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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. Acknowledgments
  9. References

Objective: Gastric electrical stimulation (GES) has been introduced for treating obesity. The hippocampus is known to be involved in the regulation of gastrointestinal motility. Changes in hypathalumus cholecystokinin (CCK) have been observed in genetically obese rodents. This experiment was to study the effect of GES on the activities of neurons and the expression of CCK in the hippocampus.

Methods and Procedures: We investigated the effect of GES (GES-I: pulse train of standard parameters; GES-2: reduced train-on time; GES-3: increased pulse width; GES-4: reduced pulse frequency) on neurons responsive to gastric distention (GD) by recording extracellular potentials of single neurons and observing the expression of CCK in the rodent hippocampus by immunohistochemistry staining, radioimmunoassay, and real-time PCR.

Results: 92.1% of neurons in the CA2-3 region responded to GD, 53.2% of which showed excitation (GD-E), and 46.8% showed inhibition (GD-I). 64.8% GD-responsive neurons were excited by GES. The response was associated with stimulation strength, pulse width, and frequency; 70.6, 57.1, 94.4, and 66.7% of GD-E and 72.7, 57.1, 86.4, and 50% of GD-I neurons showed excitatory responses to GES-I, −2, −3, and −4, respectively. CCK immunoreactive positive neurons (P < 0.001), the content of CCK-like materials (P < 0.05) and the amount of CCK mRNA were significantly increased after GES (P < 0.05).

Discussion: These findings suggest the central, neuronal, and hormonal mechanisms of GES. GES may excite the activity of GD-sensitive neurons and increase the expression of CCK in the hippocampus. These excitatory effects of GES seem to be related to the parameters of stimulation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. Acknowledgments
  9. References

Obesity is taking on pandemic proportions. Several neural, humoral, and psychological factors control the complex process known as appetite. Currently, there are no satisfactory treatment options available for obesity. Clinically, gastric electrical stimulation (GES) has been evaluated as a potential therapy for patients with morbid obesity. The available data seem to indicate that GES may benefit some obese patients (1,2,3,4). GES was reported to decrease gastric tone, reduce food consumption, and induce weight loss in animals (5,6,7,8,9). However, the effects of GES on weight loss are not consummate (10). None of previous studies have investigated the effects of GES on weight loss with different stimulation parameters and little is known about central neuronal activity and cholecystokinin (CCK) mechanisms involved with GES. Recently, a close evolutionary relationship between the gut and the brain has become apparent. The gut hormones regulate important gastrointestinal functions such as motility, secretion, and absorption, and provide feedback to the central nervous system on the availability of nutrients, and may play a role in regulating food intake.

The brain plays a pivotal role among all organs concerning energy metabolism. It is the central organ regulating energy supply and is able to receive information from peripheral organs via peripheral (e.g., hepatic) sensors and their afferent neuronal pathways. It also controls the functions of many peripheral organs, such as the skeletal musculature, the heart, the gastrointestinal tract, or the sexual organs, via its efferent nerve pathways. In the central nervous system, the nuclei or areas involved in energy metabolism, such as the cerebral cortex, the limbic system (hippocampus, septum, hypothalamus, amygdale, and so on), and the medulla oblongata, form a neural network that plays essential roles in the processing of visceroceptive information and the control of food intake and satiety (11). The hippocampus, an element of the neural network, is an integrative relay station of the limbic system, and is involved not only in the cognitive functions but also in the control of energy balance (12). It is known that CCK is present in hippocampal neurons (13,14) and can activate central mechanisms controlling food intake and digestion via the hippocampus (15,16,17,18).

The aims of this study were (i) to investigate the effects of GES on neurons in the hippocampal CA2-3 region responsive to gastric distention (GD), (ii) to compare the responses of these neurons to GES of various stimulation parameters, and (iii) to explore whether GES-induced neuronal activation is concomitant with changes in the expression of CCK protein and CCK mRNA in the hippocampus.

Methods and procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. Acknowledgments
  9. References

Preparation of animals

Sixty-one male Wistar rats (Qingdao Institution of Marine Pharmacology, Qingdao, China), weighing 250–300 g, were used. The animals were housed in a temperature-controlled room (22 ± 2 °C) with lights on only from 8:00 am to 8:00 pm. Standard laboratory chow pellets and tap water were available ad libitum. All animal experiments were approved and carried out according to the guidelines for animal experimentation of the Institutional Animal Care and Use Committee at Qingdao University.

Surgical procedures

The rats, fasted for 20 h, were anesthetized with urethane (1 g/kg, IP) and the maintenance anesthetic was given whenever necessary. Anesthesia was confirmed by the absence of paw pinch reflex in response to punching at legs. The rat heart was continuously monitored. The trachea was cannulated to maintain an open airway. The core temperature was monitored throughout the surgical procedure using a rectal thermometer, and maintained at ∼37 °C by the manual adjustment of a heating pad. The surgical procedure was performed under the aseptic condition in the following order. After midline laparotomy, gastric contents were removed through a small incision in the fundus. A small latex balloon attached to a polyethylene tube was inserted into the gastric cavity through the incision and fixed on the edge of the incision by a ligature. The balloon had a greater volume than that of the stomach and offered no resistance to inflation when the stomach was distended. One pair of platinum electrodes (0.3 cm apart) for delivering GES was sutured onto the serosal surface in the middle of the lesser curvature of the stomach. The abdomen was then closed and a small piece of gauze was used as drainage to prevent the accumulation of secretory fluids in the abdomen.

After the abdominal surgery, the rat was positioned on a stereotaxic frame (Narashige SN-3, Tokyo, Japan) and the dorsal surface of the brain was exposed. A glass microelectrode filled with 0.5 M sodium acetate and 2% pontamine sky blue (tip 1–1.5 μm, resistance 5–20 MΩ) was advanced in 10 μm steps to the hippocampal CA2-3 region (bregma: P: 2.30–3.14 mm, L (R): 1.7–2.2 mm, H: 3.6–3.8 mm) (19). The open part of the brain was covered by 3% agar in saline to limit any displacement due to respiration or heartbeats.

Electrophysiological recordings

Once the microelectrode was advanced into the CA2-3 region of hippocampus, extracellular action potentials of single neurons were recorded (another electrode was placed on the epicranium of the rat), amplified using a high-input impedance amplifier (MEZ8201; Nihon Kohden, Tokyo, Japan), displayed on an oscilloscope (VC-11; Nihon Kohden, Tokyo, Japan), and digitized and stored in a computer for further analyses.

After the firing pattern of the neuron became stable, the neuron was tested with GD to determine whether it was responsive to GD. GD was performed by inflating the gastric balloon with 3–5 ml 37 °C water at a rate of 0.5 ml/s and maintained for 20 s. If the neuron was responsive to GD, it was further classified into GD excitatory (GD-E) or GD inhibitory (GD-I) if its action potentials were increased or decreased in frequency by at least 20%. Once a neuron was identified to be responsive to GD, it was tested for its responses to GES with different parameters. For each identified and classified neuron, GES with four sets of parameters was applied for 1 min in a randomized order: GES-1 (pulse train of standard parameters used for treating obesity: train on-time of 2 s and off-time of 3 s, pulse width of 0.3 ms, frequency of 40 Hz and amplitude of 6 mA), GES-2 (reduced train on-time of 0.1 s and increased train off-time of 4.9 s), GES-3 (increased pulse width of 3 ms), and GES-4 (reduced pulse frequency of 20 Hz). Sufficient time was given for the neuron to recover in between two consecutive applications of GES.

At the completion of the experiment, the recording site was marked by delivering a direct current (10 μA for 20 min) via the electrode followed by forming an iron deposit of pontamine sky blue for the histological validation. The animal was killed with an overdose of pentobarbital. The brain was removed and placed in a 10% buffered formalin solution. Frozen sections (50 μm) of the brain were examined to locate the lesion sites where the neuronal recordings were made. Locations were drawn on cross-sections from the cytoarchitectonic scheme of Paxinos and Watson (19).

Measurement of CCK with immunohistochemistry staining assay

The hormonal study was performed in 10 rats. In the fasting state, GES was performed for 2 h continuously with stimulation parameter set 3 (pulse trains of increased pulse width: 6 mA, 3 ms, 40 Hz, 2 s-on and 3 s-off) in five of the rats. In the other five rats, sham-GES was performed for 2 h. At the end of GES or sham-GES, the animals were deeply anesthetized with 10% chloral hydrate (0.35 ml/kg) and perfused transcardially with 100 ml saline followed by 400 ml 4% paraformaldehyde solution in 0.1 mol/l phosphate buffer for 2 h. The brains were removed and postfixed in 4% paraformaldehyde for 2 h, then transferred to 30% sucrose overnight at 4 °C. A series of 20 μm coronal brain sections were cut on a freezing microtome and examined under a microscope according to the rat brain atlas (bregma: −3.3 to −3.8 mm). For each brain, five sections were taken at an interval of at least 40 μm.

The sections were washed in a 0.1 M phosphate-buffered solution, and nonspecific binding sites were blocked by incubating in a 0.1 M phosphate-buffered solution with 0.5% Triton X-100 and 4% goat serum at 4 °C for 2 h followed by incubation with a primary anti-CCK antibody (Polyclonal, dilution: 1:200; Chemicon International, Temecula, CA) at 4 °C for ∼40 h. After incubation with a fluorochrome-labeled secondary antibody (FITC-conjugated goat anti-rabbit IgG, dilution: 1:100; Jackson Immunoresearch) for 2 h at room temperature, the sections were washed using a 0.1 M phosphate-buffered solution and mounted with Citifluor (Citifluor, London, UK). All fluorophores were visualized and photographs were taken under a Leica DMRB/Bio-Rad MRC 1024 krypton–argon laser scanning confocal microscope (Olympus, Tokyo, Japan). Negative controls included incubation with normal goat serum instead of the primary antibody, or the omission of the secondary antibody.

CCK radioimmunoassay

The CCK radioimmunoassay (RIA) study was performed in 20 rats. In the fasting state, GES was performed for 2 h continuously with stimulation parameter set 3 (pulse trains of increased pulse width: 6 mA, 3 ms, 40 Hz, 2 s-on and 3 s-off) in 10 of the rats. In the other 10 rats, sham-GES was performed for 2 h. At the end of GES or sham-GES, the animals were deeply anesthetized with 10% chloral hydrate (0.35 ml/kg). The brains were removed and boiled in 0.9% NaCl for 5 min. Water was absorbed through filter paper. The hippocampuses were isolated according to the rat brain atlas (19). The tissues were weighed by an analytical balance and homogenized in 1 mol/l ice-cold hydrochloric acid. After incubation the suspension for 120 min at room temperature, the same volume of 1 mol/l sodium hydroxide was added and centrifuged at 4,000 revolutions per minute (rpm) at 4 °C for 20 min. The supernatants were stored at −70 °C until assayed.

CCK levels in the hippocampus tissues were measured by RIA using a CCK RIA commercial kit (Phoenix Pharmaceuticals, Beijing, China) according to the manufacturer's protocol. The lowest detectable concentration was 0.1 pmol/l. The intra-assay and inter-assay shifts were 4.4–11.5 and 4.2–20.6 pmol/l, respectively. In brief, samples or standards diluted in the a perfusion medium (0.02 mol/l barbital buffer, pH 8.4, containing 1 g/l bovine serum albumin) were incubated with CCK antiserum in tubes for 24 h at 4 °C (20). After the addition of 125I-CCK (around 1.8 × 104 counts per minute, cpm), all samples were further incubated for 24 h at 4 °C to reach equilibrium. Antibody-bound and free tracers were separated by adding 0.5 ml of a suspension of 20 mg of activated charcoal. The samples were incubated in room temperature for 45 min and then centrifuged at 4,000 rpm at 4 °C for 20 min; the supernatant and sedimented charcoal were counted in automatic g-scintillation counters for 5 min. The precipitated cpm values were detected using FJ-2008 γ auto-events-per-unit-time meter (Nuclear Apparatus, Xian, China).

Assay of CCK mRNA in the rat hippocampus by real-time PCR

The assay of CCK mRNA was performed in 16 rats. Eight of the rats were treated with GES for 2 h continuously with stimulation parameter set 3. In the other 8 rats, sham-GES was performed for 2 h. At the end of GES or sham-GES, the animals were deeply anesthetized with 10% chloral hydrate (0.35 ml/kg). The brains were removed, and the hippocampuses were isolated in the same manner as mentioned above and immediately frozen in liquid nitrogen, and stored at −80 °C. The frozen specimens were homogenized with a polytron homogenizer and total RNA was extracted using the TRIzol Reagent method (GIBCO, NY). The first strand cDNA was synthesized from 1 μg total RNA using 200 units Superscript II RNAse H– Reverse Transcriptase (GIBCO, Gland Island, NY) and the oligo-dT16 anchor prime from the Rapid Amplification of cDNA Ends kit. The reaction mixture was first incubated at 42 °C for 1 h, followed by incubation at 95 °C for 10 min. Real-time quantitative PCR was performed on a GeneAmp 5700 Sequence detection system (Applied Biosystems, Warrington, UK) using SYBR Green I as double-stranded DNA-specific binding dye for continuous fluorescence monitoring. Amplification was carried out in a total volume of 25 μl containing 2× PCR Master Mix (Applied Biosystems, Warrington, UK), 2 μl of 1:4 diluted cDNA and 5 μmol/l of each specific primer. Primer sequences for CCK were 5′-AGC CGG TAG TCC CTG TAG AA-3′ (sense), 5′-GTC CCG GTC ACT TAT CCT GT-3′ (antisense) (227 bp); for housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 5′-CGG CAA GTT CAA CGG CAC AG −3′ (sense), 5′-ACT CCA CGA CAT ACT CAG CAC-3′ (antisense) (134 bp). The PCRs were cycled 40 times through denaturation (95 °C, 15 s) and annealing (60 °C, 1 min). Data was analyzed by Gene Amp 5700 SDS software (Applied Biosystems, Warrington, UK). Relative quantification was performed by calculating the difference of the threshold cycle (ΔCt = CtCCKCtGAPDH) of CCK and GAPDH in the GES or sham group (21).

Data analysis

Activity of a neuron was presented as the number of impulses per second which was counted and averaged over a period of 10 s. The neuronal response was defined as the difference in neuronal activity between the baseline spontaneous activity and the activity during GD or GES. An increase or decrease of ≥20% from the baseline spontaneous activity was considered an excitatory or inhibitory response to GD or GES. The raw tracing of neuronal responses to gastric stimuli was processed by a Spike 3 digital filter to eliminate GES artifacts.

In the immunohistochemistry study, CCK immunoreactive (CCK-IR) positive cells in the CA2-3 area of the hippocampus were counted using a laser confocal microscope and an image analysis system (Jiangsu JEDA Science and Technology Development, Nanjing, China). Only positive cells with sharp focus were counted. The numbers of CCK-IR positive cells were presented as cells per mm2.

Statistical analysis

All data are presented as mean ± s.e.m. ANOVA was applied to study the difference among four sets of stimulation parameters. Paired Student's t-test was used to study the difference between any paired data. χ2 Analysis was applied to investigate the difference in the neuronal response patterns (excitatory or inhibitory) between any pairs of the four parameters sets. P < 0.05 was considered as statistically significant. Student's t-test was used to compare the difference in the number of neurons IR to CCK, the content of CCK-like material (CCK-LM) and the amount of CCK mRNA between the stimulated rats and the control rats.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. Acknowledgments
  9. References

Neuronal responses to GES

Spontaneous unit discharges of 67 neurons in the CA2-3 region of the hippocampus were recorded from 15 rats, and classified as three different patterns: single, phasic, and continuous with 9 (9/67, 13.4%), 18 (18/67, 26.9%), and 40 (40/67, 59.7%) neurons in each class, respectively (Figure 1). Out of the 67 neurons, 62 (92.1%) responded to gastric distension with 53.2% classified as GD-E neurons (Figure 2a) and 46.8% classified as GD-I neurons (Figure 2b). Compared with the baseline, the mean firing frequency of GD-E neurons was increased by 70.1 ± 10.8% (P < 0.01) and the mean firing frequency of GD-I neurons was decreased by 57.9 ± 8.3% (P < 0.01).

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Figure 1. : Different patterns of spontaneous unit discharge of neurons in the hippocampus: single, phasic, and continuous.

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Figure 2. : Hippocampal neuronal activity with gastric distention (GD). (a) GD-E neuron increased firing frequency with GD; (b) GD-I neuron reduced firing frequency with GD. GD-E, GD excitatory; GD-I, GD inhibitory.

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Most of GD-responsive neurons (64.8%) were activated by GES and the response seemed associated with stimulation strength. GES-1, −2, −3, and −4 changed activity of 79.6, 41.2, 90.9, and 54.5% of GD-responsive neurons in the hippocampal CA2-3 region, respectively (Table 1). Neuronal activity in the hippocampus was affected more frequently with GES-3 (increased pulse width) (40/44) than GES-2 (14/34) (P < 0.01) or GES-4 (12/22) (P < 0.01). GES-1 (39/49, 79.6%) also showed more significant effect on GD-responsive neurons than GES-2 (P < 0.01) or GES-4 (P < 0.01). Although no significant difference was noted between GES-3 and GES-1, as shown in Figure 3, GES-3 was of the highest efficacy in activating the GD-responsive neurons.

Table 1. . Effect of GES with various parameters on activity of hippocampus neurons responding to GD
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Figure 3. : Effects of gastric electrical stimulation (GES) on all gastric distention–responsive neurons in the CA2-3 region of hippocampus. χ2 Analysis, compare with GES-2, *P < 0.01; χ2 analysis, compare with GES-4, **P < 0.01. NR, no response to GES; R, response to GES.

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Most of the neurons were excited instead of inhibited by GES (Table 1). Figure 4 shows excitatory responses of a GD-E neuron to GES with different parameters. In GD-E neurons, 70.6, 57.1, 94.4, and 66.7% showed excitatory responses to GES-1, −2, −3, and −4, respectively (Figure 5). More GD-E neurons were excited by GES-3 than GES-1 (P < 0.05), GES-2 (P < 0.001) or GES-4 (P < 0.001). The responses of the GD-I neurons to GES with different parameters were shown in Figure 6. In the GD-I neurons, the percentages of neurons excited by GES were 72.7, 57.1, 86.4, and 50%, respectively with GES-1, −2, −3, and −4, (Figure 7). The percent of response was higher with GES-3 than GES-1 (P < 0.05), GES-2 (P < 0.001), or GES-4 (P < 0.001). GES-1 had more excitatory effect than GES-2 (P < 0.01) or GES-4 (P < 0.05).

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Figure 4. : Response of GD excitatory (GD-E) neurons to gastric electrical stimulation (GES) with different parameters. (a–d) show increased firing frequency of GD-E neurons to GES-1, −2, −3, and −4, respectively, and GES-3 had more obviously excitatory effects. GD, gastric distention.

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Figure 5. : Excitatory and inhibitory responses of GD-E neurons to gastric electrical stimulation (GES). χ2 Analysis, compare with GES-1, *P < 0.05; χ2 analysis, compare with GES-2, **P < 0.001; χ2 analysis, compare with GES-4, ***P < 0.001. E, excitatory response; GD, gastric distention; I, inhibitory response.

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Figure 6. : Response of GD inhibitory (GD-I) neurons to gastric electrical stimulation (GES) with different parameters. (a) The firing frequency of GD-I neuron to GES-I shows increased. (b) GES-2 had no significant change for firing frequency on GD-I neurons. (d) The increased firing frequency of GD-I neuron shows more obviously to GES-3. (d) GES-4 had no significantly change for firing frequency on GD-I neurons. GD, gastric distention.

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Figure 7. : Excitatory and inhibitory responses of GD-I neurons to gastric electrical stimulation (GES). χ2 Analysis, compare with GES-2, *P < 0.001; χ2 analysis, compare with GES-4, **P < 0.001; χ2 analysis, compare with GES-1, ***P < 0.05; χ2 analysis, compare with GES-4, †P < 0.05; χ2 analysis, compare with GES-2, ††P < 0.01. E, excitatory response; GD, gastric distention; I, inhibitory response.

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Expression of CCK-IR positive neurons in rodent hippocampus

Figure 8 shows immunohistochemical localization of CCK-IR positive neurons in the hippocampus. The expression of CCK-IR was clearly seen in the cytoplasm of cells. The CCK-IR positive cells were mainly located in the pyramidal cell layer of the hippocampus. Less CCK-IR cells were observed in the stratum radiatum of the hippocampus. Increased staining of CCK-IR positive neurons was found with GES in the CA2-3 region (5.29 ± 0.50 cells/mm2 vs. 2.31 ± 0.42 cells/mm2; P < 0.001) (Figure 8b) after GES (para-3) compared to the control group (Figure 8a).

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Figure 8. : The expression of cholecystokinin immunoreactive (CCK-IR) neurons in rat hippocampus. (a) Immunohistochemical localization of CCK-IR neurons in rat hippocampus. The CCK-IR neurons were mainly located in the pyramidal cell layer of hippocampus. Less CCK-IR positive neurons were observed in the stratum radiatum of hippocampus. (b,c) show the expression of CCK-IR neurons in the CA2-3 regions. The numbers of CCK-stained cells significantly increased in CA2-3 region after gastric electrical stimulation (para-3) for 2 h (b) comparing with sham-operated group (a). Bars = 200 μm (a); 100 μm (b,c).

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Contents of CCK like-material and CCK precursor mRNA in rodent hippocampus

In order to confirm the expression change of CCK in the rat hippocampus by GES, the contents of the CCK like-material (CCK-LM) by RIA and CCK mRNA by real-time PCR were performed after 2-h GES-3 (increased pulse width). The CCK-LM was significantly increased in the hippocampus from 8.48 ± 1.34 to 14.59 ± 1.85 pg/mg tissue (P < 0.05); Similarly, the expression of CCK mRNA was significantly increased after GES-3 (ΔCt 6.81 ± 0.35 vs. ΔCt 4.15 ± 0.29, P < 0.05). Figure 9 shows analysis of real-time PCR products on agarose gel electrophoresis. The 227-bp band stands for CCK and the 134-bp for housekeeping GAPDH.

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Figure 9. : The expression of cholecystokinin (CCK) mRNA in rat hippocampus. Analysis of real-time PCR products on agarose gel electrophoresis. The 227-bp band corresponds to CCK and the 134-bp for housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The expression of CCK mRNA significantly increased (tract 2) after gastric electrical stimulation (para-3) for 2 h comparing with sham-operated group (tract 1).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. Acknowledgments
  9. References

As food is ingested, its constituents evoke a cascade of responses of sensory cells that line the alimentary canal. Resulting signals are carried by vagal afferents to the nucleus tractus solitarius (NTS) in the brain stem (22). From there, this visceral information is disseminated to various other brain sites, where it influences regulatory functions by engaging endocrine, autonomic, and behavioral effect mechanisms. The hippocampus is an integrative relay station of the limbic system that is also called “visceral brain.” It is reciprocally connected with the amygdala, the hypothalamus, and the medulla oblongata (23,24), thereby fulfilling essential roles in the processing of visceroceptive information and the control of food intake and satiety. Previous studies showed that ingestive behavior patterns in rats change following hippocampal lesion. It was reported that the lesioned rats consumed smaller meals and there was a similar change in drinking. In addition, the hippocampal lesioned rats alternated more frequently between feeding and drinking during a single bout of ingestive behavior (25). In addition to the hippocampus, the amygdala may also contribute to the food intake behavioral changes observed following traumatic brain injury (26), whereas lesions placed in the caudodorsal amygdala and ventral hippocampal formation induce obesity (27). The important role of the hippocampus in digestion is also revealed by the fact that food storing passerines have a larger hippocampus than that of nonstoring songbird species (11).

To show a possible visceral input to the hippocampus, we chosen GD as a stimulus because GD activates a relatively homogeneous and specific class of vagal afferent and efferent signals associated with food ingestion. It was demonstrated previously that neurons in various parts of the brain (lateral hypothalamic area (28), olfactory bulb (29), dorsal vagal complex (30), paraventricular nucleus (31), supraoptic nucleus (32), and parabrachial nucleus (33)) responded to mechanical stimulation of the gastrointestinal tract. This study showed that neurons in the CA2-3 region of the hippocampus were sensitive to GD and most of CA2-3 region neurons (92.1%) responded to GD. This finding strongly indicates that peripheral somatosensory afferent inputs and the inputs from gastric mechanoreceptors of the stomach ascend to the hippocampus and influence the electrical activities of CA2-3 neurons via an inferior nucleus relay pathway. These CA2-3 neurons possibly play a strong and integrative role in the eventual hippocampal response to these stimuli.

Obesity is a major public health problem in western societies including the United States. This condition affects approximately one-fifth of the US population (34) and is considered to be a contributing factor in 280,000 annual deaths in the United States (35,36), with an estimated cost of >$100 billion a year (37,38). The management of this condition remains difficult. Medical therapy results in limited weight loss in most patients, and the best success is achieved through bariatric surgery (39). However, each of the various types of bariatric surgery is associated with side effects and complications (39,40,41), hence, there is continued search for alternative treatment modalities.

GES is currently being investigated as an alternative for treating obesity. The rationale is based on the observations that GES with appropriate parameters disrupts intrinsic gastric electrical activity, delays gastric emptying, suppresses antral contractions, induces gastric dysrhythmia (41,42,43,44), and results in weight loss (1,3). However, the central mechanisms by which GES affects food intake are unclear, especially the effect of GES on superior central neuronal activity has not been examined previously. This study was carried out to explore the potential central mechanism of GES with four different parameters in the management of obesity. The major finding of this study is that GES with different parameters modulated activity of 41.2–90.9% of hippocampus neurons responsive to GD; the primary effect of GES on hippocampus neurons with gastric input was excitatory; the neuronal responses to GES was enhanced with stimulation at an increased pulse width. These results suggest that hippocampus neurons might play a role in processing afferent information from the stomach under the condition of GES. This is the first study to document the involvement of the hippocampus mechanism by which GES might affect gastric sensory and motor functions.

On the basis of the pulse width, GES can be classified into three categories: long-pulse, short-pulse, and train of short pulses (42). For the treatment of obesity, GES is performed using pulse trains. In this study, we used four sets of stimulation parameters: “standard” pulse trains (GES-1), pulse trains with a decreased train on-time (GES-2), increased pulse width (GES-3), and decreased frequency (GES-4). All four sets of GES parameters elicited an excitatory response in most of hippocampus neurons with gastric input. In addition, GES with pulse trains of increased pulse width (GES-3) was very effective in soliciting neuronal responses in the hippocampus. GES with a decreased train on-time (GES-2) and a decrease frequency (GES-4) may degrade the excitatory response to GES. These results suggest that the frequency and width of the stimulation pulse play an important role in the regulation of neuronal activity and have to be considered in the application of GES for various therapies.

It is well-known that various so-called gut–brain peptides, including CCK, exist in the hippocampus. CCK is known as a “satiety factor” that inhibits appetite, food intake, and gastric motility (7,14,45). We previously showed that CCK-8 injected into the amygdale (46) or into the hippocampus (47) inhibits gastric motility and that this effect is abolished by vagotomy. ICV administration of the CCK-A receptor antagonist devazepide increases food intake in rats (18). GD enhances CCK-8 mRNA expression in the hippocampus (48,49). It was also shown that exogenous CCK-8 in the lateral hypothalamic area decreased the firing frequency of GD-responsive neurons in the dorsal vagal complex (50). Moreover, Cigaina et al. found that GES affected regulatory peptide secretion, with evidence of GES output-related increased postprandial CCK release in obese patients (51). This is meaningful because CCK is considered to play an important role in satiety signals and meal termination (52). The present data revealed that GES increased the expression of CCK-IR, CCK-LM, and CCK mRNA in the hippocampal neurons. These findings seem to suggest the involvement of CCK in the hippocampus in the treatment of obesity with GES. We therefore hypothesize that CCK-containing neurons in the hippocampus may participate in the integration of satiety and feeding behavior within the neuronal circuit composed of nucleus of brain stem (NTS; dorsal nucleus of vagus)-hypothalamus (paraventricular nucleus; ventromedial hypothalamic)-limbic (hippocampus, amygdala) system acting on afferent gastric related signals arrived in the hippocampus and this would be an important area for future consideration.

In summary, GES, especially GES with an increased pulse width affected both neuronal and hormonal pathways in the central nervous system. GES had excitatory effects on hippocampus neurons receiving input from the stomach. The central neuronal response to GES is enhanced with stimulation at an increased pulse width, train on-time, and frequency. This modulatory effect of GES on the central neurons provides a theoretical support for GES in treating obesity. Moreover, the findings of this study demonstrate the importance of appropriate selection of stimulation parameters for GES. Before GES is applied in the long-term therapy of obesity, further research may be required to determine the optimal stimulation parameters and configurations.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. Acknowledgments
  9. References

This study was supported by a grant from the Oklahoma Center for the Advancement of Science and Technology (HR 02-034R, to J.D.Z.C.) and the National Natural Science Foundation of China (nos. 30470642 and 30670780, L.X.) and Qingdao Science and Technique Bureau (05-1-JC-93 L.X.).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. Acknowledgments
  9. References
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