (FOF), or second frontal area (Fr2) [30,40– 47]. Since the AGm receives both cortical and subcortical afferents from visual, auditory,

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somatosensory cortices, parietal cortex, anterior cingulate area, and prelimbic and infralimbic areas,

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its role in integration of information may be multimodal [32,33,47–51]. The efferent connections from the AGm are also widely distributed in cortical and subcortical structures, including the PPC, AC, and

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superior colliculus (SC) [29,31,34,35,43,49,52–54]. These physiological and anatomical neural substrates suggest that the AGm may not only integrate bottom-up multimodal inputs but also play a

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crucial role in top-down executive controls [50,52,55,56]. It has been suggested that the AGm processes evidence for behavioral choices based on perception and memory inputs and exhibits its choice outputs in the form of neuron spiking [40,41,57–59]. It has been reported that inactivation of the AGm can disrupt the learning of skilled movements [60–63], and particularly, unilateral lesions of the AGm can cause the contralateral multimodal neglect [36,41,64–66].

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A well-established method, reversible inactivation of neurons by local microinjection of the glutamate receptor antagonist, kynurenic acid (KYNA), is useful for exploring the neural substrates underlying PPI-modulation [26,28]. KYNA, which is an endogenous antagonist of excitatory amino acid receptors [67], blocks both NMDA and non-NMDA receptors, decreasing neural excitations in the injected area [68–70]. The main purpose of the present study was to compare PPI before and after bilateral injection of KYNA into the AGm for determining whether the attentional modulation of PPI

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was dependent on the AGm. If the AGm plays a role in this process, there should be a downward

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change in PPI by the chemical blockage.

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2.

MATERIALS AND METHODS

2.1 Animal Subjects Thirty male Sprague-Dawley rats aged 11 weeks and weighting 270-320 g (purchased from the Vital-River Experimental Animals Technology Ltd., Beijing, China) were randomly divided into two groups, namely the AGm-injection group as the experimental group (n = 18) and the anatomically-

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control group (n = 12). During this study, the rats were kept in a room with an average temperature of 24 ± 2°C and a 12-hour circadian rhythm, and were provided with sufficient water and food in transparent plastic cages (48 × 30 × 18 cm).

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2.2 Animal Surgery Preparation

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All the rats were anesthetized with 10% chloral hydrate (400 mg/Kg, intraperitoneal injection) during the surgical procedures of implanting microinjection-guide cannulas (C317G guide cannula;

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Plastics One Inc., Roanoak, VA, USA).

In the experimental group (i.e., AGm-injection group), the guide cannulas for microinjection were

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implanted bilaterally into the AGm. Referenced to bregma, the stereotaxic coordinates of the inner

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cannula (which was inside the guide cannula) aimed the target structure (i.e., AGm) were the following: anteroposterior, +1.2 mm; mediolateral, ±1.1 mm; depth, -1.5 mm. In the anatomically control group,

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the guide cannulas for microinjection were implanted into bilateral primary somatosensory cortex (S1BF). Referenced to bregma, the stereotaxic coordinates of the inner cannula aimed the structure (i.e., S1BF) were the following: anteroposterior, -3.1 mm; mediolateral, ±5 mm; depth, -2.5 mm. After the implantation, the rats were given at least a week to recover from surgery before the PPI test. In this study, the rats were treated in accordance with the Guidelines of the Beijing Laboratory

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Animal Center, and the Policies on the Use of Animals and Humans in Neuroscience Research approved by the Society of Neuroscience (2006). All the experimental procedures were approved by the Committee for Protecting Human and Animal Subjects in the School of Psychology and Cognitive Sciences at Peking University.

2.3 Stimuli and Apparatus

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The apparatus for PPI testing have been described in detail elsewhere [26-28,106,107]. Briefly, all the PPI tests were conducted in a soundproof room. The rats were confined to a custom-made cage [26]. The prepulse stimulus and masking noise were delivered by each of the two spatially separated

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loudspeakers, which were placed horizontally in the frontal field with a 100° separation angle and 52 cm away from the rat’s head position. The rat’s whole-body startle reflex, which was induced by an

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intense broadband noise burst delivered by a loudspeaker (PCxb 352, Blaupunkt, USA) 30 cm above

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the rat’s head, was measured by a custom-made electrical scale (the National Key Laboratory on Machine Perception, Peking University) in the soundproof chamber described above.

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The startling stimulus was a 10-ms broadband noise burst (0-10 kHz, 100 dB SPL). The prepulse stimulus was a 50-ms three-harmonic-tone complex with either lower frequency components (1.3, 2.6,

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and 3.9 kHz) or higher frequency components (2.3, 4.6, and 6.9 kHz). Both harmonic complexes were

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within the audible frequency range of rats and can be distinguished by them [26,27,28,71,72]. The prepulse was digitally generated by MATLAB software and converted by a custom-developed sound delivery system (National Key Laboratory on Machine Perception, Peking University) with the 16kHz sampling rate and 16-bit resolution. It started 100 ms before the onset of startling stimuli. Both the single-source sound level of the prepulse stimulus and that of the broadband masking noise for each of the two horizontal loudspeakers were fixed at 60 dB SPL. Calibration of sound levels was

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conducted with a Larson Davis Audiometer Calibration and a 3091 Electro-acoustic Testing System (AUDit & System 824, Larson Davis, Depew, NY, USA) whose microphone was placed at the central location of the rat’s head when the rat was absent, using a Fast/Peak meter response. Drug administration was made through the injection cannula, which was inside the guide cannula. The infusion cannula was connected to a 5 l microsyringe by a polyethylene tube (Clay Adams, division of Becton and Dickinson Company, Parsippany, NJ, USA), and a volume of 2.0 μl of either

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Locke’s solution or KYNA (2 mM in Locke’s solution; Sigma-Aldrich, St Louis, MO, USA) was delivered slowly into the AGm (on each side) over a period of 2 min on each side.

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2.4 Procedures

For the two groups of rats (Figure 1, the solid wireframe for the experimental group; the dashed

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wireframe for the anatomically-control group), they were allowed seven days of post-surgery recovery

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with ad libitum access to food and water, and then went through the 6-day (experimental group) or 5-

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day (anatomically-control group) testing procedure.

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------ Insert Figure 1 about here ------

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All rats were habituated to the restraining cage for 30 min for each of 3 consecutive days. They were exposed to a broadband noise (60 dB SPL) which was continuously presented by each of the two horizontal loudspeakers. During this adaptation period, neither prepulse nor startling stimuli were presented. On day 4, the rats underwent the baseline startle testing, the PPI testing, and the fear conditioning/conditioning-control manipulations. Specifically, PPI before conditioning (procedure

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stage BC) was measured first. The rat was placed in the restraining cage for 5 min, receiving 10 presentations of the 10-ms startling broadband noise burst (0-10 kHz, 100 dB SPL) without the prepulse presentation on the broadband-noise background whose intensity was 60 dB SPL. The interval between startling stimuli varied between 25 and 35 s (mean = 30 s). Then 2 PPI-testing blocks were introduced. The inter-loudspeaker onset delay for the broadband noise, which was continuously delivered from each of the two horizontal loudspeakers as the masking noise, was +1 ms (left leading)

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in one block and -1 ms (right leading) in the other block. Since the same (correlated) masking noise sound was delivered by the two spatially separated loudspeakers with a short inter-loudspeaker delay (1 ms), a single fused continuous noise-masker image occurred at the left loudspeaker in one block

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(when the left loudspeaker was the leading one) and at the right loudspeaker in the another block (when

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the right loudspeaker was the leading one) due to the auditory precedence effect [73],which is a type of perceptual fusion of correlated leading and lagging sounds based on the attribute-capturing process

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[74]. In addition to the masking noise, the prepulse was also presented from each of the two horizontal

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loudspeakers with the inter-loudspeaker onset delay being either +1 ms (left leading) or -1 ms (right leading) in each of the two testing blocks. The inter-loudspeaker onset delay for the prepulse caused a

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single fused prepulse image at the left loudspeaker in some trials (when the left loudspeaker led) and at the right loudspeaker in other trials (when the right loudspeaker led). Thus, two types of perceived

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spatial relationships between the prepulse and the masker (built by the four (2×2) combinations of the perceived locations between the prepulse stimulus and masking noise) were created in each block: perceptual separation (when prepulse and masker had different leading loudspeakers) and perceptual colocation (when prepulse and masker shared the same leading loudspeaker). Each testing block contained 25 trials, including 5 trials with the startling stimulus alone, 10 trials

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with the lower-frequency prepulse stimulus preceding the startling stimulus (5 trials under the condition of perceptual co-location, and 5 trials under condition of perceptual separation), and 10 trials with the higher-frequency prepulse stimulus preceding the startling stimulus (5 trials under the condition of perceptual co-location, and 5 trials under condition of perceptual separation). In other words, in each testing block, 10 trials were assigned to the condition of perceptual spatial separation (5 trials with the higher-frequency prepulse stimulus, and 5 trials with the lower-frequency prepulse

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stimulus), 10 trials were assigned to the condition of perceptual co-location (5 trials with the higherfrequency prepulse stimulus, and 5 trials with the lower-frequency prepulse stimulus), and 5 trials were

(ranging from 25 to 35 s) separated consecutive trials.

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assigned to the none-prepulse (startling stimulus only) condition. In each block, an average of 30 s

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Also, on the same day (day 4), after the procedure stage BC, all the rats received both fearconditioning and conditioning-control manipulations no less than 3 hours after the PPI testing before

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conditioning (i.e., procedure stage BC). The conditioning stimulus (CS) was one of the prepulse stimuli

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(either the lower- or the higher-frequency prepulse) delivered by each of the two horizontal loudspeakers with balanced left/right leading. Therefore, the conditioning-control stimulus for an

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individual rat was the other prepulse stimuli (either the higher- or the lower-frequency prepulse) delivered by each of the two horizontal loudspeakers with balanced left/right leading. The

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unconditioned stimulus (US) was a 6-mA rectangular-pulse foot-shock with a duration of 3 ms provided by a Grass S-88 stimulator (Grass, Quincy, MA, USA). For each rat, during the fearconditioning manipulation, 10 temporally synchronized (paired) combinations of the foot-shock (US) and the conditioning prepulse stimulus (CS) were presented every 30 s (US started 3 ms before CS ending, and co-terminated with CS). During the conditioning-control manipulation, 10 temporally

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random (unpaired) combinations of the foot-shock and the other prepulse were presented every 30 s. In each group, one half of the rats received fear conditioning with the lower-frequency prepulse and conditioning control with the higher-frequency prepulse, and the other one half of the rats received the contrary manipulations. PPI in the next 4 procedure stages was examined on day 5 and day 6: 1) PPI after conditioning, before injecting KYNA (procedure stage BK); 2) PPI after injecting KYNA (procedure stage AK); 3)

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PPI after the 2nd conditioning, before injecting Locke’s solution, which was the vehicle (procedure stage BL); and 4) PPI after injecting Locke’s solution (procedure stage AL). For the injection of KYNA and injection of Lockes’ solution in an individual rat, either the order BL-AL-BK-AK or the order BK-

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AK-BL-AL was assigned. In other words, either KYNA or Locke’s solution was injected first for an

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individual rat randomly.

On day 5 (24 h after the conditioning/conditioning-control manipulation), 2 procedure stages were

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examined, with the order of either BK-AK or BL-AL was used first, and the order of injection of the

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two chemicals was balanced across individual rats. To reduce or prevent the possible fear extinction leaning caused by the PPI testing procedures before the 2nd injection and to use the animal participants

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sufficiently, on day 5 all the rats received the 2nd fear-conditioning/conditioning-control manipulations with the same procedures as used on Day 4 no less than 3 hours after the PPI testing.

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For the injection methods, after the procedure stage BK or BL, either the KYNA (2 mM in Locke’s solution; Sigma-Aldrich, St Louis, MO, USA) or Locke’s solution was injected slowly into the bilateral AGm (2.0 μl on each side) over a period of 2 min on each side. According to our previous studies, local and slow injection of 2.0 μl of KYNA does not spread too much to the surrounding brain areas [26,28]. The blocking effect of KYNA is reversible and can vanish about 2-3 hours after the injection

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at the dose. For the brain-region-control group, only procedure stages BC, BK, and AK were performed, with the order of BC-BK-AK (see the dashed wireframe of Figure 1). After the procedure stage BK, KYNA (2 mM in Locke’s solution) was injected slowly into the bilateral S1BF (2.0 μl on each side) over a period of 2 min for each side. Other details were the same as used in the experimental group. On day 6 (24 h after the 2nd conditioning manipulation/conditioning-control manipulation), the other

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2 procedure stages were examined for individual rats as on day 4, with the order of either BL-AL or BK-AK, which was reversal compared to that on day 5. Note that on the procedure stages on days 5 and 6, PPI was measured with the procedure used as in the stage before conditioning (BC on day 4) as

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described above. Both the conditioned prepulse and the conditioning-control prepulse were always

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presented in each of the two blocks.

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2.5 Data Analyses

The magnitude of PPI was calculated with the following generally used the formula:

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PPI (%) = (amplitude to startling noise alone - amplitude to startling noise preceded by prepulse) / (amplitude to startling noise alone) × 100%.

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Mixed and within-subject repeated-measures ANOVAs followed by Bonferroni’s pairwise

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comparisons (for comparisons between procedure stages) and Bonferroni’s pairwise comparisons (for comparisons between perceived colocation and spatial separation) were performed using SPSS 21.0 software. Multivariate tests were conducted, and the null-hypothesis rejection level was set at 0.05.

2.6 Histology Upon completion of the experiments, rats were deeply anaesthetized and euthanized with an overdose of 10% chloral hydrate (no less than 600 mg/Kg, intraperitoneal injection). Lesion marks

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were made via the cannula by an anodal DC current (500 μA, 10 s). Brains were stored in 10% formalin with 35% sucrose, and then sectioned at 50 μm in the frontal plane in a cryostat (-20°C). Sections were examined to determine locations of injection cannulas.

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RESULTS

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3.1 Histology

Histological analysis revealed that the tips of the cannulas were situated within the bilateral AGm in 18 rats and the bilateral S1BF in 12 rats as shown in Figure 2. Thus, descriptions and statistical

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analyses were based on data from 18 rats in the experimental group and 12 rats in the anatomically

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control group.

3.2 Responses to the Startling Stimulus Alone

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The mean amplitudes of startle responses to the startling stimulus alone across procedure stages

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(when the prepulse was not presented) in the experimental group (the AGm-injection group, n = 18) are shown in Figure 3. Data was averaged across the 2 testing blocks in each of the procedure stages (BC, BK, AK, BL, AL) in the experimental group. A repeated-measures ANOVA with one withinsubjects factor (procedure stage: BC, BK, AK, BL, AL) showed that the effect of the procedure stage on the startle response to the startling stimulus alone was not significant in the experimental group (F(4,14)= 1.299, p > 0.05). Injecting either KYNA or Locke’s solution into the bilateral AGm, and

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either the conditioning manipulation or the conditioning control manipulation did not significantly influence the startle amplitude to the startling stimulus alone (all ps > 0.05).

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3.3 Effects of Blocking the AGm on PPI Figure 4 shows PPI at the procedure stage BC and the effects of fear conditioning (procedure stage BK and BL) and perceived spatial separation on PPI for the experimental group with the injection of

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either KYNA (procedure stage AK) or Locke’s solution (procedure stage AL) into the AGm.

A 5 (procedure stage: BC, BK, AK, BL, AL) × 2 (perceived spatial relationship, simply called

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separation type: co-location, separation) within-subject repeated-measures ANOVA showed that the main effects of the procedure stage and separation type were significant (procedure stage: F(4,14) =

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16.347, p < 0.001, ηp2 = 0.824; separation type: F(1,17) = 9.670, p = 0.006, ηp2 = 0.363), the interaction

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between the two factors was significant (F(4,14) = 4.920, p = 0.011, ηp2 = 0.584). Then, pairwise comparisons (for comparisons between procedure stages) and Bonferroni’s pairwise comparisons (for comparisons between separation types) were carried out. The results showed that the PPI level changed between some procedure stages and between the two perceived spatial relationships at some procedure stages (see below and Figure 4). Specifically, at procedure stage BC, the effect of separation type on PPI was not significant (F(1,17)

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= 1.403, p = 0.252, ηp2 = 0.076). The PPI level at procedure stage BK and BL were significantly larger than that at procedure stage BC (procedure stage BC vs BK: p = 0.010; procedure stage BC vs vBL: p = 0.001), showing the fear-conditioning effect on PPI. Also, there was no significant difference between procedure stage BK and BL on PPI level (p > 0.05). At procedure stage BK and BL (after fear conditioning), the effect of separation type on PPI were significant (procedure stage BK F(1,17) = 16.947, p = 0.001, ηp2= 0.499; procedure stage BL F(1,17) = 13.841, p = 0.002, ηp2 = 0.449), showing

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that the perceived spatial separation between the conditioned prepulse and the noise masker enhanced the PPI.

Following the injection of KYNA into the bilateral AGm (procedure stage AK), the perceived spatial

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separation-induced PPI enhancements disappeared (AK F(1,17) = 0.006, p = 0.938, ηp2 < 0.001). Also,

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the PPI level at procedure stage AK became significantly smaller than that at procedure stage BK and BL (procedure stage AK vs procedure stage BK: p < 0.01; procedure stage AK vs procedure stage BL:

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p = 0.001), but not significantly different from that at procedure stage BC (p > 0.05).

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On the other hand, the injection of Locke’s solution into the AGm did not significantly change either the conditioning-induced PPI enhancement (procedure stage AL vs procedure stages BK and procedure

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stage BL, all ps > 0.05) or the separation-induced PPI enhancement (at the procedure stage AL, PPI under the separation condition was still larger than that under co-location condition, F(1,17) = 7.947,

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p = 0.012, ηp2 = 0.319). Also, the PPI level at procedure stage AL was significantly larger than that at procedure stage BC (p = 0.001) and procedure stage AK (p = 0.006).

3.4 PPI Induced by the Conditioning-Control Prepulse Figure 5 shows PPI at the procedure stage BC and the effects of the conditioning-control manipulation (procedure stage BK and BL) on PPI for the experimental group with the injection of

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KYNA (procedure stage AK) or Locke’s solution (procedure stage Al) into the AGm. The results showed that the conditioning-control manipulation did not significantly affect PPI. A 5 (procedure stage: BC, BK, AK, BL, AL) × 2 (perceived spatial relationship, simply called separation type: co-location, separation) within-subject repeated-measures ANOVA confirmed that the main effects of the procedure stage and separation type were not significant (procedure stage: F(4,14) = 2.533, p = 0.087, ηp2 = 0.420; separation type: F(1,17) = 1.525, p = 0.234, ηp2 = 0.082), and the

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interaction between the two factors was not significant (F(4,14) = 0.459, p = 0.765, ηp2 = 0.116). In other words, after the conditioning-control manipulation, the PPI level was not enhanced.

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3.5 Effects of Blocking the S1BF in the Brain-Region Control Group To examine the anatomical specificity of KYNA injection into the AGm, PPI was tested in 12 rats

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with KYNA injection into the S1BF area.

The mean amplitude of the startle response to the startling stimulus alone (when the prepulse was

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not presented) was shown in Figure 6. It was averaged across the 2 blocks of PPI testing in each of the

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procedure stage (BC, BK, AK) in the brain-region control group. A repeated-measures ANOVA with one within-subjects factor (procedure stage: BC, BK, AK) showed that the effect of procedure stages was not significant (F(2,10) = 2.752, p > 0.05).

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Figure 7 shows PPI at the procedure stage BC and the effects of fear conditioning (left panel, procedure stage BK) /conditioning-control manipulations (right panel, procedure stage BK) on PPI for the brain-region control group with the injection of KYNA (procedure stage AK) into the S1BF. The results showed that PPI was enhanced by fear conditioning of the prepulse, and further enhanced by the perceived spatial separation. However, injection of KYNA into the S1BF area appears not to affect PPI.

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A 3 (procedure stage: BC, AC, AK) × 2 (separation type) within-subject repeated-measures ANOVA showed that the main effect of procedure stage was significant (F(2,10) = 6.938, p = 0.013, ηp2 = 0.581), the main effect of separation type was significant (F(1,11) = 1.525, p = 0.001, ηp2 = 0.626), and the

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interaction between the two factors was not significant (F(2,10) = 3.064, p = 0.092, ηp2 = 0.38).

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Pairwise comparisons (for comparisons between procedure stages) and Bonferroni’s pairwise comparisons (for comparisons between separation types) showed that (1) at the procedure stage BC,

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the effect of separation type on PPI was not significant (F(1,11) = 1.212, p = 0.294, ηp2 = 0.099); (2)

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the PPI level at procedure stages BK and that at the stage AK were all significantly larger than that at procedure stage BC (AC: p = 0.008; KY: p = 0.008); (3) at procedure stages BK and AK, the effect of

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separation type on PPI was still significant (BK: F(1,11) = 20.469, p = 0.001, ηp2 = 0.65; AK: F(1,11) = 5.764, p = 0.035, ηp2 = 0.344).

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For the prepulse used for the fear conditioning-control manipulation, a 3 (procedure stage: BC, AC, KY) × 2 (separation type) within-subject repeated-measures ANOVA showed that the main effects of procedure stage and separation type, and the interaction between the two factors were all not significant (all ps > 0.05).

19

------ Insert Figure 7 about here ------

4. DISCUSSION The present study, for the first time, demonstrates that the AGm is an essential brain region involved in both the conditioning-induced PPI enhancement and the perceptual separation-induced PPI

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enhancement in rats. More in detail, PPI was significantly enhanced after fear conditioning of the prepulse stimulus, and further enhanced by perceived spatial separation between the conditioned prepulse stimulus and a noise masker.

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KYNA is a reversible and broad-spectrum antagonist of glutamate receptors. It can block both NMDA receptors and non-NMDA receptors without affecting axonal information transmission [67–

re

70]. The effect of the KYNA dose used in this study has been verified by previous studies [26,28].

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Then, these two PPI enhancements were abolished completely by bilateral injection of KYNA into the AGm, while no changes were observed after bilateral injection of Locke’s solution into this region.

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In summary, at the procedure stage BC, the perceived spatial separation between the prepulse and noise masker did not enhance PPI. However, when the prepulse became fear conditioned (procedure

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stage BK and BL), PPI was remarkably enhanced, and the enhancement was further increased by the

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perceived spatial separation. And then, injection of KYNA into the bilateral AGm markedly eliminated the two types of PPI enhancements (procedure stage AK), leading to that PPI reduced to the level at the procedure stage BC. In contrast, the injection of Locke’s solution into the AGm did not influence the two types of PPI enhancements (procedure stage AL). Also, no enhancements were significant for PPI elicited by the conditioning-control prepulse at all procedure stages, before and after bilateral infusion of either KYNA or Locke’s solution into the AGm under either co-location or separation

20

conditions. For the anatomically-control group, there were no significant changes in startle amplitude after the conditioning/conditioning control manipulations compared with startle amplitude at procedure stage BC. Also, injection of KYNA into S1BF did not significantly influence the startle amplitude to the startling stimulus alone (see Figure 6). At the procedure stage BC, the perceived spatial separation between the prepulse and noise masker did not enhance PPI (see Figure 7). However, when the

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prepulse became fear conditioned, PPI was remarkably enhanced, and the enhancement was further increased by the perceived spatial separation (see Figure 7 left panel, procedure stage BK). Meanwhile, the conditioning-control manipulation did not affect the PPI level (see Figure 7 right panel, procedure

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stage BK). Injection of KYNA into the bilateral S1BF was not same as the injection of KYNA into the

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bilateral AGm. It did not significantly eliminate the two types of PPI enhancements after the conditioning manipulation, and PPI was not significantly changed after the conditioning control

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manipulation was introduced. (see Figure 7 procedure stage AK).

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In this study, since bilateral KYNA blockage of the AGm did not affect the baseline PPI but reduced the two attentional modulations of PPI, the results suggested that the attentional modulation of PPI,

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but not the mediation of the baseline PPI, depends on the AGm. PPI occurs in all mammals, and is observed in laboratory rats after decerebration or chemical

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suppression of the cortex [14-16,26,75,78]. For example, bilateral infusion of KYNA into the primary auditory cortex or the posterior parietal cortex does not affect the PPI if the prepulse stimulus is not conditioned [26]. PPI can even be observed in laboratory rats with acutely surgical decerebration or chemical suppression of the cortex [75–78], indicating that the basic neural circuitry mediating PPI resides in the brainstem, including the inferior colliculus (IC) [17–19], pedunculopontine tegmental

21

nucleus (PPTg) [20–22], and deeper layers of the superior colliculus (DpSC) [28,79]. Therefore, the AGm blockage by KYNA infusion does not affect PPI baseline because this structure is not located in the basic circuitry mediating PPI. The PPI response can be enhanced by facilitating selective attention to the prepulse [23]. For example, an emotionally conditioned (such fear conditioned) prepulse stimulus or a prepulse stimulus with emotional content can induce greater PPI than an emotionally neutral prepulse stimulus.

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Specifically, PPI induced by a neutral image prepulse becomes significantly stronger when prepulse image comes to be emotional (such as a fearful image) [81]. For prepulse stimuli combined with paired electrical shocks, the induced PPI is significantly higher compared to randomly presented shock threats

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or non-shock conditions [82]. Moreover, the expectation of shock can also significantly enhance PPI

re

[83]. Finally, the perceived spatial separation between the background noise and the conditioned prepulse stimulus (i.e., the background noise and the prepulse stimulus appear to come from different

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locations due to the precedence effect) can enhance PPI by the facilitation of spatially selective

of research [85–88].

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attention [23,26–28,84]. The results of this study generally agree with the previous studies in this line

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To the best of our knowledge, this is the first work showing that the AGm plays a role in PPI enhancements based on facilitating selective attention to prepulse. When the AGm is not blocked under

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the condition with the infusion of Locke’s solution, two types of attentional enhancements of PPI are not affected. KYNA infusion into the AGm completely disrupts the enhancements of the PPI without affecting its baseline. In rats, unilateral blocking the AGm leads to the multimodal neglect contralateral to the inactivated AGm, indicating that the AGm is crucial for attentional functions [37,64,65,89]. It is well known that

22

the cholinergic system located in the basal forebrain (BF) is critical for the attentional processes [90,91]. Neurophysiological studies have shown that increased cholinergic transmission in sensory areas is associated with enhanced cortical processing of thalamic inputs. It has been shown that activation of BF enhances the release of acetylcholine (ACh) in the cortex, causing enhancement of the processing of target signals, and at the same time, suppression of interferences of non-attention masker on target signals [90,92,93].

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The ACh release in cortical regions can be altered by regulating the activity of the AGm. For example, some previous studies have shown that attentional processing is "top-down" regulated by the medial prefrontal cortex (mPFC) [94,95], which is also the brain area directly projecting to BF

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cholinergic cells [56,96]. As a subregion of the mPFC, the AGm has a large number of nerve fibers

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connecting to other brain regions [51]. Thus, the AGm may directly or indirectly regulate the excitability of the BF, controlling cholinergic release into other cortices and affecting selective

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attention to target signals [97]. Nelson et al. (2005) have reported that chemical activation of the AGm

na

can significantly increase ACh release in posterior parietal cortex (PPC) [94]. Moreover, Rasmusson et al. (2007) have shown that the increase in ACh release in the auditory cortex (AC) induced by

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stimulation of the auditory thalamus (medial geniculate nucleus, MGN) can be completely eliminated by inactivation of the AGm [98]. On the other hand, activation of the BF can enhance responses of the

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AC to stimulation of MGN, suggesting that ACh promotes auditory processing in the AC, and this increase of activation enhances the information transmission from the thalamus to the cortex [92,99]. Therefore, attention deficits caused by cholinergic projection damage may be related to denervation of axonal connections between the AGm and the basal forebrain cholinergic system [100]. The AGm may also be involved in the interaction between top-down cognitive modulation and

23

bottom-up signal-driven signal detection [30,40,50,59,101]. Inactivation of the AGm achieved by means of destruction, pharmacological inhibition, or photogenetic silencing, etc., causes decreased behavioral performance of learning, impaired ability to perceive target signals [36,43,64–66], and weakened preparation for action choices related to decision-making [57–59,102]. The AGm is also recognized as the cortical region where “sensory” signals meet “motor” signals [30]. The AGm blocking does not affect the movement of rats, suggesting that it is only involved in the transmission

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of action intention, but not the movement outputs [40]. It is of interest to understand whether the AGmmediated attentional modulation of PPI is caused by the increase of ACh release in the AC and PPC due to the AGm-depended activation of cholinergic cells located in the BF.

-p

The AGm may also participate in evaluating the signal value and selecting the outputs. Particularly

re

the AGm may pass the selected outputs to the DpSC [37,39], which is a critical brainstem structure mediating both the baseline PPI and attentional enhancements of PPI [28].

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In healthy humans, paying attention to the prepulse stimulus enhances PPI [84]. However in people

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with schizophrenia, the both the attentional enhancement of PPI and the baseline PPI are impaired, and particularly the impairment of the

attention-enhancement PPI is significantly correlated to some

ur

positive symptoms [103–105]. Similar deficiency can also be observed in animals models of schizophrenia [23,25,106,107].

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It should be noted that although the rat AGm is a homolog of the premotor cortex, supplementary motor area, or frontal eye field in primate, it is difficult to achieve a strict one-to-one correspondence between the two species [34,41,51,56,58,62,66]. It has been confirmed that PPI in normal people is positively correlated with grey matter volume of premotor cortex and supplementary motor area, while the deficiency of PPI in people with schizophrenia may be associated with abnormalities in these brain

24

regions [108]. In fact, some previous studies have demonstrated that these brain regions are altered in schizophrenic patients compared to healthy controls, in terms of morphology, functional connectivity, and integrity [109–111]. Kynurenine 3-monooxygenase (KMO) enzyme activity in the postmortem frontal eye field is lower in the patient group than the control group, which could be the reason of that schizophrenia is related to high KYNA concentration [112]. Therefore, the AGm may be a brain region

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in future studies for establishing new animal models of schizophrenia.

SUMMARY

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This study, for the first time, provides evidence showing the dependence on the AGm in two types

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of attentional enhancements of PPI.

Fear conditioning of the prepulse stimulus enhances PPI induced by the conditioned prepulse. Under

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the noise-masking condition, the perceived spatial separation between the fear-conditioned prepulse stimulus and the noise masker further enhances PPI, confirming that PPI in rats can be top-down

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enhanced by attentional processes.

Blocking excitatory glutamate neurotransmissions in the AGm reduces each of the two types of

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attentional enhancements of PPI without affecting baseline PPI, suggesting that both the fear-

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conditioning-induced PPI enhancement and the perceptual-separation-induced PPI enhancement depend on the activation of the AGm. Statement

Qingxin Meng: investigation (including data cellection), manusprit writing (original); Yu Ding: Methodology, investigation, manusprit writing (review and editing); Liangjie Chen: Methodology,

investigation, manusprit writing (review and editing); Liang Li: Conceptualization, supervision,

25

manusprit writing (review and editing), Funding acquisition.

ACKNOWLEDGMENT This work was supported by the National Natural Sciences Foundation of China (31771252) and the Beijing Municipal Science & Tech Commission (Z161100002616017).

[1]

C.

Landis,

W.

Hunt,

The

startle

ro of

REFERENCES

pattern.,

(1939)

1976.

doi:http://scholar.google.de/scholar?hl=de&q=Landis+C%2C+Hunt+WA.+The+startle+patter

L. Li, B.J. Frost, Azimuthal sensitivity of rat pinna reflex. EMG recordings from

re

[2]

-p

n.+New+York%3A+Farrar+and+Rinehart+Inc%3B+1939.&btnG=&lr=#2.

cervicoauricular muscles, Hear. Res. 100 (1996) 192–200. doi:10.1016/0378-5955(96)00119-0. B.W. Scott, P.W. Frankland, L. Li, J.S. Yeomans, Cochlear and trigeminal systems contributing

lP

[3]

to the startle reflex in rats, Neurosci. 91 (1999) 1565–1574. doi:10.1016/S0306-4522(98)00708-

[4]

na

8.

M. Davis, The mammalian startle response, in: Neural Mech. Startle Behav., Springer, 1984:

J.S. Yeomans, L. Li, B.W. Scott, P.W. Frankland, Tactile, acoustic and vestibular systems sum

Jo

[5]

ur

pp. 287–351. doi:10.1007/978-1-4899-2286-1_10.

to elicit the startle reflex, Neurosci. Biobehav. Rev. 26 (2002) 1–11. doi:10.1016/S01497634(01)00057-4.

[6]

M. Koch, The neurobiology of startle, Prog. Neurobiol. 59 (1999) 107–128. doi:10.1016/S03010082(98)00098-7.

[7]

L. Li, S. Steidl, J.S. Yeomans, Contributions of the vestibular nucleus and vestibulospinal tract

26

to the startle reflex, Neurosci. 106 (2001) 811–821. doi:10.1016/S0306-4522(01)00324-4. [8]

J.A. Foss, J.R. Ison, J.P. Torre Jr, S. Wansack, The acoustic startle response and disruption of aiming: II. Modulation by forewarning and preliminary stimuli, Hum. Factors. 31 (1989) 319– 333. doi:10.1177/001872088903100306.

[9]

H.S. Hoffman, W. Overman, Performance disruption by startle-eliciting acoustic stimuli, Psychon. Sci. 24 (1971) 233–235. doi:10.3758/BF03331807.

ro of

[10] J.A. Foss, J.R. Ison, J.P. Torre Jr, S. Wansack, The acoustic startle response and disruption of aiming: I. Effect of stimulus repetition, intensity, and intensity changes, Hum. Factors. 31 (1989) 307–318. doi:10.1177/001872088903100305.

-p

[11] H.S. Hoffman, J.L. Searle, Acoustic variables in the modification of startle reaction in the rat.,

re

J. Comp. Physiol. Psychol. 60 (1965) 53–58. doi:10.1037/h0022325. [12] H.S. Hoffman, J.R. Ison, Reflex modification in the domain of startle: I. Some empirical

lP

findings and their implications for how the nervous system processes sensory input., Psychol.

na

Rev. 87 (1980) 175–189. doi:10.1037/0033-295X.87.2.175. [13] F.K. Graham, The more or less startling effects of weak prestimulation, Psychophysiology. 12

ur

(1975) 238–248. doi:10.1111/j.1469-8986.1975.tb01284.x. [14] N.R. Swerdlow, M.A. Geyer, D.L. Braff, Neural circuit regulation of prepulse inhibition of

Jo

startle in the rat: current knowledge and future challenges., Psychopharmacology(Berl). 156 (2001) 194–215. doi:10.1007/s002130100799.

[15] N.R. Swerdlow, V.A. Keith, D.L. Braff, M.A. Geyer, Effects of spiperone, raclopride, SCH 23390 and clozapine on apomorphine inhibition of sensorimotor gating of the startle response in the rat., J. Pharmacol. Exp. Ther. 256 (1991) 530–536.

27

[16] N.R. Swerdlow, D.L. Braff, M.A. Geyer, B.K. Lipska, D.R. Weinberger, G.E. Jaskiw, Increased sensitivity to the sensorimotor gating-disruptive effects of apomorphine after lesions of medial prefrontal cortex or ventral hippocampus in adult rats, Psychopharmacology (Berl). 122 (1995) 27–34. doi:10.1007/BF02246438. [17] D.S. Leitner, M.E. Cohen, Role of the inferior colliculus in the inhibition of acoustic startle in the rat, Physiol. Behav. 34 (1985) 65–70. doi:10.1016/0031-9384(85)90079-4.

ro of

[18] L. Li, L.M. Korngut, B.J. Frost, R.J. Beninger, Prepulse inhibition following lesions of the inferior colliculus: prepulse intensity functions, Physiol. Behav. 65 (1998) 133–139. doi:10.1016/S0031-9384(98)00143-7.

-p

[19] L. Li, R.R.M. Priebe, J.S. Yeomans, Prepulse inhibition of acoustic or trigeminal startle of rats

re

by unilateral electrical stimulation of the inferior colliculus, Behav. Neurosci. 112 (1998) 1187– 1198. doi:10.1037//0735-7044.112.5.1187.

lP

[20] N.R. Swerdlow, M.A. Geyer, Prepulse Inhibition of Acoustic Startle in Rats After Lesions of

na

the Pedunculopontine Tegmental Nucleus, Behav. Neurosci. 107 (1993) 104–117. doi:10.1037/0735-7044.107.1.104.

ur

[21] M. Koch, M. Kungei, H. Herbert, Cholinergic neurons in the pedunculopontine tegmental nucleus are involved in the mediation of prepulse inhibition of the acoustic startle response in

Jo

the rat, Exp. Brain Res. 97 (1993) 71–82. doi:10.1007/BF00228818.

[22] M.H. Kodsi, N.R. Swerdlow, Regulation of prepulse inhibition by ventral pallidal projections, Brain Res. Bull. 43 (1997) 219–228. doi:10.1016/S0361-9230(96)00440-6. [23] L. Li, Y. Du, N. Li, X. Wu, Y. Wu, Top-down modulation of prepulse inhibition of the startle reflex

in

humans

and

rats,

Neurosci.

Biobehav.

Rev.

33

(2009)

1157–1167.

28

doi:10.1016/j.neubiorev.2009.02.001. [24] J. Huang, Z. Yang, J. Ping, X. Liu, X. Wu, L. Li, The influence of the perceptual or fear learning on rats’ prepulse inhibition induced by changes in the correlation between two spatially separated noise sounds, Hear. Res. 223 (2007) 1–10. doi:10.1016/j.heares.2006.09.012. [25] N. Li, J. Ping, R. Wu, C. Wang, X. Wu, L. Li, Auditory fear conditioning modulates prepulse inhibition in socially reared rats and isolation-reared rats, Behav. Neurosci. 122 (2008) 107–

ro of

118. doi:10.1037/0735-7044.122.1.107.

[26] Y. Du, X. Wu, L. Li, Differentially organized top-down modulation of prepulse inhibition of startle, J. Neurosci. 31 (2011) 13644–13653. doi:10.1523/JNEUROSCI.1292-11.2011.

-p

[27] M. Lei, L. Luo, T. Qu, H. Jia, L. Li, Perceived location specificity in perceptual separation-

re

induced but not fear conditioning-induced enhancement of prepulse inhibition in rats, Behav. Brain Res. 269 (2014) 87–94. doi:10.1016/j.bbr.2014.04.030.

lP

[28] Y. Ding, N. Xu, Y. Gao, Z. Wu, L. Li, The role of the deeper layers of the superior colliculus

na

in attentional modulations of prepulse inhibition, Behav. Brain Res. 364 (2019) 106–113. doi:10.1016/j.bbr.2019.01.052.

ur

[29] P.L.A. Gabbott, T.A. Warner, P.R.L. Jays, P. Salway, S.J. Busby, Prefrontal cortex in the rat: Projections to subcortical autonomic, motor, and limbic centers, J. Comp. Neurol. 492 (2005)

Jo

145–177. doi:10.1002/cne.20738.

[30] F. Barthas, A.C. Kwan, Secondary Motor Cortex: Where ‘Sensory’ Meets ‘Motor’ in the Rodent Frontal Cortex, Trends Neurosci. 40 (2017) 181–193. doi:10.1016/j.tins.2016.11.006. [31] H. Hintiryan, N.N. Foster, I. Bowman, M. Bay, M.Y. Song, L. Gou, S. Yamashita, M.S. Bienkowski, B. Zingg, M. Zhu, X.W. Yang, J.C. Shih, A.W. Toga, H.W. Dong, The mouse

29

cortico-striatal projectome, Nat. Neurosci. 19 (2016) 1100–1114. doi:10.1038/nn.4332. [32] W.B. Hoover, R.P. Vertes, Anatomical analysis of afferent projections to the medial prefrontal cortex in the rat, Brain Struct. Funct. 212 (2007) 149–179. doi:10.1007/s00429-007-0150-4. [33] R.L. Reep, G.S. Goodwin, J. V. Corwin, Topographic organization in the corticocortical connections of medial agranular cortex in rats, J. Comp. Neurol. 294 (1990) 262–280. doi:10.1002/cne.902940210.

ro of

[34] S.L. Stuesse, D.B. Newman, Projections from the medial agranular cortex to brain stem visuomotor centers in rats, Exp. Brain Res. 80 (1990) 532–544. doi:10.1007/BF00227994. [35] T. Tsumori, S. Yokota, K. Ono, Y. Yasui, Organization of projections from the medial agranular

-p

cortex to the superior colliculus in the rat: A study using anterograde and retrograde tracing

re

methods, Brain Res. 903 (2001) 168–176. doi:10.1016/S0006-8993(01)02437-4. [36] K.J. Burcham, J. V. Corwin, M.L. Stoll, R.L. Reep, Disconnection of medial agranular and

lP

posterior parietal cortex produces multimodal neglect in rats, Behav. Brain Res. 86 (1997) 41–

na

47. doi:10.1016/S0166-4328(96)02241-3.

[37] R.L. Reep, J. V. Corwin, Posterior parietal cortex as part of a neural network for directed

ur

attention in rats, Neurobiol. Learn. Mem. 91 (2009) 104–113. doi:10.1016/j.nlm.2008.08.010. [38] K.S. Bohon, M.C. Wiest, 2014. Role of medio-dorsal frontal and posterior parietal neurons auditory

Jo

during

detection

performance

in

rats.

PLoS

One.

9,

e114064.

doi:10.1371/journal.pone.0114064.

[39] G. Felsen, Z.F. Mainen, Neural Substrates of Sensory-Guided Locomotor Decisions in the Rat Superior Colliculus, Neuron. 60 (2008) 137–148. doi:10.1016/j.neuron.2008.09.019. [40] J.H. Sul, S. Jo, D. Lee, M.W. Jung, Role of rodent secondary motor cortex in value-based action

30

selection., Nat. Neurosci. 14 (2011) 1202–8. doi:10.1038/nn.2881. [41] J.C. Erlich, M. Bialek, C.D. Brody, A cortical substrate for memory-guided orienting in the rat, Neuron. 72 (2011) 330–343. doi:10.1016/j.neuron.2011.07.010. [42] R.L. Reep, B. Kirkpatrick, Forebrain connections of medial agranular cortex in the prairie vole, Microtus ochrogaster, Exp. Brain Res. 126 (1999) 336–350. doi:10.1007/s002210050741. [43] R.L. Reep, H.C. Chandler, V. King, J. V Corwin, Rat posterior parietal cortex: topography of and

thalamic

connections.,

Exp.

Brain

doi:10.1007/BF00227280.

Res.

100

(1994)

67–84.

ro of

corticocortical

[44] T. Shinba, Medial agranular cortex activity related to event-related potential generation in the

-p

rat, Cogn. Brain Res. 14 (2002) 264–268. doi:10.1016/S0926-6410(02)00127-1.

re

[45] T. Shinba, L. Briois, S.J. Sara, Spontaneous and auditory-evoked activity of medial agranular cortex as a function of arousal state in the freely moving rat: Interaction with locus coeruleus

lP

activity, Brain Res. 887 (2000) 293–300. doi:10.1016/S0006-8993(00)03009-2.

cortex

of

na

[46] D. Ongür, J.L. Price, The organization of networks within the orbital and medial prefrontal rats,

monkeys

and

humans.,

Cereb.

Cortex.

10

(2000)

206–219.

ur

doi:10.1093/cercor/10.3.206.

[47] C.G. van Eden, V.A.F. Lamme, H.B.M. Uylings, Heterotopic Cortical Afferents to the Medial

Jo

Prefrontal Cortex in the Rat. A Combined Retrograde and Anterograde Tracer Study, Eur. J. Neurosci. 4 (1992) 77–97. doi:10.1111/j.1460-9568.1992.tb00111.x.

[48] B. Zingg, H. Hintiryan, L. Gou, M.Y. Song, M. Bay, M.S. Bienkowski, N.N. Foster, S. Yamashita, I. Bowman, A.W. Toga, H.W. Dong, Neural networks of the mouse neocortex, Cell. 156 (2014) 1096–1111. doi:10.1016/j.cell.2014.02.023.

31

[49] R.L. Reep, J. V. Corwin, Topographic organization of the striatal and thalamic connections of rat medial agranular cortex, Brain Res. 841 (1999) 43–52. doi:10.1016/S0006-8993(99)017795. [50] S. Zhang, M. Xu, W.-C. Chang, C. Ma, J.P. Hoang Do, D. Jeong, T. Lei, J.L. Fan, Y. Dan, Organization of long-range inputs and outputs of frontal cortex for top-down control., Nat. Neurosci. 19 (2016) 1733–1742. doi:10.1038/nn.4417.

ro of

[51] F. Condé, E. Maire‐ lepoivre, E. Audinat, F. Crépel, Afferent connections of the medial frontal cortex of the rat. II. Cortical and subcortical afferents, J. Comp. Neurol. 352 (1995) 567–593. doi:10.1002/cne.903520407.

-p

[52] R.L. Reep, J. V Corwin, A. Hashimoto, R.T. Watson, Efferent connections of the rostral portion

re

of medial agranular cortex in rats, Brain Res. Bull. 19 (1987) 203–221. doi:10.1016/03619230(87)90086-4.

lP

[53] S.R. Sesack, A.Y. Deutch, R.H. Roth, B.S. Bunney, Topographical organization of the efferent

Phaseolus

na

projections of the medial prefrontal cortex in the rat: an anterograde tract‐ tracing study with vulgaris

leucoagglutinin,

J.

Comp.

Neurol.

290

(1989)

213–242.

ur

doi:10.1002/cne.902900205.

[54] R.P. Vertes, Analysis of projections from the medial prefrontal cortex to the thalamus in the rat, emphasis

Jo

with

on

nucleus

reuniens,

J.

Comp.

Neurol.

442

(2002)

163–187.

doi:10.1002/cne.10083.

[55] Y. Ueta, Y. Hirai, T. Otsuka, Y. Kawaguchi, 2013. Direction- and distance-dependent interareal connectivity of pyramidal cell subpopulations in the rat frontal cortex. Front. Neural Circuits. 7, 164. doi:10.3389/fncir.2013.00164.

32

[56] H.B.M. Uylings, H.J. Groenewegen, B. Kolb, Do rats have a prefrontal cortex?, Behav. Brain Res. 146 (2003) 3–17. doi:10.1016/j.bbr.2003.09.028. [57] J.C. Erlich, B.W. Brunton, C.A. Duan, T.D. Hanks, C.D. Brody, 2015. Distinct effects of prefrontal and parietal cortex inactivations on an accumulation of evidence task in the rat. Elife. 4, e05457. doi:10.7554/elife.05457. [58] T.D. Hanks, C.D. Kopec, B.W. Brunton, C.A. Duan, J.C. Erlich, C.D. Brody, Distinct

ro of

relationships of parietal and prefrontal cortices to evidence accumulation, Nature. 520 (2015) 220–223. doi:10.1038/nature14066.

[59] M. Murakami, M.I. Vicente, G.M. Costa, Z.F. Mainen, Neural antecedents of self-initiated

-p

actions in secondary motor cortex, Nat. Neurosci. 17 (2014) 1574–1582. doi:10.1038/nn.3826.

re

[60] K.R. Bailey, R.G. Mair, Effects of frontal cortex lesions on action sequence learning in the rat, Eur. J. Neurosci. 25 (2007) 2905–2915. doi:10.1111/j.1460-9568.2007.05492.x.

lP

[61] V.Y. Cao, Y. Ye, S. Mastwal, M. Ren, M. Coon, Q. Liu, R.M. Costa, K.H. Wang, Motor

na

Learning Consolidates Arc-Expressing Neuronal Ensembles in Secondary Motor Cortex, Neuron. 86 (2015) 1385–1392. doi:10.1016/j.neuron.2015.05.022.

ur

[62] S.B. Ostlund, N.E. Winterbauer, B.W. Balleine, Evidence of action sequence chunking in goaldirected instrumental conditioning and its dependence on the dorsomedial prefrontal cortex., J.

Jo

Neurosci. 29 (2009) 8280–8287. doi:10.1523/JNEUROSCI.1176-09.2009.

[63] H. Yin, 2009. The role of the murine motor cortex in action duration and order. Front. Integr. Neurosci. 3, 23. doi:10.3389/neuro.07.023.2009. [64] K.J. Burcham, J. V Corwin, Effects of delay and duration of light deprivation on recovery of function from neglect induced by unilateral medial agranular prefrontal cortex lesions in rats,

33

Psychobiology. 26 (1998) 216–230. doi:10.3758/BF03330610. [65] W.L. Conte, H. Kamishina, J. V. Corwin, R.L. Reep, Topography in the projections of lateral posterior thalamus with cingulate and medial agranular cortex in relation to circuitry for directed attention and neglect, Brain Res. 1240 (2008) 87–95. doi:10.1016/j.brainres.2008.09.013. [66] M. Brecht, Movement, Confusion, and Orienting in Frontal Cortices, Neuron. 72 (2011) 193– 196. doi:10.1016/j.neuron.2011.10.002.

ro of

[67] K.J. Swartz, M.J. During, A. Freese, M.F. Beal, Cerebral synthesis and release of kynurenic acid: an endogenous antagonist of excitatory amino acid receptors, J. Neurosci. 10 (1990) 2965– 2973. doi:10.1523/JNEUROSCI.10-09-02965.1990.

-p

[68] T.W. Stone, J.H. Connick, Quinolinic acid and other kynurenines in the central nervous system,

re

Neuroscience. 15 (1985) 597–617. doi:10.1016/0306-4522(85)90063-6. [69] M. Kessler, T. Terramani, G. Lynch, M. Baudry, A glycine site associated with N‐ methyl‐

lP

D‐ aspartic acid receptors: characterization and identification of a new class of antagonists, J.

na

Neurochem. 52 (1989) 1319–1328. doi:10.1111/j.1471-4159.1989.tb01881.x. [70] A.M. Thomson, V.E. Walker, D.M. Flynn, Glycine enhances NMDA-receptor mediated

ur

synaptic potentials in neocortical slices, Nature. 338 (1989) 422–444. doi:10.1038/338422a0. [71] J.B. Kelly, B. Masterton, Auditory sensitivity of the albino rat., J. Comp. Physiol. Psychol. 91

Jo

(1977) 930–936. doi:10.1037/h0077356.

[72] J.B. Kelly, S.L. Glenn, C.J. Beaver, Sound frequency and binaural response properties of single neurons in rat inferior colliculus, Hear. Res. 56 (1991) 273–280. doi:10.1016/03785955(91)90177-B. [73] R.Y. Litovsky, H.S. Colburn, W.A. Yost, S.J. Guzman, The precedence effect, J. Acoust. Soc.

34

Am. 106 (1999) 1633–1654. doi:10.1121/1.427914. [74] L. Li, Q. Yue, Auditory gating processes and binaural inhibition in the inferior colliculus, Hear. Res. 168 (2002) 98–109. doi:10.1016/S0378-5955(02)00356-8. [75] L. Li, B.J. Frost, Azimuthal directional sensitivity of prepulse inhibition of the pinna startle reflex in decerebrate rats, Brain Res. Bull. 51 (2000) 95–100. doi:10.1016/S03619230(99)00215-4.

ro of

[76] M. Davis, P.M. Gendelman, Plasticity of the acoustic startle response in the acutely decerebrate rat., J. Comp. Physiol. Psychol. 91 (1977) 549–563. doi:10.1037/h0077345.

[77] J.E. Fox, Habituation and prestimulus inhibition of the auditory startle reflex in decerebrate rats,

-p

Physiol. Behav. 23 (1979) 291–297. doi:10.1016/0031-9384(79)90370-6.

rat is lost

re

[78] J.R. Ison, K. O’connor, G.P. Bowen, A. Bocirnea, Temporal resolution of gaps in noise by the with functional decortication., Behav. Neurosci. 105 (1991) 33–40.

lP

doi:10.1037//0735-7044.105.1.33.

na

[79] M. Fendt, M. Koch, H.U. Schnitzler, Sensorimotor gating deficit after lesions of the superior colliculus, Neuroreport. 5 (1994) 1725–1728. doi:10.1097/00001756-199409080-00009.

ur

[80] A. Elden, M.A. Flaten, The relationship of automatic and controlled processing to prepulse inhibition, J. Psychophysiol. 16 (2002) 46–55. doi:10.1027//0269-8803.16.1.46.

Jo

[81] M.M. Bradley, M. Codispoti, P.J. Lang, A multi-process account of startle modulation during affective

perception,

Psychophysiology.

43

(2006)

486–497.

doi:10.1111/j.1469-

8986.2006.00412.x. [82] B.R. Cornwell, A.M. Echiverri, M.F. Covington, C. Grillon, Modality-specific attention under imminent but not remote threat of shock: evidence from differential prepulse inhibition of startle,

35

Psychol. Sci. 19 (2008) 615–622. doi:10.1111/j.1467-9280.2008.02131.x. [83] C. Grillon, M. Davis, Effects of stress and shock anticipation on prepulse inhibition of the startle reflex, Psychophysiology. 34 (1997) 511–517. doi:10.1111/j.1469-8986.1997.tb01737.x. [84] M. Lei, C. Zhang, L. Li, 2018. Neural correlates of perceptual separation-induced enhancement of prepulse inhibition of startle in humans. Sci. Rep. 8, 472. doi:10.1038/s41598-017-18793-x. [85] S. Carlson, J.F. Willott, The behavioral salience of tones as indicated by prepulse inhibition of

ro of

the startle response: Relationship to hearing loss and central neural plasticity in C57BL/6J mice, Hear. Res. 99 (1996) 168–175. doi:10.1016/S0378-5955(96)00098-6.

[86] J.C. Franklin, N.A. Moretti, T.D. Blumenthal, Impact of stimulus signal-to-noise ratio on inhibition

of

acoustic

startle,

44

(2007)

339–342.

re

doi:10.1111/j.1469-8986.2007.00498.x.

Psychophysiology.

-p

prepulse

[87] J.R. Ison, G.P. Bowen, J. Pak, E. Gutierrez, Changes in the strength of prepulse inhibition with

lP

variation in the startle baseline associated with individual differences and with old age in rats

na

and mice, Psychobiology. 25 (1997) 266–274. doi:10.3758/BF03331936. [88] S. Roskam, M. Koch, Enhanced prepulse inhibition of startle using salient prepulses in rats, Int.

ur

J. Psychophysiol. 60 (2006) 10–14. doi:10.1016/j.ijpsycho.2005.04.004. [89] M.M. Brenneman, M.J. Hylin, J. V Corwin, The time-dependent and persistent effects of

Jo

amphetamine treatment upon recovery from hemispatial neglect in rats, Behav. Brain Res. 293 (2015) 153–161. doi:10.1016/j.bbr.2015.07.032.

[90] Y. Fu, J.M. Tucciarone, J.S. Espinosa, N. Sheng, D.P. Darcy, R.A. Nicoll, Z.J. Huang, M.P. Stryker, A Cortical Circuit for Gain Control by Behavioral State, Cell. 156 (2014) 1139–1152. doi:10.1016/j.cell.2014.01.050.

36

[91] M. Sarter, M.E. Hasselmo, J.P. Bruno, B. Givens, Unraveling the attentional functions of cortical cholinergic inputs: interactions between signal-driven and cognitive modulation of signal detection, Brain Res. Rev. 48 (2005) 98–111. doi:10.1016/j.brainresrev.2004.08.006. [92] M. Goard, Y. Dan, Basal forebrain activation enhances cortical coding of natural scenes, Nat. Neurosci. 12 (2009) 1444–1449. doi:10.1038/nn.2402. [93] L. Pinto, M.J. Goard, D. Estandian, M. Xu, A.C. Kwan, S.-H. Lee, T.C. Harrison, G. Feng, Y.

16 (2013) 1857–1863. doi:10.1038/nn.3552.

ro of

Dan, Fast modulation of visual perception by basal forebrain cholinergic neurons, Nat. Neurosci.

[94] C.L. Nelson, M. Sarter, J.P. Bruno, Prefrontal cortical modulation of acetylcholine release in parietal

cortex,

Neuroscience.

132

-p

posterior

347–359.

re

doi:10.1016/j.neuroscience.2004.12.007.

(2005)

[95] R.P. Kesner, J.C. Churchwell, An analysis of rat prefrontal cortex in mediating executive

lP

function, Neurobiol. Learn. Mem. 96 (2011) 417–431. doi:10.1016/j.nlm.2011.07.002.

na

[96] R.P.A. Gaykema, R. Van Weeghel, L.B. Hersh, P.G.M. Luiten, Prefrontal cortical projections to the cholinergic neurons in the basal forebrain, J. Comp. Neurol. 303 (1991) 563–583.

ur

doi:10.1002/cne.903030405.

[97] S. Zhang, M. Xu, T. Kamigaki, J.P.H. Do, W.C. Chang, S. Jenvay, K. Miyamichi, L. Luo, Y.

Jo

Dan, Long-range and local circuits for top-down modulation of visual cortex processing, Science. 345 (2014) 660–665. doi:10.1126/science.1254126.

[98] D.D. Rasmusson, S.A. Smith, K. Semba, Inactivation of prefrontal cortex abolishes cortical acetylcholine release evoked by sensory or sensory pathway stimulation in the rat, Neuroscience. 149 (2007) 232–241. doi:10.1016/j.neuroscience.2007.06.057.

37

[99] R. Metherate, J.H. Ashe, Nucleus basalis stimulation facilitates thalamocortical synaptic transmission

in

the

rat

auditory

cortex,

Synapse.

14

(1993)

132–143.

doi:10.1002/syn.890140206. [100] J.L. Muir, B.J. Everitt, T.W. Robbins, The cerebral cortex of the rat and visual attentional function: Dissociable effects of mediofrontal, cingulate, anterior dorsolateral, and parietal

doi:10.1093/cercor/6.3.470.

ro of

cortex lesions on a five-choice serial reaction time task, Cereb. Cortex. 6 (1996) 470–481.

[101] J.H. Sul, H. Kim, N. Huh, D. Lee, M.W. Jung, Distinct roles of rodent orbitofrontal and medial prefrontal

cortex

in

decision

making,

66

(2010)

449–460.

-p

doi:10.1016/j.neuron.2010.03.033.

Neuron.

re

[102] M.J. Siniscalchi, V. Phoumthipphavong, F. Ali, M. Lozano, A.C. Kwan, Fast and slow transitions in frontal ensemble activity during flexible sensorimotor behavior, Nat. Neurosci. 19

lP

(2016) 1234–1242. doi:10.1038/nn.4342.

na

[103] D. Braff, C. Stone, E. Callaway, M. Geyer, I. Glick, L. Bali, Prestimulus effects on human startle reflex in normals and schizophrenics, Psychophysiology. 15 (1978) 339–343.

ur

doi:10.1111/j.1469-8986.1978.tb01390.x. [104] E.A. Hazlett, M.J. Romero, M.M. Haznedar, A.S. New, K.E. Goldstein, R.E. Newmark, L.J.

Jo

Siever, M.S. Buchsbaum, Deficient attentional modulation of startle eyeblink is associated with symptom severity in the schizophrenia spectrum, Schizophr. Res. 93 (2007) 288–295. doi:10.1016/j.schres.2007.03.012.

[105] N.-B. Yang, Q. Tian, Y. Fan, Q.-J. Bo, L. Zhang, L. Li, C.-Y. Wang, 2017. Deficits of perceived spatial separation induced prepulse inhibition in patients with schizophrenia: relationships to

38

symptoms and neurocognition. BMC Psychiatry. 17, 135. doi:10.1186/s12888-017-1276-4. [106] Y. Du, X. Wu, L. Li, Emotional learning enhances stimulus-specific top-down modulation of sensorimotor gating in socially reared rats but not isolation-reared rats, Behav. Brain Res. 206 (2010) 192–201. doi:10.1016/j.bbr.2009.09.012. [107] Z.-M. Wu, Y. Ding, H.-X. Jia, L. Li, Different effects of isolation-rearing and neonatal MK-801 treatment

on attentional

modulations

of prepulse

inhibition of startle

in rats,

ro of

Psychopharmacology. 233 (2016) 3089–3102. doi:10.1007/s00213-016-4351-5.

[108] T.B. Hammer, B. Oranje, A. Skimminge, B. Aggernaes, B.H. Ebdrup, B. Glenthoj, W. Baare, Structural brain correlates of sensorimotor gating in antipsychotic-naive men with first-episode

-p

schizophrenia, J. Psychiatry Neurosci. 38 (2013) 34–42. doi:10.1503/jpn.110129.

re

[109] J.H. Yoon, M.J. Minzenberg, S. Ursu, R. Walters, C. Wendelken, J.D. Ragland, C.S. Carter, Association of dorsolateral prefrontal cortex dysfunction with disrupted coordinated brain

function,

Am.

J.

Psychiatry.

165

(2008)

1006–1014.

na

global

lP

activity in schizophrenia: Relationship with impaired cognition, behavioral disorganization, and

doi:10.1176/appi.ajp.2008.07060945.

ur

[110] C. Exner, G. Weniger, C. Schmidt-Samoa, E. Irle, Reduced size of the pre-supplementary motor cortex and impaired motor sequence learning in first-episode schizophrenia, Schizophr. Res. 84

Jo

(2006) 386–396. doi:10.1016/j.schres.2006.03.013.

[111] C. Wu, Y. Zheng, J. Li, H. Wu, S. She, S. Liu, Y. Ning, L. Li, Brain substrates underlying auditory speech priming in healthy listeners and listeners with schizophrenia, Psychol. Med. 47 (2017) 837–852. doi:10.1017/S0033291716002816. [112] I. Wonodi, O.C. Stine, K. V Sathyasaikumar, R.C. Roberts, B.D. Mitchell, L.E. Hong, Y. Kajii,

39

G.K. Thaker, R. Schwarcz, Downregulated Kynurenine 3-Monooxygenase Gene Expression and Enzyme Activity in Schizophrenia and Genetic Association With Schizophrenia Endophenotypes,

Arch.

Gen.

Psychiatry.

68

(2011)

665–674.

doi:10.1001/archgenpsychiatry.2011.71.

Figure 1. Illustration of the surgery and testing procedure for the experimental group (solid wireframe,

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n = 18) and the anatomically-control group (dashed wireframe, n = 12). BC: Procedure Stage BC, before conditioning; BK: Procedure Stage BK, after conditioning and before injecting KYNA; AK: Procedure Stage AK, after injecting KYNA; BL: Procedure Stage BL, after conditioning and before

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injecting Locke’s solution; AL: Procedure Stage AL, after injecting Locke’s solution. K: KYNA; L:

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Locke’s solution. Note that for the rats in the anatomically-control group, only KYNA was injected,

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which means only procedure stages BC, BK, and AK were performed.

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Figure 2. Histological locations of injection cannulaes in the brains of all 30 rats, showing bilateral injection cannula placements in the medial agranular cortex (AGm, n = 18, Left panel) and the primary

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somatosensory cortex (S1BF, n = 12, right panel).

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Figure 3. Group mean startle response amplitudes to the startling stimulus alone in the experimental group (the AGm- injection group, n = 18). Procedure Stage BC: before conditioning; Procedure Stage BK: after conditioning, before injecting KYNA; Procedure Stage AK: after injecting KYNA; Procedure Stage BL: after conditioning, before injecting Locke’s solution; and Procedure Stage AL: after injecting Locke’s solution. Note that half of the rats were tested in the order of “BC-BL-AL-BKAK”, and the other half were in another order of “BC-BK-AK-BL-AL”. Data was averaged across the

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2 testing blocks in each of the procedure stages (BC, BK, AK, BL, AL) in the experimental group. A repeated-measures ANOVA with one within-subjects factor (procedure stage: BC, BK, AK, BL, AL) showed that the effect of the procedure stage on the startle response to the startling stimulus alone was

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not significant in the experimental group (F(4,14)= 1.299, p > 0.05). Injecting either KYNA or Locke’s

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solution into the bilateral AGm, and either the conditioning manipulation or the conditioning control manipulation did not significantly influence the startle amplitude to the startling stimulus alone (all

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ps > 0.05). Error bars represent the standard errors of the mean (SEM).

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Figure 4. PPI induced by the fear-conditioned prepulse at different procedure stages in the experimental group (AGm, n = 18). The black bars represent the group-mean PPI magnitudes when the prepulse was perceptually co-located with the noise masker, while the diagonal bars represent the group-mean PPI magnitudes when the prepulse was perceptually separated from the noise masker. Procedure Stage BC: before conditioning; Procedure Stage BK: after conditioning, before injecting KYNA; Procedure Stage AK: after injecting KYNA; Procedure Stage BL: after conditioning, before

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injecting Locke’s solution; and Procedure Stage AL: after injecting Locke’s solution. Note that half of the rats were tested in the order of “BC-BL-AL-BK-AK”, and the other half were in another order of “BC-BK-AK-BL-AL”. Error bars represent the standard errors of the mean (SEM). *: p < 0.05; **: p

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< 0.01, ***: p < 0.001 (by repeated-measures ANOVA, Bonferroni’s pairwise comparisons).

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Figure 5. PPI induced by the conditioning-control prepulse at different procedure stages in the experimental group (with injections into the AGm, n = 18). After the conditioning-control manipulation, the PPI level was not significantly enhanced (the main effect of the procedure stage was not significant (F(4,14) = 2.533, p = 0.087, ηp2 = 0.420). The black bars represent the group-mean PPI magnitudes when the prepulse was perceptually co-located with the noise masker, while the diagonal bars represent the group-mean PPI magnitudes when the prepulse was perceptually separated with the

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noise masker. Procedure Stage BC: before conditioning; Procedure Stage BK: after conditioning, before injecting KYNA; Procedure Stage AK: after injecting KYNA; Procedure Stage BL: after conditioning, before injecting Locke’s solution; and Procedure Stage AL: after injecting Locke’s

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solution. Note that half of the rats were tested in the order of “BC-BL-AL-BK-AK”, and the other half

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were in another order of “BC-BK-AK-BL-AL”. Error bars represent the standard errors of the mean

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(SEM).

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Figure 6. Startle response amplitudes to the startling stimulus alone in the anatomically-control group (with injections into the S1BF, n = 12). Procedure Stage BC: before conditioning; Procedure Stage BK: after conditioning, before injecting KYNA; and Procedure Stage AK: after injecting KYNA. Note that all the rats were tested in the order of “BC-BK-AK”. Error bars represent the standard errors of the

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mean (SEM).

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Figure 7. PPI magnitudes at different procedure stages in the anatomically control group (with injections into the S1BF, n = 12). The black bars represent the group-mean PPI magnitudes when the prepulse stimulus was perceptually co-located with the noise masker, while the diagonal bars represent the group-mean PPI magnitudes when the prepulse was perceptually separated with the noise masker. Left panel: PPI induced by the conditioned prepulse; right panel: PPI induced by the conditioningcontrol prepulse. Procedure Stage BC: before conditioning; Procedure Stage BK: after conditioning,

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before injecting KYNA; and Procedure Stage AK: after injecting KYNA. Note that all the rats were tested in the order of “BC-BK-AK”. Data for all rats are shown in the figure. Error bars represent the standard errors of the mean (SEM). *: p < 0.05; **: p < 0.01 (by repeated-measures ANOVA,

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Bonferroni’s pairwise comparisons).

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