Focused Ultrasound Stimulates the Prefrontal Cortex and Prevents MK-801-Induced Psychiatric Symptoms of Schizophrenia in Rats

Abstract

Background and Hypothesis

Treatment of schizophrenia remains a major challenge. Recent studies have focused on glutamatergic signaling hypoactivity through N-methyl-D-aspartate (NMDA) receptors. Low-intensity pulsed ultrasound (LIPUS) improves behavioral deficits and ameliorates neuropathology in dizocilpine (MK-801)-treated rats. The aim of this study was to investigate the efficacy of LIPUS against psychiatric symptoms and anxiety-like behaviors.

Study Design

Rats assigned to 4 groups were pretreated with or without LIPUS for 5 days. The open field and prepulse inhibition tests were performed after saline or MK-801 (0.3 mg/kg) administration. Then, the neuroprotective effects of LIPUS on the MK-801-treated rats were evaluated using western blotting and immunohistochemical staining.

Study Results

LIPUS stimulation of the prefrontal cortex (PFC) prevented deficits in locomotor activity and sensorimotor gating and improved anxiety-like behavior. MK-801 downregulated the expression of NR1, the NMDA receptor, in rat medial PFC (mPFC). NR1 expression was significantly higher in animals receiving LIPUS pretreatment compared to those receiving only MK-801. In contrast, a significant increase in c-Fos-positive cells in the mPFC and ventral tegmental area was observed in the MK-801-treated rats compared to those receiving only saline; this change was suppressed by pretreatment with LIPUS.

Conclusions

This study provides new evidence for the role of LIPUS stimulation in regulating the NMDA receptor and modulating c-Fos activity, which makes it a potentially valuable antipsychotic treatment for schizophrenia.

Introduction

Schizophrenia is a major neuropsychiatric disorder, with positive, negative, and cognitive symptoms. The N-methyl-D-aspartate (NMDA) hypofunction theory of psychosis suggests that hypoactivity of NMDA receptors on gamma-aminobutyric acid (GABA) interneurons in the prefrontal cortex (PFC) leads to overactive downstream glutamate signaling. A number of studies have demonstrated morphological abnormalities in the cortex and dysfunction of the medial PFC (mPFC) in patients with schizophrenia.^1–3 In the cortex, glutamatergic neurons project axons to GABAergic interneurons. Furthermore, the cortical pyramidal glutamatergic neurons that project to dopaminergic neurons in the ventral tegmental area (VTA), in turn, project to the nucleus accumbens (NAc), forming the cortex–VTA–NAc circuit.^4–6 Changes in c-Fos expression have been observed in animal models of schizophrenia and have proved to be a useful parameter in mapping the distribution of neurons activated by pharmacological stimuli.⁷ Dizocilpine (MK-801)-treated animals show increased c-Fos expression in cortical regions and the VTA.^8,9 The c-Fos protein, the marker of neuronal activity, has been suggested to play a crucial role in the schizophrenic model process induced by the NMDA receptor antagonist.¹⁰

MK-801, a noncompetitive NMDA receptor antagonist, causes locomotor hyperactivity, anxiety-like behavior, and sensory-motor gating deficits in rodents.^11,12 The action of MK-801 on interneurons reduces the inhibition of excitatory pyramidal neurons, leading to hyperexcitation in the PFC neuronal circuit.^13,14 In addition, evidence suggests that NR1 is crucial for the formation and synaptic expression of NMDA receptors among various NMDA receptor subunits.¹⁵ MK-801 induced blunted hyperlocomotor activity in parvalbumin (PV) interneuron-specific NR1 knockout mice, suggesting that NMDA receptors in PV interneurons may be the site of MK-801.^16,17 Furthermore, NMDA receptors are involved in transcriptional regulation of the GABA-synthesizing enzyme, glutamate decarboxylase (GAD65 and GAD67).¹⁸

Vesicular glutamate transporters (VGLUTs) play an important role in neuronal glutamate delivery.¹⁹ VGLUT levels determine presynaptic vesicle filling and affect glutamate release. VGLUT1 is the main isotype, with the highest proportion and number of functions, accounting for most of the excitatory glutamatergic terminals in the central nervous system.²⁰ Vesicular GABA transporter (VGAT) is localized to vesicles of inhibitory terminals of the GABAergic neurons. A balanced ratio of VGLUT1 to VGAT is important for normal brain function.²¹ Dopamine transporter (DAT) expression, which is selective to dopaminergic neurons within the brain, is critical to the regulation of dopamine reuptake from extracellular space. DAT is a dynamic contributor to the regulation of dopamine activity through its regulation of dopaminergic function.²²

Our previous findings indicate that low-intensity pulsed ultrasound (LIPUS) improves cognitive deficits and restores the levels of expression of calcium-binding proteins in the MK-801-treated rats.²³ Accordingly, in the present study, we evaluated the effects of pretreatment by LIPUS on positive symptoms of schizophrenia in the MK-801-treated rats. Our results suggest that LIPUS prevents hyperlocomotor activity and anxiety-like behavior, as well as sensory-motor gating deficit by acting on its receptors, to suppress neuronal activity in the mPFC of rats.

Materials and Methods

Ultrasound System and Setup

Low-intensity pulsed ultrasound was generated by a therapeutic ultrasound generator (ME740, Mettler Electronics, Anaheim, CA, USA) and a 1-MHz plane transducer (ME7413, Mettler Electronics; with a 4.4 cm² effective radiating area) with 2-miliseconds burst lengths at a 20% duty cycle and a repetition frequency of 100 Hz. The spatial average intensity over the plane transducer head was 500 mW/cm². The transducer was mounted on a removable aluminum cone 10 mm in diameter at the tip. The focused ultrasound was positioned using a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA) in order to direct the acoustic beam to the PFC (3.0 mm anterior to the bregma) of the brain (figure 1A). To examine the spatial precision of LIPUS stimulation on PFC, activated neurons with c-Fos were quantified in the cingulate cortex 1(Cg), prelimbic cortex (PrL), infralimbic cortex (IL), primary motor cortex (M1), and primary visual cortex (V1) (supplementary figure S1). In the 3 subregions of mPFC following LIPUS treatment, the expressions of c-Fos have the trend of increase, although there was no significant difference. On the contrary, LIPUS stimulation induced significantly fewer NeuN-positive cells with c-Fos expression than MK-801 treatment in the PrL and IL of mPFC (supplementary figure S1B). However, no changes were found in c-Fos immunoreactivity patterns in the unsonicated regions of M1 and V1 between LIPUS-treated group and LIPUS-untreated group (supplementary figure S1C). In order to reduce the thermal effect of ultrasound, the duration of each sonication was 5 minutes, with a 5-minute interval between each sonication. The total sonication time of the LIPUS stimulation was 15 minutes every day. Several parameters were used in the animal studies, such as intensity, duty cycle, and duration time. Different combinations of parameters lead to various modulatory effects. The parameters of the TUS exposures were selected based on the results of our previous studies.^23,24

Fig. 1.

Effects of LIPUS stimulation on the behavior of rats treated with MK-801. (A) Schematic diagram of the ultrasound transducer and treatment of the rat brain with LIPUS. The open field test was performed for 120 minutes for measuring (B) distance traveled, (C) progression in a 10-minute bin, and (D) time spent in the center area by the Sham group of rats and those treated with LIPUS or MK-801 or MK-801 plus LIPUS. (E) Effects of LIPUS on MK-801-induced disruption of prepulse inhibition. *, ^#, and ^† denote significant differences from the Sham group, LIPUS group, and MK-801 group, respectively (*^,†, P < .05; **^,##,††, P < .01; ***^,###, P < .001; n = 10). .

Animal Model and Experimental Protocols

Male Sprague-Dawley rats weighing 230–280 g were purchased from BioLASCO Taiwan Co., Ltd. (Yilan City, Taiwan). All procedures involving animals were performed in accordance with the guidelines for the Care and Use of Laboratory Animals. This study protocol was approved by the Animal Care and Use Committee of National Yang Ming Chiao Tung University (No. 1080206). The animals were randomly assigned to 4 groups: The Sham group, the LIPUS group, the MK-801 group, and the LIPUS+MK-801 group. All animals were habituated in a home cage for 7 days. After the habituation period, animals in the LIPUS group and LIPUS+MK-801 group were treated with LIPUS daily for 5 days. One day after the last LIPUS stimulation, the LIPUS group and the Sham group were intraperitoneally injected with saline, while the MK-801 group and the LIPUS+MK-801 group were injected with MK-801 (0.3 mg/kg). During LIPUS stimulation, 4 groups were anesthetized in the prone position through inhalation of 2% isoflurane in 2 L/min oxygen; body temperature was maintained at 37 °C using a heating pad. The head of each rat was positioned on a stereotaxic apparatus, and the top of the hair around the cranium was removed with an electric shaver and hair removal cream for LIPUS stimulation. The only different step of LIPUS stimulation between 4 groups, is that, LIPUS was turned on or not. Thirty minutes after MK-801 administration, the animals were assessed for their behavior, evaluated histologically, and analyzed biochemically.

It has been demonstrated that MK-801 induces hyperlocomotion, anxiety-like behavior, and prepulse inhibition (PPI) disruption in rodents.^11,25,26 The hypermotility induced by MK-801 was no longer observed during chronic treatment.²⁷ PPI was reduced after acute MK-801 administration. However, the sensorimotor gating impairments may be reduced by MK-801 tolerance effect.²⁶ Therefore, an acute MK-801-induced schizophrenia model was used in this study.

Analysis of Behavior

Locomotor activity and anxiety-like behavior of the rats were assessed using the open field test. The system consisted of clear plastic chambers (100 × 100 × 30 cm), a computer interface board, and a computer for recording and analyzing data. Each rat was placed in the center of the field. Total distance traveled was measured for 120 minutes. All tests were monitored using a computerized motion tracking apparatus and software equipped with a CCD camera using Etho Vision software.

The PPI test was used to assess changes in sensorimotor gating. Attentional processing was examined by testing prepulse inhibition of the acoustic startle response. The rats were placed in startle chambers for a 30-min adaptation period. After acclimatization to background white noise (65 dB for 30 seconds), the rats received a startle pulse at 120 dB for 40 ms and the prepulse, a pure tone at 72, 78, or 84 dB, for 20 ms. Each trial had an average intertrial interval of 15 seconds. The PPI response was calculated for each of the 3 prepulse intensities using the following formula: PPI (%) = (mean peak amplitude of pulse-only sessions—mean peak amplitude of prepulse/pulse sessions)/mean peak amplitude of pulse-only sessions ×100.

Immunofluorescence

The rats were intracardially perfused with 4% paraformaldehyde in phosphate buffer under anesthesia. Frozen 10-μm sections were cut coronally using a microtome. After incubation in blocking solution with fetal bovine serum (SKU 04-001-1A, Biological Industries, USA) for 30 minutes at 37 °C, the sections were immunostained with the following primary antibodies: rabbit anti-NMDAR1 (NR1, 1:200; GeneTex, California, USA), goat anti-parvalbumin (1:200; GeneTex, California, USA), mouse anti-c-Fos (1:200; Abcam, Cambridge, USA), rabbit anti-neuronal nuclei (NeuN, 1:500; GeneTex, California, USA), and rabbit anti-tyrosine hydroxylase (TH, 1:200; GeneTex, California, USA). The brain sections were incubated with primary antibodies overnight and then washed and incubated with either Alexa Fluro 488- or Alexa Fluro 594-tagged secondary antibodies (1:500; Abcam, Cambridge, MA, USA) at room temperature. Three sections per rat were analyzed at the same exposure level. At least 3 randomly distributed fields from the mPFC were captured from each section. Double immunofluorescence was observed under a fluorescence microscope (Leica DM 6000B, Mannheim, Germany) and photographed. The number of cells positive for the NR1–PV double label and c-Fos–NeuN double label was counted in 3 non-overlapping fields in the mPFC at a magnification of 400× (318 × 237 μm²) and 200× (635 × 475 μm²), respectively. The number of cells positive for the c-Fos–TH double label was counted in an area 635 × 475 μm² in 3 non-overlapping fields in the VTA at a magnification of 200×.

Western Blot Analysis

Fresh brain tissue from the mPFC and NAc was homogenized using T-Per extraction reagent supplemented with the Halt Protease Inhibitor Cocktail (Pierce Biotechnology, Inc.). Samples containing 30 μg of protein were resolved using 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to Immun-Blot® polyvinyldifluoride membranes (Bio-Rad, CA, USA). After blotting, the membranes were blocked for at least 1 hour in milk (Anchor), and then the blots were incubated overnight at 4 °C in a solution with rabbit antibodies against GAD65/GAD67 (GeneTex, 1:2000), c-Fos (Abcam, 1:2000), VGLUT1 (Abcam, 1:2000), VGAT (GeneTex, 1:2000), DAT (GeneTex, 1:2000), β-actin (GeneTex, 1:5000), and GAPDH (GeneTex, 1:5000). The gel image was captured using an ImageQuant™ LAS 4000 biomolecular imager (GE Healthcare Life Sciences, Pennsylvania, USA) and analyzed using Image J software (Image J, National Institute of Health, Bethesda, MD, USA) to estimate the integral optical density of the protein bands.

Statistical Analysis

All data are shown as mean ± standard error of the mean (SEM). One-way ANOVA, followed by Tukey’s post hoc test, was used to determine significant differences between groups. The level of statistical significance was set at P < .05.

Results

LIPUS Stimulation Ameliorates Behavioral Impairments in Rats With Schizophrenia

Consistent with published data,^28,29 a significant increase was observed in the total distance traveled by the MK-801 group compared to the Sham group (P < .001; figure 1B), which indicated that MK-801 induced the increased spontaneous activity, suggesting that the schizophrenia animal model was established. Direct group comparisons of the distance traveled every 10 min showed that locomotor activity of the MK-801-treated rats was significantly higher in time blocks 3–12 than that of the Sham group (all P < .05; figure 1C). In time blocks 7–9, LIPUS significantly reduced the distance traveled scores of MK-801-treated rats (all P < .05; figure 1C). Additional information was obtained from open field activity by assessing the time spent in the center area. The tendency to avoid the open field center has been used as a standard measure of anxiety-like behavior. Compared to the rats in the Sham group, the MK-801-treated rats spent significantly less time in the center area (P < .05; figure 1D).

At all 3 prepulse intensities, no significant change was observed in the mean PPI percentage in the LIPUS group compared to the Sham group (all P > .05; figure 1E). Mean PPI percentage was significantly reduced; however, compared to the Sham group, the decreased PPI percentage of the MK-801-treated rats was significantly attenuated by LIPUS treatment (all P < .05; figure 1E).

LIPUS Reversed MK-801-Induced Downregulation of NR1 and GD65 Expression in MK-801-Treated Rats

The puncta localized in individual neurons were counted (Figure 2A). The MK-801-treated group showed significantly reduced puncta numbers in the NR1 subunit in PV interneurons compared to the Sham group (14.49 ± 1.94 vs 26.25 ± 0.97, P < .001; Figure 2B). Compared to the MK-801 group, in the LIPUS+MK-801 group, the MK-801-induced decrease in the puncta numbers of NR1 subunits in PV interneurons was significantly attenuated by LIPUS (23.72 ± 1.88 vs 14.49 ± 1.94, P < .001; figure 2B). Furthermore, no significant changes were observed in PV interneuron numbers in the mPFC of rats treated with saline or MK-801 with or without LIPUS stimulation (figure 2C). GABA is synthesized from glutamate by GAD, which exists as 2 isoforms, GAD65 and GAD67. Both isoforms are expressed in GABAergic neurons. In agreement with a previous study,³⁰ we observed a decrease in GAD65 levels in the mPFC after MK-801 administration (figure 2D). LIPUS stimulation significantly reversed the levels of GAD65 in the MK-801+LIPUS group. Although quantitative analysis revealed a slight decrease in GAD67 levels in the mPFC of the MK-801-treated group compared to the Sham group, this decrease was not statistically significant (figure 2E). No significant differences were observed between the 4 groups.

Fig. 2.

LIPUS attenuates the MK-801-induced decrease in NR1 subunits and GAD65 in the mPFC of MK-801-treated rats. (A) Representative images of immunofluorescent labeling of NR1 subunits (red), PV (green), and DAPI (blue) in the mPFC of Sham, LIPUS-treated, MK-801-treated, and MK-801 + LIPUS-treated rats. The arrow indicates NR1- and PV-labeled cells. Images at high magnification show the methods used for quantification of NR1 puncta in PV interneurons. (B) Density of NR1 subunit puncta in PV interneurons in the mPFC. (C) Density of PV interneurons in the mPFC. Nuclei were counterstained with DAPI. Scale bars = 50 μm for all images. (D) Representative western blot bands and their quantification of GAD65 levels. (E) Representative western blot bands and their quantification of GAD67 levels. *, ^#, and ^† denote a significant difference from the Sham group, LIPUS group, and MK-801 group, respectively (**^,††, P < .01; ***^{,###,†††}, P < .001; n = 5). N.S., no significance; PV, parvalbumin; GAD, glutamic acid decarboxylase.

LIPUS Reversed MK-801-Induced Upregulation of c-Fos Expression in the mPFC

The c-Fos protein expression in the mPFC was significantly higher in the MK-801-treated group than in the Sham group (1.46 ± 0.14 vs 1.00 ± 0.07, P < .01; figure 3A). LIPUS stimulation prevented the MK-801-induced increase in c-Fos expression (1.08 ± 0.15 vs 1.46 ± 0.14, P < .05; figure 3A). To ascertain whether LIPUS stimulation can reverse c-Fos expression in neurons in the mPFC, we used immunohistochemistry to further examine the effects of LIPUS on c-Fos expression in mPFC neurons (figure 3B). Similar to the results of a previous study, we showed that treatment with MK-801 significantly increased the number of NeuN-positive cells with c-Fos expression compared to the Sham group (136.34 ± 13.10 vs 89.08 ± 9.21, P < .01; figure 3C). LIPUS stimulation induced significantly fewer NeuN-positive cells with c-Fos expression than MK-801 treatment (84.85 ± 7.50 vs 136.34 ± 13.10, P < .01; figure 3C). Furthermore, no significant changes were observed in the number of NeuN-positive cells in the mPFC of rats treated with saline or MK-801 with or without LIPUS stimulation (figure 3D).

Fig. 3.

Effects of LIPUS stimulation on MK-801-induced c-Fos expression in the mPFC. (A) Western blot validation of c-Fos protein levels. (B) Representative images of c-Fos (red)- and NeuN (green)-positive cells in the mPFC. The arrow indicates c-Fos- and NeuN-labeled cells. Images at high magnification show the methods used for quantification of c-Fos in neurons. (C) A quantitative analysis shows that the LIPUS-increased expression of c-Fos is identifiable with neurons in the mPFC. (D) Density of NeuN+ cells in the mPFC. Nuclei were counterstained with DAPI (blue). Scale bars = 100 μm for all images.*, ^#, and ^† denote significant differences from the Sham group, LIPUS group, and MK-801 group, respectively (**^,##,††, P < .01; n = 5). NeuN, Neuronal nuclei; N.S., no significance.

Effects of LIPUS Treatment on the VGLUT/VGAT Ratio and DAT Level in MK-801-Treated Rats

As shown in figure 4, MK-801 treatment significantly upregulated VGLUT1 expression (P < .05; figure 4A), without affecting VGAT (figure 4B), in the mPFC. The VGLUT1/VGAT ratio was significantly higher in rats treated with MK-801 (P < .05; figure 4C). LIPUS stimulation significantly reversed the VGLUT1 level and VGLUT1/VGAT ratio in the MK-801+LIPUS group (both p < 0.05) compared to the MK-801 group. A key regulator of dopamine activity is DAT.³¹ As shown in figure 4D, MK-801 treatment significantly decreased the DAT level in the NAc (P < .01). However, LIPUS significantly reversed this reduction (P < .05; figure 4D).

Fig. 4.

Effects of LIPUS stimulation on the VGLUT1/VGAT ratio and DAT level in MK-801-treated rats. Representative western blot bands and their quantification of (A) VGLUT1 levels and (B) VGAT levels. (C) The VGLUT1/VGAT ratio is derived from (A) and (B). (D) Representative western blot bands and their quantification of DAT levels. *, ^#, and ^† denote significant differences from the Sham group, LIPUS group, and MK-801 group, respectively (*^,†, P < .05; ^††, P < .01; ***^,###, P < .001; n = 5). DAT, dopamine transporter; VGAT, vesicular gamma-aminobutyric acid transporter; VGLUT1, vesicular glutamate transporter 1.

LIPUS Reversed MK-801-Induced Upregulation of c-Fos Expression in TH-Positive Dopamine Neurons

Double immunostaining for c-Fos and TH was performed to assess the excitability of VTA neurons (figure 5A). Immunocytochemical analysis showed that c-Fos expression in TH+ dopamine neurons in the VTA, which may preferentially project to NAc, was significantly higher in the MK-801-treated group than in the Sham group (270.90 ± 7.0 vs 132.30 ± 24.2, P < .001; figure 5B). The MK-801-induced increase in the number of c-Fos puncta in TH-positive dopamine neurons was significantly attenuated by pretreatment with LIPUS compared to the MK-801-only treatment (270.9 ± 7.0 vs 131.4 ± 39.6, P < .001; figure 5B). However, no significant changes were observed in the numbers of TH-positive dopamine neurons in the VTA of rats treated with saline or MK-801 with or without LIPUS stimulation (figure 5C).

Fig. 5.

Effects of LIPUS stimulation on MK-801-induced c-Fos expression in tyrosine hydroxylase (TH)-positive dopamine neurons in the ventral tegmental area (VTA). (A) Representative images of immunofluorescent labeling of c-Fos (red), TH (green), and DAPI (blue) staining in the Sham, LIPUS, MK-801, and MK-801 + LIPUS groups of rats in the VTA. The arrow indicates c-Fos- and TH-labeled cells. Images at high magnification show the methods used for quantifying c-Fos puncta in TH-positive dopamine neurons. (B) Quantitative analyses show that the MK-801-increased expression of c-Fos is identifiable with dopamine neurons in the VTA. (C) Density of TH-positive dopamine neurons in the VTA. Nuclei were counterstained with DAPI (blue). Scale bars = 100 μm for all images. *, ^#, and ^† denote significant differences from the Sham group, LIPUS group, and MK-801 group, respectively (*^,#,†, P < .05; n = 5). N.S., no significance; TH, tyrosine hydroxylase.

Discussion

In this study, we demonstrated that pretreatment of the PFC with LIPUS stimulation prevented deficits in locomotor activity, sensorimotor gating, and anxiety-like behavior induced by MK-801. This effect was associated with changes in c-Fos in the mPFC and VTA, where LIPUS attenuated NMDA receptor hypofunction and influenced downstream signaling.

The NR1 subunit has a central role in NMDA receptor folding and assembly by potentially providing stable support for receptor folding and oligomerization. Low NR1 levels are believed to cause a reduction in NR2 and NR3 levels because protein degradation experiments have shown that the NMDA receptor subunit NR1 is relatively stable and undergoes further folding, but the NR2 and NR3 subunits break down more quickly.³² Our results showed that MK-801 treatment caused downregulation of NR1 in PV interneurons in the mPFC (figure 2). However, this reduction could be reversed by LIPUS stimulation. In addition, GAD65 is important in tonic inhibition and constitutes a prominent part of GABAergic transmission.^33,34 The deficits in GAD65, rather than in GAD67, could be a developmental feature of schizophrenia.³⁰ We validated the MK-801-induced downregulation of GAD65 but not of GAD67 (figure 2). Similarity, LIPUS stimulation attenuated the decrease in GAD65 in the MK-801-treated rats.

c-Fos expression in the brain serves as an indicator of neuronal activation.³⁵ To examine the mechanism of the protective effect of LIPUS in the mPFC and VTA, activated neurons with c-Fos were quantified. Similar to previous studies,^36,37 MK-801 caused a significant increase in c-Fos expression in the mPFC (figure 3). Interestingly, c-Fos expression in the mPFC is not correlated with GAD67 expression (figure 2E).³⁷ Therefore, in this study, we investigated the effects of LIPUS on c-Fos expression in the mPFC and VTA of rats after acute administration of MK-801. Excitatory projections from the PFC to VTA play a crucial role in regulating the activity VTA neurons.³⁸ Our data revealed that pretreatment of rats with LIPUS in mPPFC significantly decreased c-Fos expression in both brain regions after acute administration of MK-801 (figure 3 and 5). The effects may be mediated by the monosynaptic projection from the PFC to DA neurons within the VTA.³⁹ A previous study suggested that NMDA receptor hypofunction induces cortical excitation by disinhibition of pyramidal neurons.⁴⁰ As LIPUS could alleviate the decrease in NR1 (figure 2), it may prevent MK-801-induced c-Fos expression in the mPFC through inhibition of pyramidal neurons.

A balanced excitatory/inhibitory input is important for maintaining homeostasis of neurotransmission in the brain and thus for preventing over-excitation of circuits.⁴¹ The increased VGLUT1/VGAT ratio in MK-801-treated rats was driven by an increase in VGLUT1 levels in the mPFC (figure 4). The increase in VGLUT1 indicates an increase in glutamate release from excitatory pyramidal neurons because VGLUT1 directly influences presynaptic glutamate release.⁴² Furthermore, TH and DAT are modulators of the dopamine concentration in the reward system. TH is involved in the synthesis of dopamine and DAT modulates dopamine concentrations in the synaptic terminals.^43,44 Our data showed that MK-801 treatment reduced DAT expression in the NAc but did not change TH expression in the mPFC (figure 4D and 5C). These findings suggest that MK-801 treatment induces a reinforcing effect by inhibiting dopamine reuptake in dopaminergic terminals of the NAc. However, LIPUS may reduce this effect because the decrease in DAT can be reversed by pretreatment with LIPUS.

Schizophrenia is notorious for relapses, which are likely to lead to a vicious cycle of illness.⁴⁵ Relapse prevention in schizophrenia is of utmost clinical relevance. Although maintenance of antipsychotic agents for prevention of relapse of schizophrenia is important, various adverse effects may well discourage patients from adhering to antipsychotic regimens, which is an established risk factor of relapse. In the present study, we demonstrated that pretreatment with LIPUS prevents psychiatric symptoms in the acute MK-801 animal model of schizophrenia. These findings indicate the potential of LIPUS for prevention of schizophrenia and offer a future avenue of research on a new neuromodulation method for treatment of the disease. LIPUS is a technique of non-invasively delivering mechanical forces to cells deep within the brain. The mechanical forces can result in several neural functions, such as signaling and differentiation.^46,47 The mechanical effects provide a physical basis of ultrasound neuromodulation. There are some possible mechanisms by which LIPUS could lead to triggering of action potentials, including the activation of mechanosensitive channels and the generation of capacitive currents due to membrane displacements.⁴⁸ In contrast to antipsychotic medications with systemic administration, LIPUS may provide local brain stimulation with minimal side effects for prevention and treatment of schizophrenia. However, MK-801-treated animal model only can mimic particular symptoms of schizophrenia and the mechanisms of schizophrenia remain unknown. Here, the effects of LIPUS may be only against MK-801 treatment but not to the real schizophrenia.

To conclude, pretreatment with LIPUS stimulation partially reversed MK-801-induced psychiatric symptoms and region-specific hypofunction of NMDA receptor subunits. Moreover, LIPUS may prevent MK-801-induced c-Fos expression in the mPFC and VTA. The excitatory/inhibitory imbalance in the mPFC and the downregulated DAT expression in the NAc in the MK-801-treated rats could be prevented by LIPUS.

Funding

This study was supported by grants from the National Science and Technology Council of Taiwan (no. NSTC 111-2218-E-A49-033-MY3 and NSTC 111-2314-B-A49-045-MY3), the FEMH-NYCU Joint Research Program (no. 111DN27 and 112DN27).

Conflicts of Interest

The authors declare that they have no competing interests.