Prenatal cyanuric acid exposure induced spatial learning impairments associated with alteration of acetylcholine-mediated neural information flow at the hippocampal CA3-CA1 synapses of male rats

Abstract

Cyanuric acid (CA) is reported to induce nephrotoxicity but its toxic effect is not fully known. Prenatal CA exposure causes neurodevelopmental deficits and abnormal behavior in spatial learning ability. Dysfunction of the acetyl-cholinergic system in neural information processing is correlated with spatial learning impairment and was found in the previous reports of CA structural analogue melamine. To further investigate the neurotoxic effects and the potential mechanism, the acetylcholine (ACh) level was detected in the rats which were exposed to CA during the whole of gestation. Local field potentials (LFPs) were recorded when rats infused with ACh or cholinergic receptor agonist into hippocampal CA3 or CA1 region were trained in the Y-maze task. We found the expression of ACh in the hippocampus was significantly reduced in dose-dependent manners. Intra-hippocampal infusion of ACh into the CA1 but not the CA3 region could effectively mitigate learning deficits induced by CA exposure. However, activation of cholinergic receptors did not rescue the learning impairments. In the LFP recording, we found that the hippocampal ACh infusions could enhance the values of phase synchronization between CA3 and CA1 regions in theta and alpha oscillations. Meanwhile, the reduction in the coupling directional index and the strength of CA3 driving CA1 in the CA-treated groups was also reversed by the ACh infusions. Our findings are consistent with the hypothesis and provide the first evidence that prenatal CA exposure induced spatial learning defect is attributed to the weakened ACh-mediated neuronal coupling and NIF in the CA3-CA1 pathway.

Keywords

Acetylcholine cyanuric acid hippocampal CA1 local field potential neural information

Introduction

Cyanuric acid (CA, 2,4,6-trihydroxy-1,3,5-triazine; CAS No. 108–80-5) is an important industrial chemical, and it is widely used as an ingredient in the production of scouring powders, household bleaches, industrial cleansers and automatic dishwasher compounds.^1,2 Earlier studies suggested that CA is no more than slightly toxic. But later research pointed out that daily oral injection of CA may cause serious damage to animals.³ Therefore, CA has been listed in Drinking Water Contaminant Candidate List by US Environmental Protection Agency.

So far, CA has been reported to mainly cause kidney damage.^4-6 In addition, CA taken orally could also disrupt the amino acid metabolism of tryptophan, disrupt polyamine and tyrosine metabolisms, alter the TCA and cycle, change the gut microfloral population structure,⁷ change the ratio of Th1/Th2 spleen lymphocytes⁸ and enhance erythrocyte membrane permeability and increase hemolysis.⁹ Therefore, the aforementioned findings have attracted long-time public attention, leading to the strict determination of CA in food and further research about its toxicity.

CA can dose-dependently pass the placental barrier to reach the embryo or fetus.^10,11 Importantly, the accumulations of CA in the developing brain lead to a risk of neurodevelopmental impairment, such as depressing spontaneous excitatory postsynaptic currents and decreasing phosphorylated expression of N-methyl-D-aspartate (NMDA) receptors,¹² which are essential mediators of controlling synaptic function and memory formation. Our previous studies found that prenatal exposure to melamine, a structural analogue of CA, induced spatial learning acquisition deficits and reversal learning disability.^13,14 These impairments are attributed to the dysfunction of acetyl-cholinergic transmission in the hippocampus.^15,16 Recently, spatial learning and memory defects were also observed following prenatal CA treatment.¹⁷ The cholinergic tone can modulate ongoing hippocampal activities by enhancing the excitatory and depressing inhibitory transmissions in the hippocampus, thus increasing the excitatory output to promote neural correlate with learning processing.^18,19 However, whether the destructive effect of CA is associated with the cholinergic system is still unknown.

Synchronous neural activity at particular frequencies is often seen as humans and animals engage in the learning process.^20,21 Like many brain regions, the hippocampal circuit supports multiple modes of information processing, which are essential for rapidly encoding new memories, retrieving stored memories and consolidating memories for long-term storage.^22,23 Indeed, encoding of new spatial information within the hippocampus is thought to depend primarily on the internal drive from CA3 to CA1.^24,25 The relative strengths of the CA3 input to CA1 govern, to a large extent, the ongoing information processing state of the hippocampal circuit.^26,27 One major influence on hippocampal rhythmicity is from cholinergic afferents.^28,29 Disruptions to cholinergic transmission impair spatial learning while increasing acetylcholine (ACh) can reverse these impairments.^30–32 In particular, ACh levels are increased when learning new information and thus facilitate encoding while hindering retrieval.^19,30 It is widely accepted that, in both humans and rodents, aging is linked to impairments in hippocampus-dependent function along with degradation of cholinergic function while cholinomimetics can reverse some age-related memory impairments and modulate oscillations in the hippocampus.^33,34 More importantly, prenatal CA exposure compromises learning and memory encoding by functionally disrupting synaptic transmission.^17,35 Meanwhile, intrahippocampal infusion with ACh could mitigate the weakened neural information flow (NIF) induced by prenatal melamine treatment.¹⁵ Little is known about whether prenatal CA exposure influences ACh expression or whether this change is sufficient to obstruct hippocampal NIF.

In this study, rats were prenatally exposed to CA the whole of gestation as previously described.¹⁷ In the eighth postnatal week, the expression of ACh was detected and the learning performance was accessed in the Y-maze task. Meanwhile, ACh was systemically injected and the neural activity was recorded when rats’ were trained in the behavioral test. Multiple studies in rats have shown that the integrity of hippocampal CA1 field activity is necessary for the encoding and long-term storage of spatial information.^36–38 Similarly, effects of CA3 neurotoxic lesions, or other experimentally-induced dysfunctions of CA3, have been interpreted as either impairing spatial long-term memory^39,40 or working memory.^41,42 Thus, to identify the relative effects of CA on the hippocampal CA3-CA1 pathway, ACh was intra-hippocampal infused into CA3 and CA1, respectively. Furthermore, cholinergic receptor agonist was also infused into two sub-regions respectively, to investigate the postsynaptic effect. Our findings provide new insight into the neurotoxic mechanism by which CA affects spatial cognition.

Experimental procedure

Experimental animals

Male and nulliparous female Sprague-Dawley rats were purchased from the Laboratory Animal Center, Academy of Military Medical Science of People’s Liberation Army, and reared in plastic cages in a colony room (21 ± 2°C; 45 ± 5% humidity; lights on at 7:00). Unless specifically stated otherwise, the rats were paired housed in clear plastic cages with ad libitum access to food and water. Animal care and experimental procedures were approved by the Care and Use of Animals Committee of Guizhou University of Chinese Medicine (SCXK-2013–0020).

Two females were housed with one male for mating. Vaginal plug check and smear observation by microscope were carried out each morning. A positive sign of mating was confirmed by the presence of copulatory plugs and sperm in vaginal lavage fluid. On day 0 of gestation (GD 0), which was determined by the observation of a vaginal plug and/or spermatozoids in the vaginal smear, female rats were then housed separately from the male. After confirmed copulation, pregnant dams were daily received one of the following by i. p. Injection during the whole of gestation: 1 mL artificial cerebral spinal fluid treatment (ACSF, Control group); the low dose of 10 mg/kg CA (Cat#A15447, Thermo Fisher Scientific) in 1 mL ACSF (CA (10)); the moderate dose of 20 mg/kg CA in 1 mL ACSF (CA (20)); the high dose of 40 mg/kg CA in 1 mL ACSF (CA (40)). The doses of CA were used according to the doses used by the previous studies.^12,17,35 Each litter was standardized on the day after birth to 8–13 pups, with the similar number of males and females. Since the sex-specific effect of CA in the CNS has not yet well been defined, only male offspring were selected for this study. Only one or two offspring from the same litter were chosen for each group and the number of rats in each group was indicated in each figure legend.

Additionally, 30 min before the behavioral test, acetylcholine chloride (5.0 nM; Cat#A-6625, Sigma-Aldrich) and muscarinic cholinergic receptor agonist oxotremorine sesquifumarate salt (10.0 μM; Cat#O-9126, Sigma-Aldrich) were bilaterally infused into the HPC of rats (0.25 μL/side) in the CA + ACh groups and CA + XO groups, respectively. ACSF was used as vehicle for the acetylcholine chloride and oxotremorine sesquifumarate salt infusions in the Control group. The doses of acetylcholine chloride and oxotremorine sesquifumarate salt were determined from published studies.^16,43-46 For the systemic studies, rats were intraperitoneal injected (i.p.) with 3.0 mg/kg acetylcholine chloride dissolved in saline 30 min before the initiation of the behavioral test as previous reports.^47–49

Spatial learning in the Y-maze task

Around postnatal day (PND) 54 and PND62, rats were trained in the Y-maze task as described previously.^50–52 Three black Plexiglas arms (40 cm × 15 cm×8 cm) separated with 120° angles were built the maze. Two of arms were the test arms and one was the start arm. Briefly, the rat was placed in the start arm and allowed to visit the end of the reward arm. For each animal, the reward location was fixed, however, the start arm and test arms were pseudo-randomly selected but counter-balanced across rats of groups. After reaching the end of an arm, the rat was returned to its home cage that served as the inter-trial box. A visit was defined as the animal placing all four paws. The inter-trial interval was about 20 s. On one single learning day, rats were trained till they got six correct trials in a row. Total trials to reach the criterion, total time spent in reaching the criterion and velocity to reach the reward cup were quantified.

ACh level in the hippocampus

Separate groups of rats were deeply anesthetized and the brains were removed as described previously.^53–55 Subsequently, the HPC on the side of the neuronal recording was isolated on an ice-cold operation table. After being weighed, it was rinsed in 0.1 M phosphate buffer (pH = 7.4) and homogenized with ice-cold saline to be 10% (w/v) homogenates. The mixtures were homogenized using a glass homogenizer for 5 min and centrifuged at 3000 r/min at 4°C for 15 min. The supernatant was collected and stored at −70°C. The levels of acetylcholine (Ach; Jiancheng Bioengineer Institute, Nanjing, PR China, cat. Number A105-1) were determined according to the methods described in the references by using commercial ELISA kits. Briefly, the samples were first diluted with coating buffer (10 mM phosphate pH 7.8 containing 144 mM NaCl and 0.02% NaN₃). Wells were coated with 100 μL of the diluted specimens at 4°C for 24 h and then rinsed twice with washing buffer (10 mM sodium phosphate pH 7.2 containing 0.05% Tween-20). Post-coating was carried out at 37°C for 60 min by the addition of 200 μL/well of the coating buffer containing 1% bovine serum albumin washing three times, 100 μL/well of mAb diluted in washing buffer was added and incubated at 37°C for 60 min. Following washing three times, 100 μL/well of the second antibody solution was added to the plate and incubated at 37°C for 60 min. After washing three times, 100 μL/well of a solution of 0.1% o-phenylenediamine dihydrochloride containing 0.03% H₂O₂ in substrate buffer was added to the wells. The reaction was allowed to proceed at room temperature for 15 min. Optical density was read at 490 nm. The protein levels of samples were measured by the Coomassie Brilliant Blue G-250 method with bovine serum albumin as standard.

Bilateral microinjection

Rats were anesthetized with isoflurane and prepared for surgery as previously described.^56-58 Stainless steel guide cannulae (22-Ga; Plastics One, Inc.) were bilaterally implanted to the dorsal HPC (AP -3.3 mm, ML ± 2.2 mm, DV 2.4–2.8 mm from the dorsal surface of the brain). Obdurators (30-gauge, Plastics One Inc.) were inserted into the guide cannula to prevent obstruction. Rats were treated with Anafen immediately following surgery and allowed to recover for at least 7 days.

Infusions were performed by inserting custom needles (30-Ga, Small Parts Inc.) connected through PE-50 tube into an infusion pump (Harvard Apparatus), extended 1.0 mm pass the end of the cannulae. Acetylcholine chloride, oxotremorine or ACSF was infused into the HPC (0.5 μL/min/side for 2 min) 30 min before testing began. The needles were left for 3–5 min to allow the diffusion of the drug. One week before the treatment, the infusion procedure was habituated on two separate days. The infusions were conducted on the same animals of each group at most twice, with randomly assigned to receive the infusion drugs.

LFPs recording

As described previously,^59–61 microelectrodes were arranged in two four by four matrix using 25-μm-diameter platinum/iridium wire, coated with polyimide (California Fine Wire Company) in a 16-gauge silica tube (World Precision Instruments). It was then attached via gold pins to an EIB-36-PTB board (Neuralynx Inc.), which was assembled to a microdrive (Harlan 8-drive; Neuralynx). The electrode tips were gold-plated to maintain the impedance to 200–600 kΩ measured at 1.0 kHz (NanoZ, Neuralynx).

Rats were anesthetized with isoflurane and prepared for surgery. Two electrode arrays were chronically implanted: one located at the CA1 region (AP -3.5 mm, ML 2.5 mm, DV 2.0 mm) and the other one was located at the CA3 region (AP -4.2 mm, ML 3.5 mm, DV 2.5 mm) of the HPC. The left or right hemisphere was implanted randomly but counterbalanced across groups.

The recording was conducted with a Digital Cheetah system (Cheetah software, Neuralynx Inc.), sampled at 32 kHz and filtered at 0.1–9000 Hz. The animals’ behavior was monitored by a digital ceiling camera (Neuralynx Inc.) and the CCD camera’s signal was fed to a frame grabber (sampling rate, 64 Hz) with the experimental time superimposed for offline analysis. Fast movements, which were defined as any movements with a velocity higher than 5 cm/s, were scored using the EthoVision XT software (Noldus Information Technology). Local field potentials (LFPs) were recorded during the whole behavioral tests. Since the hippocampal LFP is highly sensitive to the behavioral state, the signal from the behavioral test and homecage environment was analyzed only when rats’ velocity was higher than 5 cm/s. Before analysis, DC offsets and slow fluctuations were eliminated by applying the locdetrend function in the Chronux 2.00 toolbox,⁶² which subtracts the linear regression line fit with the following parameters: 1-s window size, 50-ms time step, the time-bandwidth product of 5, and taper count of 9. The Butterworth band-pass filter was used for all bands of interest including delta (0.5–3 Hz), theta (4–7 Hz), alpha (8–12 Hz), beta (13–35 Hz), gamma (52–100 Hz) and high-frequency (HF, 100–250 Hz). The power spectral densities were calculated using the fast Fourier transform based on Welch’s method (1024 frequencies between 1 and 200 Hz, smoothed with a Gaussian Kernel with bin width 3). The analyses were performed with Neuroexplorer and Matlab (MathWorks) software as our previous studies.^63–65

Phase locked value

PLV is defined to analyse the strength of phase synchronization. Extracting the phase of two signals, $ϕ_{a}$ and $ϕ_{b}$ were obtained. PLV is defined as following,

P L V = | \frac{1}{N} \sum_{j = 1}^{N} \exp (i [ϕ_{a} (j Δ t) - ϕ_{b} (j Δ t)]) |

with N stands for the length of time series,

\frac{1}{Δ t}

is the sampling frequency. The value of PLV is between 0 and 1, meaning that one indicates fully synch and 0 no syncing at all.

General partial directed coherence algorithm

PDC, whose defination is based on the notion of linear Granger causality, is proposed to describe the causal relationship between multivariate time series. Its core meaning is based on the decomposition of multivariate partial coherences computed from multivariate autoregressive models. 2-Variate process PDC algorithm was introduced as following.

Considering a two dimensional process

X (t) : = [x_{1} (t) x_{2} (t)]

(1)

Granger causality within a 2-variate process defined by $X (t)$ is assessed by modeling them through a vector autoregressive (VAR) model of the form:

X (t) = \sum_{r = 1}^{p} A_{r} X (t - r) + E (t)

(2)

with

A_{r} = [\begin{array}{c} a_{11} (r) & a_{12} (r) \\ a_{21} (r) & a_{22} (r) \end{array}]

Taking the Fourier Transformation of the VAR coefficients:

A (f) = \sum_{r = 1}^{p} A_{r} \cdot \exp (- i 2 π f r)

(3)

yields a frequency-domain representation of the VAR model.

Defining the matrix: $\bar{A} (f) = I - \sum_{r = 1}^{p} A_{r} \cdot \exp (- i 2 π f r) = [\bar{a_{1}} (f) \bar{a_{2}} (f)]$ as the difference between the identity matrix.

And then PDC from variable $x_{j}$ to $x_{i}$ is defined as:

π_{i j} (f) = \frac{{\bar{a}}_{i j} (f)}{\sqrt{{\bar{a}}_{j}^{H} (f) {\bar{a}}_{j} (f)}}

(4)

It has been shown that large differences in the variances of the modeled time series can lead to distortions in the resulting PDC values.^66,67 To avoid this, a variation of the original PDC which is called generalized PDC (gPDC)^67,68 is presented. In gPDC, the coefficients ${\bar{A}}_{i j} (f)$ are normalized by the standard deviation of the $E (t)$ model residuals:

{π_{i j}}^{g} (f) = \frac{\frac{1}{σ_{i}} {\bar{a}}_{i j} (f)}{\sqrt{\frac{1}{{σ_{1}}^{2}} {\bar{A}}_{1 j} (f) {\bar{A}}_{1 j}^{H} (f) + \frac{1}{{σ_{2}}^{2}} {\bar{A}}_{2 j} (f) {\bar{A}}_{2 j}^{H} (f)}}

(5)

The denominator in (5) is a normalization that bounds the gPDC coefficients to values from 0 to 1. The choice of scaling means that $| {π_{i j}}^{g} (f) |$ measures the outflow of information from signal $x_{j}$ to signal $x_{i}$ with respect to the total outflow of information from $x_{j}$ to all signals.

Data and statistical analysis

All the data were expressed as Mean ± SEM. Two- or one-way ANOVA was applied for analysis of PLV, gPDC and behavioral test, followed by Tukey’s post hoc test. Independent sample T-test was used to analyze the ACh level. The analyses were performed using SPSS 16.0 software and the significant level was set at 0.05.

Results

The ACh levels in the CA groups

As shown in Figure 1(a), the hippocampal ACh levels were statistically lower in the CA (20) and CA (40) groups compared with those in the control and CA (10) groups (one-way ANOVA, F_{(3, 22)} = 7.93, p < .001; post hoc: CA (20) vs. control and CA (10), both p < .05, CA (40) vs. control and CA (10), both p < .05). Furthermore, the expression of CA (40) group was significantly reduced compared with those of CA (20) group (CA (40) vs. CA (20), p < .05), indicating a dose-dependent effect of CA treatment. Previously, prenatal exposure to CA at the doses of 20 mg/kg and 40 mg/kg undoubtedly impaired spatial learning ability .^{17, 35} Therefore, we only employed the dose of 20 mg/kg in the following tests.

Figure 1.

ACh levels in the hippocampus and performance in the Y-maze task (A) The expression of ACh is reduced following prenatal CA exposure. #, p < .05, vs. CA (20); %, p < .05, vs. others. (B) The total trials to reaching the criterion (the insets are the schematic representation of the cannula placements), (C) the total time spent in reaching the criterion, and (D) the average speed in the Y-maze task. *, p < .05, vs. control, CA (20)+ACh(SYS), CA (20)+ACh(CA1) or ACh(CA1).

Additionally, there was no statistical difference in the gestational duration among groups (control group: 21.2 ± 0.6; the low dose of CA group: 21.7 ± 0.7; the moderate dose of CA group: 21.4 ± 0.4; the high dose of CA group: 21.6 ± 0.6. One-way ANOVA, F_{(3, 40)} = 0.21, p > .05). Thus, there was no difference in the duration of CA treatment in this study.

The hippocampus-dependent spatial learning

To assess the effects of ACh on spatial learning performance, CA-treated rats were subjected to intra-hippocampal ACh or cholinergic receptor agonist injections 30 min before the Y-maze training. Rats in the CA (20) group took more trials to reach the criterion than control group (Figure 1(b), one-way ANOVA, F_{(7, 43)} = 6.33, p < .001; post hoc: CA (20) vs. control, p < .05). Meanwhile, CA (20)-treated rats spent more time in learning to find the reward arm than control rats (Figure 1(c), one-way ANOVA, F_{(7, 43)} = 7.05, p < .001; post hoc: CA (20) vs. control, p < .05). Both systemic (Trials: CA (20)+ACh(SYS) vs. CA (20), p < .05; Time: CA (20)+ACh(SYS) vs. CA (20), p < .05) and intra-hippocampal CA1 (Trials: CA (20)+ACh(CA1) vs. CA (20), p < .05; Time: CA (20)+ACh(CA1) vs. CA (20), p < .05) injections of ACh could effectively reduce total trials and time to reaching the criterion of CA (20)-treated rats. However, this effect did not find when ACh was infused into the hippocampal CA3 region of the CA (20) group (Trials: CA (20)+ACh(CA3) vs. CA (20), p > .05; Time: CA (20)+ACh(CA3) vs. CA (20), p > .05). Furthermore, neither the CA3 (Trials: CA (20)+XO(CA3) vs. CA (20), p > .05; Time: CA (20)+XO(CA3) vs. CA (20), p > .05) nor the CA1 (Trials: CA (20)+XO(CA1) vs. CA (20), p > .05; Time: CA (20)+XO(CA1) vs. CA (20), p > .05) infusions of the cholinergic receptor agonist could alleviate the learning deficits. Additionally, the infusion of ACh into the hippocampal CA1 of the control animals did not change trials (Trials: ACh(CA1) vs. Control, p > .05) or time spent during the training (Time: ACh(CA1) vs. Control, p > .05). Among groups, the moving velocity remained constant throughout the training, with no statistical difference was found (Figure 1(d), one-way ANOVA, F_{(7, 43)} = 0.33, P > .05).

The strength of phase synchronization

The basal values of PLV, which were collected in rats’ homecage environment, were comparable among groups (Figure 2(a), two-way ANOVA, interaction effect between treatment and band: F_{(15, 100)} = 0.59, p > .05). This index at the low-frequency bands, including theta (Figure 2(b), two-way ANOVA, interaction effect between treatment and band: F_{(15, 100)} = 2.97, p < .001; post hoc: CA (20) vs. control, p < .05) and alpha (CA (20) vs. control, p < .05), was significantly lower in CA (20) group than the control group during the learning task. Meanwhile, infusions of ACh into the hippocampal CA1 region could effectively elevate the declined PLV of CA-treated rats (CA (20) vs. CA (20)+ACh(CA1), both theta and alpha bands p < .05). To evaluate the NIF in the hippocampal CA3-CA1 pathway, the directionality index d of gPDC was employed. Compared with the control group, the values of directionality index d at theta and alpha bands were distinctly lower in the CA (20) group (Figure 2(c); two-way ANOVA, interaction effect between treatment and band: F_{(15, 100)} = 2.77, p < .01; post hoc: CA (20) vs. control, both theta and alpha bands p < .05). Meanwhile, the values of the unidirectional influence c₂, indicating the unidirectional coupling from CA3 to CA1, was markedly diminished by prenatal CA exposure (Figure 2(d); two-way ANOVA, interaction effect between treatment and band: F_{(15, 100)} = 2.69, p < .01; post hoc: CA (20) vs. control, both theta and alpha bands p < .05). However, reversible effects of intra-hippocampal ACh infusion were found in the CA (20)+ACh(CA1) group, as indicated by that both d (CA (20) vs. CA (20)+ACh(CA1), both theta and alpha bands p < .05) and c₂ (CA (20) vs. CA (20)+ACh (CA1), both theta and alpha bands p < .05) indexes were increased in the CA (20)+ACh(CA1) group.

Figure 2.

The values of PLV and the directional coupling index d and c₂ between CA3 and CA1 regions (A) The basal values of PLV among groups are comparable. (B) The value of PLV during the behavioral training in the CA group is significantly declined in the theta and alpha bands, but hippocampal ACh infusion effectively enhanced the PLV value. (C) Coupling direction index d between CA1 and CA3, indicating CA3 predominantly drives CA1. (D) Unidirectional influence index c₂, indicating unidirectional NIF from CA3 to CA1 area. The values of d and c₂ are declined in the CA group and the reversible effects of ACh infusions are found. *, p < .05, vs. others.

To eliminate the probable side effects of ACh infusions on NIF, ACh was injected into the CA1 region of control rats before each recording. No statistical difference in basal PLV, test PLV, index d or c2 of gPDC was found between control and ACh(CA1) groups. Our findings support that prenatal CA exposure depresses the hippocampal NIF from CA3 to CA1, and ACh inactivation is crucial for the declined NIF, especially low-frequency bands (4–12 Hz).

Discussion

Our initial hypothesis was that prenatal CA exposure during the whole of gestation would cause a reduction in the hippocampal ACh level with subsequent learning deficits. This hypothesis was based on recent work showing that chronic exposure to CA during early postnatal development resulted in synaptic dysfunction¹² and early studies showing an association between prenatal exposure to the nitrogen-ring structural analogue of CA and acetylcholinergic pathway in the immature brain.^15,16 To our surprise, prenatal CA exposure did reduce NIF at the hippocampal CA3-CA1 synapses, which is involved in spatial learning disability. Intra-hippocampal injection of ACh into the CA1 but not the CA3 region could effectively enhance the decreased directional and unidirectional coupling indexes of the gPDC, thereby rescuing the learning defects. Therefore, our findings confirm previous reports of neurotoxic effects of prenatal CA treatment on hippocampus-dependent learning ability and further support the potential involvement of ACh-mediated NIF in CA-induced neuronal and cognitive dysfunction.

Our behavioral findings are compatible with the extensive literature demonstrating when administered near the time of training or testing, pharmacological agents that augment cholinergic functions in the hippocampus enhance learning and memory and agents that interfere with cholinergic functions in the hippocampus impair learning and memory.^18,19,31,69 The active CA1 population comes to consist of a relatively small group of cells with strong spatial tuning.⁷⁰ This process is not evident in CA3, indicating that a region-specific and long timescale process operates in CA1 to create a sparse, spatially informative population of neurons. Other findings also demonstrate that functional heteromeric nicotinic receptors are present on CA1 pyramidal neurons during a period of major hippocampal development, placing these receptors in a prime position to play an important role in the establishment of hippocampal cognitive networks.^69,71,72 Mice lacking metabotropic glutamate receptor five show impaired learning and reduced CA1 NMDA receptor–dependent long-term potentiation (LTP) but normal CA3 LTP, that LTP in the CA1 region may underlie spatial learning and memory.⁷³ Indeed, limited subfields of region CA1, but not CA3, were activated in association with the execution of spatially defined learned behaviors.^36,37,68,74 Recently, CA treatment disturbs glutamatergic neurotransmission and excitatory postsynaptic currents in the CA1 pyramidal neurons.¹² Therefore, it is possible that several mechanisms are acting in parallel to disrupt CA3-CA1 synaptic function across learning while the dysfunction of CA1 but not CA3 is associated with the behavioral impairments. Furthermore, consistent with our previous findings,^12,17,75 the similar moving velocity for CA control and other groups indicates that these behavioral findings are not caused by decreased locomotor activity.

Cholinergic hippocampal projections are critical for generating and phasing hippocampal theta, alpha and gamma oscillatory activity,^28,29,76 therefore playing a pivotal role in processes associated with learning and memory consolidation.^18,31 Acetylcholine has been demonstrated to enhance the persistent spiking of individual cortical neurons, which could provide an excellent mechanism for the active maintenance of novel information both for short-term memory and for encoding information into long-term memory.^30,77,78 For example, when an animal engages in exploratory behavior, as during the acquisition of hippocampus-mediated spatial memories, the hippocampus undergoes an oscillation in its electrical activity at frequencies between 4 and 12 Hz,^79–81 indicating that acetylcholine can enhance encoding through its role in increasing rhythm oscillations (4–12 Hz) within the hippocampal formation.^82,83

Neural information flow is inherently directional. The phase synchronization analysis suggested that the connection strength between hippocampal CA3 and CA1 regions was weakened in the CA group but reversed by ACh treatments, which manifested that there was a disturbance in directional neural information flow in the CA3-CA1 pathway. In order to evaluate the coupling directionality of NIF in the hippocampal CA3-CA1 pathway, the coupling directional index and the strength were calculated in the gPDC algorithm. It can be seen that a more predominant driving effect occurred from CA3 to CA1 in these two groups, which was in line with the anatomy synaptic projections from CA3 to CA1. Moreover, it showed that unidirectional indexes from CA3 to CA1 in the CA-treated rats were significantly declined, suggesting that prenatal CA exposure considerably wakened directional information transmission in the hippocampal CA3-CA1 pathway. The NIF disruption is attributed to the reduction in ACh expression in the hippocampus, since the intra-hippocampal infusion can elevate the declined NIF. Furthermore, hippocampal synaptic plasticity in the CA1 region can be induced by cholinergic theta oscillation.⁸⁴ It’s worth noting that a higher threshold following prenatal CA exposure was found, as the evidence that LTP could be reinstated only by increasing the intensity of theta-burst stimulation.¹⁷ The cholinergic drive to the hippocampus is critically involved in the control of the LTP induction threshold in vivo.⁸⁵ The high rate of spiking in CA1 could serve to signal the presence of new spatial information and facilitate plasticity in downstream structures.⁷⁰ This modulation in synaptic strength during the learning process has been confirmed here for the CA3-CA1 synapse, suggesting a requirement of the activity-dependent modification of the neural network for learning to occur.

Most of the experiments were conducted with isoflurane because it is a clinically widely used volatile anesthetic. Although it has been disputed by others,^86-89 isoflurane has been linked to memory deficits in rodents.^90-92 The current study chose to examine the surgical procedure, thus the choice to control animals to anesthesia could be a point of contention. While not specifically designed to investigate the effect of anesthesia, this work does support the notion that there is no lasting learning and memory impairments caused by isoflurane. However, we could not exclude that isoflurane anesthesia exacerbated spatial learning impairments in CA-treated rats. Therefore, further researches are needed to explore these effects.

In conclusion, this study demonstrates that prenatal CA exposure induces the hippocampus-dependent learning disability and this deficit is associated with the declined expression of ACh in the CA1 region. Furthermore, the ACh-mediated NIF at the hippocampal CA3-CA1 pathway is attributed to the learning deficits, since enhancement in the hippocampal CA1 level can significantly strengthen the coupling directional index and the strength of CA3 driving CA1. Our findings provide new evidence for the involvement of the hippocampal cholinergic network in CA-induced neurotoxicity, as well as in spatial learning mechanisms.

Footnotes

Author contribution

WS, XLL, YWW, XC and YZM conducted experiments and analyzed the data; WS, XYZ, YY, XXL and LA conceived and designed the experiments; XYZ, YY, XLL and LA guided research direction; WS, XYZ, YY, XLL and LA wrote the manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from the National Natural Science Foundation of China (32160196; 31700929) to LA.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethical approval

All experiments and procedures were reviewed and approved by the Experimental Animal Care Committee of Guizhou University of Traditional Chinese Medicine (SCXK-2013–0020).

ORCID iD

Lei An

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