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Keywords:

  • neurogenic bladder;
  • basal forebrain;
  • cystometry;
  • rat

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Despite the significant clinical problems of bladder dysfunction, specifically affecting the individual's quality of life, our understanding of this disease is limited. The main problem relates to the lack of clinically relevant models. The paper by Shimizu et al. describes the development of a neurogenic bladder model in the rat by an electrolytic unilateral lesion of the right basal forebrain. This model mimics the clinical situation of increased voiding frequency and decreased micturition threshold pressure. The group also uses this model to test the effects of AH-9700, a potent sigma receptor-agonist, with some success. The overall develop of clinically relevant models has important implications in future drug development and testing.

OBJECTIVE

To investigate the changes in bladder function in rats with an electrolytic lesion of the right basal forebrain (RBF) and to determine the effects of AH-9700, a novel sigma receptor ligand, on cystometry in RBF-lesioned rats.

MATERIALS AND METHODS

A lesion was made electrolytically in the RBF of male Wistar rats. At 7 or 8 days after the lesion or sham surgery, continuous cystometry was performed in awake rats. In addition, contractile responses to electrical field stimulation or carbachol were measured in isolated bladder strips, as were the forebrain contents of acetylcholine, monoamine neurotransmitters and their metabolites.

RESULTS

RBF-lesioned rats showed a remarkable increase in voiding frequency, with a decrease in voiding threshold pressure but no change in voiding pressure, compared with sham-operated rats. However, contractile responses in bladder strips isolated from RBF-lesioned rats were no different from those in strips isolated from sham-operated rats. In RBF-lesioned rats, the contents of acetylcholine, dopamine, 4-dihidroxyphenylacetic acid and homovanillic acid were significantly decreased in the right forebrain. AH-9700 dose-dependently decreased the voiding frequency and increased the threshold pressure in RBF-lesioned rats. Anti-muscarinic agents (oxybutynin and propiverine) also decreased the voiding frequency, but their effects were less potent than that of AH-9700.

CONCLUSIONS

The RBF-lesioned rat may be a useful model for the neurogenic bladder of supraspinal origin. Moreover, AH-9700 effectively improves bladder dysfunction in this model.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

More patients are presenting with urge incontinence and urinary frequency attributed to a neurogenic or unstable bladder. While this condition is not lethal it can seriously compromise the quality of life. However, despite its importance, the pathophysiological basis of the neurogenic bladder remains poorly understood.

Among the major causes of neurogenic bladder are brain insults, e.g. Alzheimer's disease and apoplectic stroke, that interfere with the central regulation of urination [1,2]. Alzheimer's disease may be attributable to a decline in the cholinergic system of basal forebrain (BF) [3–5], which projects to the cerebral cortex and is thought to participate in learning and memory [6,7]. This ‘cholinergic hypothesis’ has stimulated many studies focused on developing animal models of dementia, based on cholinergic blockage or lesion of the BF cholinergic system [8]. There is also evidence that the BF is inhibitory in the central regulation of voiding [9–11]. However, urodynamics in animals with BF lesions have rarely been investigated. In particular, there has been no report of bladder function in rats with a unilateral lesion of the BF, a model distinct from the general dementia model produced by bilateral electrolytic or neurotoxic lesioning of the BF [8].

All existing drugs for treating urge incontinence and frequency are characterized by their common ability to relax the detrusor smooth muscle, based on their antimuscarinic and/or calcium-antagonistic properties. However, the effects of these drugs are not necessarily adequate and their pharmacological properties frequently cause systemic adverse effects such as dry mouth, blurred vision and hypotension, which restrict their clinical use [12]. Accordingly, drugs with a different mode of action are needed.

AH-9700 (1-[2-(3,4-dihydro-6,7-dimethyl-2-naphthalenyl)ethyl]pyrrolidine fumarate) is a potent σ-receptor agonist with moderate antimuscarinic activity [13,14]. AH-9700 has a marked anti-micturition reflex effect, mediated presumably through central σ-receptors, and a moderate spasmolytic effect based on its peripheral antimuscarinic activity [14,15].

The purpose of the present study was to develop a neurogenic bladder model of urinary frequency by electrolytic unilateral lesion of right BF (RBF) in rats, and to determine the effects of AH-9700 on micturition in this model.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

All experiments were carried out in accordance with the Guiding Principles for the Care and Use of Laboratory Animals provided by the Japanese Pharmacological Society. Male Wistar rats (Japan SLC Inc., Shizuoka, Japan), weighing 280–320 g at the start of experiments, were used. They were housed in a room kept at 22–24 °C under a 12-h light-dark cycle (lit from 06.00 to 18.00 hours) with free access to food and water.

The electrolytic lesion of the RBF was administered using the method of Miyamoto et al.[16] with some modifications. Rats were anaesthetized with sodium pentobarbital (45 mg/kg, intraperitoneal) and fixed on a stereotaxic apparatus. The uninsulated tip (1 mm) of a stainless-steel electrode (0.5 mm in diameter) was inserted into the RBF (1.3 mm posterior to the bregma, 2.7 mm right lateral to the midline, 7.0 mm below the surface of the dura mater) according to a brain atlas [17]. The electrolytic lesion was caused by passing an anodal direct current (2 mA for 20 s) using a lesion-producing device (Stoelting Co., Wood Dale, IL, USA). The circuit was completed by attaching a cathode to the wound edge. Sham-operated rats underwent similar surgical procedures with insertion of the electrode, but with no delivery of current. Afterward, in some cases, one end of a polyethylene cannula (PE 60) was implanted into the stomach for drug administration, and the other end passed through the subcutaneous tissue, exiting through the skin at the back of the neck. The following experiments were conducted 7 or 8 days after these procedures.

CYSTOMETROGRAPHY

Under halothane anaesthesia, the abdomen of RBF-lesioned or sham-operated rats was opened. A PE-60 cannula was inserted through a small incision at the apex of the bladder into the bladder lumen and ligated. After the abdomen was sutured, the rat was placed in a Bollman's cage and allowed to recover from anaesthesia. The bladder cannula was connected to a pressure transducer (Nihon Kohden, TP-400T, Tokyo, Japan) and an infusion pump via a polyethylene T-tube. Room-temperature saline was infused into the bladder at 5 mL/h and the intravesical pressure recorded continuously. After the cystometrogram stabilized (about 2 h after starting the bladder infusion), drugs or vehicle (0.5% tragacanth solution) were administered at a volume of 2 mL/kg through the pre-implanted gastric cannula (hereafter referred to as oral). The following cystometric variables were measured: voiding frequency, voiding pressure (maximum bladder pressure during voiding) and voiding threshold pressure (bladder pressure immediately before a sharp bladder micturitional contraction). The voiding frequency and the means of voiding pressure and threshold pressure in each 30-min period for 150 min were determined from consecutive cystometrograms. Values during the first period (pre-treatment values) were taken as 100% of each variable and the percentage change calculated for every 30-min period.

FOREBRAIN CONTENT OF ACETYLCHOLINE, MONOAMINE NEUROTRANSMITTERS AND THEIR METABOLITES

On the seventh day after RBF lesioning, both groups of rats were killed by head-focused microwave irradiation (4.8 kW, 1.3 s; model TMW 6402 A, Toshiba Co., Ltd, Tokyo, Japan). The concentrations of dopamine, 3,4-dihidroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), noradrenaline, serotonin, 5-hydroxyindoleacetic acid (5-HIAA) and tyrosine in the forebrain were determined by HPLC with electrochemical detection, as described by Oka et al.[18]. Acetylcholine was separated on an analytical column (AC-GEL, Eicom Co., Kyoto, Japan) using 0.05 mol/L pyrophosphoric acid buffer solution (pH 8.2) and detected using a platinum electrode (WE-PT, Eicom).

IN VITRO BLADDER STRIP CONTRACTILITY

The bladder was removed from both groups of rats 7 days after RBF lesioning and longitudinally dissected in half. Hemispheric bladder strips were suspended longitudinally under a resting tone of 1 g in a 10-mL organ bath at 37 °C containing Krebs-Henselit solution (in mmol/L; 118 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2.5 CaCl2, 25 NaHCO3 and 10 glucose) oxygenated with a mixture of 95% O2 and 5% CO2. Contractions were recorded isometrically by a force-displacement transducer (Nihon Kohden, ST-1B, Tokyo, Japan). After the strips were allowed to stand for at least 30 min, the cumulative concentration-response curve to carbachol (3 × 10−7–10−4 mol/L) or frequency-response curve to electrical field stimulation (EFS, 0.5 ms duration, supramaximal voltage, square plus, 0.3–30 Hz) was determined in respective preparations. EFS was administered using two parallel wire platinum electrodes connected to an electrical stimulator (Nihon Kohden, SEN-1101).

DRUGS

AH-9700, propiverine hydrochloride and oxybutynin hydrochloride were synthesized in our laboratories. Carbamylcholine chloride (carbachol) was purchased from Sigma Chemical Co., St. Louis, MO, USA. AH-9700, propiverine and oxybutynin were suspended in 0.5% tragacanth solution; carbachol was dissolved in saline.

The unpaired Student's t-test was used to compare the two groups, with Dunnett's multiple range test for multiple comparisons. The dose causing 30% inhibition (ID30) of AH-9700 of the voiding frequency was determined by the logit method.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

ELECTROLYTIC LESION OF RBF AND ITS EFFECT IN CYSTOMETRY

Figure 1 and Table 1 show the consecutive cystometrograms and cystometric variables in conscious RBF-lesioned or sham-operated rats. After the cystometrogram stabilized (1.5–2 h after starting saline infusion), the voiding frequency in RBF-lesioned rats was greater than that in sham-operated rats (Table 1). The threshold pressure in RBF-lesioned rats was significantly lower than that in sham-operated rats (P < 0.001), but the voiding pressure in RBF-lesioned rats was similar (P = 0.4485). The greater frequency in RBF-lesioned rats continued for at least 2 h after 0.5% tragacanth solution was administered orally through an intragastric cannula (Fig. 2).

image

Figure 1. Representative traces of the cystometrograms in conscious sham-operated (A) and RBF-lesioned (B) rats. Closed circle, voiding.

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Table 1.  Effect of electrolytic lesion of the RBF on cystometry in conscious rats
Mean (sem) variableShamRBF-lesion
  • P  < 0.01;

  • P  < 0.001, significantly different from the corresponding value in sham-operated rats (unpaired Student's t-test).

No. of rats  6  7
Voiding
frequency, cycles/30 min  1.8 (0.2)  7.0 (1.2)
pressure, mmHg20.6 (1.1)18.2 (2.6)
threshold pressure, mmHg  8.2 (0.3)  4.9 (0.4)
image

Figure 2. The time course of changes in cystometric variables; A, voiding frequency; B, voiding pressure (triangles) and threshold pressure (squares) after oral administration of 0.5% tragacanth solution (2 mL/kg) in sham-operated (six, green open symbols) and RBF-lesioned rats (seven, red closed symbols). Each point represents the mean (sem). *P < 0.05, **P < 0.01, ***P < 0.001: significantly different from the corresponding value in sham-operated rats (unpaired Student's t-test).

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CONTRACTILE RESPONSES IN ISOLATED BLADDER DETRUSOR STRIPS

Carbachol (3 × 10−7−10−4 mol/L) and EFS (0.3–30 Hz) evoked concentration- and frequency-related contractions in isolated bladder detrusor strips, respectively. Neither contractile response was altered in RBF-lesioned rats (Fig. 3).

image

Figure 3. Contractile responses to EFS (A) and carbachol (B) in isolated bladder detrusor strips of sham-operated (green open circles) and RBF-lesioned rats (red closed circles). Each point represents the mean (sem) of five rats. There was no significant difference between the groups (unpaired Student's t-test).

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BRAIN CONTENT OF ACETYLCHOLINE, MONOAMINE AND THEIR METABOLITES

The content of acetylcholine, dopamine and its metabolites (DOPAC and HVA) in the right forebrain of RBF-lesioned rats were significantly lower than those in the RBF of sham-operated or the left brain (intact side) of RBF-lesioned rats, respectively. However, there was no significant difference in the contents of noradrenaline, serotonin, tyrosine and 5-HIAA between the groups (Table 2). Electrode insertion alone decreased the content of dopamine and DOPAC, and increased tyrosine in the right brain of sham-operated rats, but much less than that produced by electrolytic lesion (Table 2).

Table 2.  Changes in brain levels of acetylcholine, monoamine and their metabolites in sham-operated and RBF-lesioned rats
Mean (sem) metabolite (nmol/g wet wt)Right forebrainLeft forebrain (intact)
ShamlesionShamlesion
  • *

    P  < 0.01,

  • P  < 0.001: significantly different from the corresponding value in sham-operated rats (unpaired Student's t-test).

  • P  < 0.05,

  • P  < 0.01,

  • §

    P  < 0.001: significantly different from the corresponding value of the left forebrain in sham-operated or RBF-lesioned rats, respectively (unpaired Student's t-test). Values obtained from the left forebrains of sham-operated rats were not statistically different from the corresponding values in RBF-lesioned rats.

Acetylcholine19.78 (0.67)16.1 (0.41)*21.02 (0.65)19.74 (0.67)
Noradrenaline  3.36 (0.20)  3.47 (0.15)  3.06 (0.15)  3.09 (0.13)
Dopamine  9.79 (0.67)  3.50 (0.89)§13.32 (1.11)12.64 (0.24)
DOPAC  1.06 (0.04)  0.49 (0.08)§  1.20 (0.05)  0.95 (0.04)
HVA  0.85 (0.04)  0.54 (0.05)*  0.94 (0.04)  0.86 (0.04)
Tyrosine21.81 (0.55)22.05 (1.07)20.03 (0.24)19.97 (0.93)
Serotonin  5.45 (0.20)  5.19 (0.23)  5.45 (0.20)  5.45 (0.08)
5-HIAA  2.48 (0.21)  2.55 (0.14)  2.48 (0.09)  2.42 (0.08)

EFFECTS OF AH-9700, OXYBUTYNIN AND PROPIVERINE IN CONSCIOUS RBF-LESIONED RATS

The effects of AH-9700 and drugs administered orally on cystometrograms were examined using conscious RBF-lesioned rats. AH-9700 (5–20 mg/kg) decreased the voiding frequency and increased the threshold pressure in these animals, with no effect on voiding pressure (Fig. 4, Table 3). There was a statistically significant effect of AH-9700 on voiding frequency within 30 min of administration (10 or 20 mg/kg) that lasted for ≥ 120 min (Fig. 4). The ID30 (95% CI) of AH-9700 for voiding frequency was 9.2  (6.8–12.6) mg/kg. When administered orally, oxybutynin (2 mg/kg) and propiverine (5 mg/kg) also decreased the voiding frequency in RBF-lesioned rats (Fig. 5, Table 3). Propiverine and oxybutynin, which have a potent antimuscarinic activity, reduced the voiding pressure, although not significantly (Fig. 5, Table 3).

image

Figure 4. Effect of AH-9700 on cystometric variables; A, voiding frequency; B, voiding pressure; C, threshold pressure in conscious RBF-lesioned rats. Green open circle, vehicle; closed green triangle, 5 mg/kg; closed red circle, 10 mg/kg; closed red square, 20 mg/kg. Each point represents the mean (sem) of four to six experiments. P *< 0.05, **< 0.01, ***< 0.001, significantly different from the corresponding value of the group given vehicle (0.5% tragacanth solution), respectively (Dunnett's multiple range test).

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Table 3.  Effects of AH-9700, oxybutynin and propiverine on the voiding frequency, voiding pressure and threshold pressure in conscious RBF-lesioned rats; each maximum change was determined from the results shown in Figs 4 and 5.
GroupMean (sem) % maximum change from initial value
Voiding frequencyVoiding pressureThreshold pressure
  • *

    P  < 0.05,

  • P  < 0.01,

  • P  < 0.001, significantly different from the corresponding value of the group given vehicle only (Dunnett's multiple range test, AH-9700; unpaired Student's t-test, oxybutynin and propiverine).

Vehicle   −  0.2 (6.6)     4.7 (4.8) −  12.6 (6.4)
AH-9700, mg/kg
5 −  12.4 (8.2)   11.7 (5.9)    10.6 (11.7)
10 −  37.8 (2.3)   −  0.7 (8.9)    27.7 (10.5)
20 −  45.5 (4.5)   −  0.5 (11.6)    63.4 (27.2)*
Oxybutynin, 2 −  19.7 (5.6) −  15.1 (12.0)    15.8 (5.1)*
Propiverine, 5 −  24.2 (6.8)*   −  6.8 (13.5)    13.0 (12.8)
image

Figure 5. Effects of oxybutynin (green closed circle) and propiverine (red square) on A, voiding frequency; B, voiding pressure; and C, threshold pressure in conscious RBF-lesioned rats. Green open circle, vehicle. Each point represents the mean (sem) of four to six experiments. *P < 0.05, significantly different from the corresponding value of the group given vehicle (unpaired Student's t-test).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

The present study shows that a unilateral electrolytic BF lesion in rats produces deficits in rat bladder function; cystometry in conscious RBF-lesioned rats showed markedly higher voiding frequency and lower voiding threshold pressure than in sham-operated rats. However, contractile responses to EFS or carbachol in bladder strips isolated from RBF-lesioned rats were no different from those in the strips from sham-operated rats. Moreover, there were no histopathological changes in the bladder tissue of RBF-lesioned rats compared with the control (data not shown). These results suggest that the frequent urination in RBF-lesioned rats may be caused by abnormalities in the central regulation of micturition, rather than by changes in the bladder detrusor itself. Furthermore, the BF has been considered to be an overall inhibitory area in the central regulation of micturition [9–11]. Therefore, it is likely that the bladder dysfunction in RBF-lesioned rats is caused by lack of inhibitory control in the micturition reflex, and not an acceleration of the evocation system.

Electrolytic or neurotoxic (kainic or ibotenic acid treatment) lesioning of both BF sites has been extensively used to cause dysfunction of cholinergic neurones which project to the cortical area and are important in learning and memory [6,7]. Rats with bilateral lesions of the BF are used as a model of dementia in the elderly or in Alzheimer's disease [4,6,8]. Electrolytic lesioning of the BF decreased both dopamine and acetylcholine content in the forebrain, whereas neurotoxic lesions by kainic or ibotenic acid decreased only acetylcholine content [19–21]. The present study shows that the contents of acetylcholine, dopamine, DOPAC and HVA were decreased in the right forebrain of RBF-lesioned rats. This result indicates that dysfunction of cholinergic or dopaminergic neurones, or both, might cause frequent urination. It has been well documented that Parkinsonian patients with dysfunctions of dopaminergic neurones, which originate in the substantia nigra pars compacta and project to neostriatal neurones in the basal ganglia, produce urinary frequency and urgency [22,23]. These observations suggest that dysfunctions of dopaminergic neurones on the lesion side of RBF-lesioned rats may contribute to the frequent urination observed in this model. However, others [24,25] reported that rats subjected to neurotoxic (ibotenic acid treatment) lesions of both BF sites showed small bladder volume capacity (i.e. frequent urination), despite dopaminergic neurones in this model being unlikely to be affected. Moreover, Yokoyama et al.[25] showed that i.c.v. oxotremorine M, a muscarinic receptor agonist, increased bladder capacity in bilateral neurotoxic BF-lesioned rats, but not sham-operated rats, suggesting that changes in forebrain inhibitory mechanisms, which are related to the muscarinic receptor(s) activation in the micturition reflex, cause the frequent urination. Given these data it is reasonable to assume that the frequent urination observed in RBF-lesioned rats is caused by dysfunctions of both cholinergic and dopaminergic neurones.

The increase in urination frequency in the RBF-lesioned model is probably more severe than that reported for bilateral neurotoxic BF lesion, as the bladder volume capacity in the present and the other rat models were about a quarter and a half that in the corresponding sham-operated rats, respectively [24,25]. While we cannot accurately compare data obtained under such different experimental conditions, the apparently more frequent urination in the present model may result from the superimposed dopaminergic dysfunction.

AH-9700, a putative drug for treating urge incontinence and frequency, has both a marked anti-micturition reflex effect, mediated presumably through central σ receptors, and a moderate spasmolytic effect, based on its peripheral antimuscarinic activity [13,14]. In cystometry testing using conscious RBF-lesioned rats as a model of neurogenic bladder, AH-9700 markedly decreased voiding frequency, increased bladder volume capacity, and raised the threshold pressure. Oxybutynin (2 mg/kg) and propiverine (5 mg/kg) (peripherally acting anti-frequency drugs with potent antimuscarinic activity [26,27]) also decreased the voiding frequency. However, in the present model, at higher doses (10 and 20 mg/kg, respectively), they unexpectedly increased voiding frequency (data not shown). The basis of this biphasic effect is unclear but the results appear to be consistent with the findings of Kontani et al.[28,29], that antimuscarinic agents such as atropine and oxybutynin increase voiding frequency in anaesthetized and/or conscious rats. In RBF-lesioned rats, the bladder capacity-increasing effect of AH-9700 was more potent than that of oxybutynin or propiverine. This may reflect the fact that AH-9700 has a marked central anti-micturition reflex effect in addition to a moderate antimuscarinic effect, as noted, and that most neurogenic bladder contractions of rats, but not humans, are atropine-resistant [30]. Taken together, these data suggest that centrally acting drugs such as AH-9700 would be useful for treating urge incontinence related to disturbances of the BF. However, further study is needed to clarify this issue, e.g. applying the RBF-lesioned model to another animal whose bladder is atropine-sensitive.

Antimuscarinic drugs significantly suppress voiding pressure in normal rats [14,15,28,29]. In the RBF-lesioned model, the antimuscarinic drugs, especially oxybutynin, reduced voiding pressure, although the reduction was not statistically significant. This might be because the reduction in voiding pressure was variable among RBF-lesioned rats. However, AH-9700 barely suppressed voiding pressure in this model; the reason is unclear, but it may be assumed that the doses of AH-9700 used were insufficient to reduce voiding pressure, as the antimuscarinic activity of AH-9700 is less potent than that of oxybutynin or propiverine [13,14,26,27].

In conclusion, the RBF-lesioned rat represents a promising model for studying the neurogenic bladder. In particular, the robust effect of AH-9700 on urinary frequency in this model suggests that it will be useful for identifying and evaluating agents for treating the neurogenic bladder of supraspinal origin.

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

The authors thank Dr T. Morie for synthesizing AH-9700, oxybutynin and propiverine. We also thank Dr Michael J. Caterina (Johns Hopkins University) for critical reading of this manuscript.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
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Abbreviations
RBF

right basal forebrain

DOPAC

dopamine 3,4-dihidroxyphenylacetic acid

HVA

homovanillic acid

5-HIAA

5-hydroxyindoleacetic acid

EFS

electrical field stimulation.