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Endocrinology Vol. 139, No. 9 3730-3735
Copyright © 1998 by The Endocrine Society

ARTICLES

Intracisternal Antisense Oligodeoxynucleotides to the Thyrotropin-Releasing Hormone Receptor Blocked Vagal-Dependent Stimulation of Gastric Emptying Induced by Acute Cold in Rats1

V. Martinez2, S. V. Wu and Y. Taché

CURE: Digestive Diseases Research Center, West Los Angeles Veterans Administration Medical Center, and the Department of Medicine, Digestive Disease Division, and Brain Research Institute, University of California School of Medicine, Los Angeles, California 90073

Address all correspondence and requests for reprints to: Y. Taché, Ph.D., CURE: Digestive Diseases Research Center, Veterans Administration Medical Center, Building 115, Room 203, 11301 Wilshire Boulevard, Los Angeles, California 90073. E-mail: ytache{at}ucla.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cold exposure increases TRH gene expression in hypothalamic and raphe nuclei and results in a vagal activation of gastric function. We investigated the role of medullary TRH receptors in cold (4–6 C, 90 min)-induced stimulation of gastric motor function in fasted conscious rats using intracisternal injections of TRH receptor (TRHr) antisense oligodeoxynucleotides (100 µg twice, -48 and -24 h). The gastric emptying of a methyl-cellulose solution was assessed by the phenol red method. TRH (0.1 µg) or the somatostatin subtype 5-preferring analog, BIM-23052 (1 µg), injected intracisternally increased basal gastric emptying by 34% and 47%, respectively. TRHr antisense, which had no effect on basal emptying, blocked TRH action but did not influence that of BIM-23052. Cold exposure increased gastric emptying by 64%, and the response was inhibited by vagotomy, atropine (0.1 mg/kg, ip), and TRHr antisense (intracisternally). Saline or mismatched oligodeoxynucleotides, injected intracisternally under similar conditions, did not alter the enhanced gastric emptying induced by cold or intracisternal injection of TRH or BIM-23052. These results indicate that TRH receptor activation in the brain stem mediates acute cold-induced vagal cholinergic stimulation of gastric transit, and that medullary TRH may play a role in the autonomic visceral responses to acute cold.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
COLD exposure triggers the increased synthesis and release of hypothalamic TRH and stimulation of pituitary TSH secretion (1, 2). In addition to this specific endocrine response, cold stimulates autonomic outflow to the viscera (2, 3, 4). In particular, cold exposure for 2–3 h increases vagal efferent activity (5) and induces vagal-dependent stimulation of gastric secretion, contractions, and erosion formation in rats (6, 7, 8). Growing evidence indicates that brain TRH pathways may be involved in mediating the vagal activation induced by cold in addition to its established role in the hypophyseal-thyroid axis response (1, 2, 3). A single class of TRH receptor (TRHr) has been cloned (9, 10, 11) and shown to be expressed in the dorsal motor nucleus (DMN) of the vagus (12), where TRH acts to stimulate the firing rate of neurons (13, 14, 15) and vagal outflow to the stomach (16). Consistent findings indicate that chemical or electrical activation of the raphe pallidus and obscurus nuclei containing TRH neurons projecting to the DMN (17) results in a vagal cholinergic stimulation of gastric secretory and motor function and alteration of the resistance of the gastric mucosa to injury through endogenous TRH actions in the DMN in urethane-anesthetized rats (3, 18, 19, 20, 21, 22). We recently reported that acute cold exposure activated neurons in the raphe pallidus, raphe obscurus, and DMN, as shown by Fos expression (23, 24) and increased pro-TRH messenger RNA (mRNA) expression in the raphe pallidus and obscurus (7). However, the demonstration of a causal relationship between activation of medullary TRH neurons and the vagus-dependent gastric functional changes induced by the action of acute cold exposure is still missing.

Antisense oligodeoxynucleotides targeted to inhibit specific peptide receptor expressions have been useful tools to uncover the physiological action of peptides (25, 26), especially in cases such as TRH (21, 27) when blocking receptor function, traditionally achieved by specific receptor antagonists, cannot be realized due to their unavailability (11). For instance, intracisternal injections of antisense oligodeoxynucleotides to the TRHr abolished the increase in intragastric pressure and pyloric motility induced by TRH microinjected into the dorsal vagal complex or chemical stimulation of raphe obscurus cell bodies in anesthetized rats (21).

In the present study, we first established that acute exposure to cold induces a vagal cholinergic stimulation of gastric emptying. Second, we determined whether intracisternal pretreatments with antisense phosphorothioate-modified oligodeoxynucleotides to TRHr suppress the gastric motor response to cold. Different aspects of specificity were examined using similar pretreatments with mismatch oligodeoxynucleotides in cold-exposed rats. We also compared the influence of TRHr antisense oligodeoxynucleotides on intracisternal (ic) TRH- and the somatostatin receptor subtype 5-preferring analog, BIM-23052, (28, 29)-induced stimulation of gastric emptying in rats maintained at room temperature (30, 31).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male Sprague-Dawley rats (250–290 g, Harlan Laboratories, San Diego, CA) were housed under controlled illumination (12-h light, 12-h dark cycle, starting at 0600 h), temperature (20–22 C), and humidity. They were maintained ad libitum on a standard rat diet (Purina Laboratory Chow) and tap water. Gastric emptying measurements were performed in animals fasted for 18–20 h but allowed free access to water. Studies were conducted under the V.A. Animal Component of Research Protocol 95-085-10.

Drugs and treatments
Rat TRH (Peninsula Laboratories, Belmont, CA) in powder form was freshly dissolved in sterile saline (Sigma Chemical Co., St. Louis, MO) before use. The somatostatin receptor subtype 5 ligand, BIM-23052 (28) [D-Phe-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-NH2] (D. H. Coy, Peptide Research Laboratories, Tulane University, New Orleans, LA), was synthesized and purified as previously described (32). BIM-23052 was dissolved in 0.01% acetic acid to a concentration of 1 µg/µl and further diluted in sterile saline to a concentration of 1 µg/10 µl before administration. Atropine, as sulfate salt (Sigma), was dissolved in saline.

Antisense oligodeoxynucleotides complementary to the first 18 bases downstream from the initiation codon of the rat TRH receptor mRNA (10) were synthesized with phosphorothioate derivatives of each nucleotide (5'-GAC GGT TTC ATT CTC CAT-3'; UCLA Molecular Biology Core, Los Angeles, CA). Mismatch oligodeoxynucleotides (5'-GAT GGT CTC ACT CTC TAT-3') mutated at four different positions (underlined nucleotides), but kept identical in composition to the TRHr antisense, were also synthesized with phosphorothioate derivatives and used as one of the control treatments. The mismatch sequence has neither significant complementarity to any part of the TRH receptor mRNA nor significant complementarity to any other gene sequences in the GenBank database. The oligodeoxynucleotides were purified by PAGE and diluted in sterile saline to a final concentration of 10 µg/1 µl, and aliquots (20 µl) were maintained at -70 C until use.

Intracisternal injections were performed acutely in rats under short enflurane anesthesia (2–3 min, 5.5% vapor in O2; Ethrane-Anaquest, Madison, WI). Animals were placed in ear bars of stereotaxic equipment, and the occipital membrane was punctured with a 50-µl Hamilton syringe (Hamilton, Reno, NV). The presence of cerebrospinal fluid in the Hamilton syringe upon aspiration before the injection insured correctness of needle placement into the cisterna magna.

Cold exposure was performed as previously described (23). Conscious rats were semirestrained in individual stainless steel cylindrical cages with flat bottoms (16 x 5.5 x 5.5 cm) and perforations to allow ventilation, then placed in a cold room (4–6 C) for 90 min or maintained at room temperature (20–23 C).

Measurement of gastric emptying
Gastric emptying of a nonnutrient viscous solution was determined by the phenol red method, as previously described (33). A suspension of continuously stirred 1.5% methyl-cellulose (Sigma) and phenol red (0.5%, Sigma) was given intragastrically (1.5 ml) to conscious rats. After a 20-min period, rats were killed by CO2 inhalation. The abdominal cavity was opened, the gastroesophageal junction and the pylorus were clamped, and the stomach was removed, rinsed in 0.9% saline, placed in 100 ml 0.1 N NaOH, and homogenized (Polytron, Brinkmann Instruments, Westbury, NY). The suspension was allowed to settle for 1 h at room temperature, and then 5 ml of the supernatant were added to 0.5 ml 20% trichloroacetic acid (wt/vol) and centrifuged at 3000 rpm at 4 C for 20 min. The supernatant was mixed with 4 ml 0.5 N NaOH, and the absorbance of the sample was read at 560 nm (Shimazu UV-260, Cole Scientific, Moorpark, CA). Phenol red recovered from animals killed immediately after administration of the methyl-cellulose solution was used as the standard (0% emptying). The percent emptying in the 20-min period was calculated according to the following equation: % emptying = 1 - (absorbance of test sample/absorbance of standard) x 100.

Experimental protocols
Cold exposure-induced stimulation of gastric emptying: effects of vagotomy and atropine. Groups of rats were placed singly in semirestraining cages either at room temperature or in a cold room (4–6 C) for a total period of 90 min. After 70 min, the phenol red solution (1.5 ml) was administered intragastrically, and rats were killed by CO2 inhalation 20 min after the administration of the marker. The stomach was quickly removed, and the rate of gastric emptying determined as described above. One control group was maintained freely moving in home cages, and the 20-min rate of gastric emptying was determined.

Subdiaphragmatic vagotomy (achieved by a circular seromuscular myotomy of the esophagus, ~2 cm from the gastroesophageal junction) or sham vagotomy (laparotomy and manipulation of abdominal viscera) was performed in fasted rats anesthetized with ketamine hydrochloride (75 mg/kg, ip; Ketaset, Fort Dodge Laboratories, Fort Dodge, IA) and xylazine (25 mg/kg, ip; Rompun, Mobay Co., Shawnee, KS). After 48 h, vagotomized or sham-operated groups were positioned in semirestrained cages and exposed for 90 min to cold or were maintained at room temperature in home cages. Gastric emptying was assessed during the 70- to 90-min period after the onset of cold exposure. Other groups were injected ip with either atropine sulfate (0.1 mg/kg) or vehicle (0.5 ml), and 30 min later were positioned in semirestraint cages and exposed for 90 min to cold or were maintained at room temperature in home cages. The 20-min rate of gastric emptying was assessed during the 70- to 90-min period of cold exposure.

Effect of TRHr antisense oligodeoxynucleotides on intracisternal TRH- and BIM-23052- and cold-induced stimulation of gastric emptying. Animals were injected intracisternally with a total dose of 200 µg TRHr mismatch or antisense oligodeoxynucleotides in two injections (100 µg each), 48 and 24 h before measurement of gastric emptying. An additional control group was injected intracisternally with sterile saline following the same protocol (two injections, 10 µl each). Rats pretreated with saline, TRHr mismatch, or TRHr antisense oligodeoxynucleotides were either exposed to cold or maintained at room temperature for 90 min. The rate of gastric emptying of the phenol red solution was determined during the 70- to 90-min period of cold exposure.

Separate groups of rats pretreated with saline, TRHr mismatch, or TRHr antisense oligodeoxynucleotides, as described above, were injected intracisternally with vehicle (sterile saline or 0.01% acetic acid solution appropriately diluted in saline, 10 µl), TRH (0.1 µg), or BIM-23052 (1 µg). After 10 min, the phenol red-methyl-cellulose solution was administered intragastrically, and the 20-min rate of gastric emptying was determined in rats maintained at room temperature in home cages. Peptide doses were chosen based on previous reports showing the stimulation of gastric emptying through vagal pathways in conscious rats (30, 31).

Statistical analysis
Results are expressed as the mean ± SEM. Comparisons between groups were performed using one-way ANOVA, followed, when required, by a Student-Newman-Keuls multiple comparisons test. Differences between two groups were determined by two-tailed Student’s t test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cold exposure-induced stimulation of gastric emptying: effects of vagotomy and atropine
At room temperature, the 20-min rate of gastric emptying was similar in rats maintained in home cages (57.3 ± 3.7%; n = 8) and those kept in semirestraint cages for 90 min (54.0 ± 2.1%; n = 6; P > 0.05; Fig. 1Go). Cold exposure for 90 min in semirestraint increased the rate of gastric emptying to 95.1 ± 1.1% [n = 11; P < 0.05 compared with room temperature with or without semirestraint; F(2, 22) = 104.93; Fig. 1Go].



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  		Figure 1. Acute exposure to cold stimulates basal gastric emptying in conscious rats. Rats were maintained in semirestraint cages for 90 min at room temperature or 4–6 C, and the 20-min rate of gastric emptying of a nonnutrient viscous solution was determined during the 70- to 90-min period after the onset of the semirestraint. The control group was maintained at room temperature in their home cages. Data represent the mean ± SEM of 8 (room temperature), 6 (room temperature and semirestraint), and 11 (cold and semi-restraint) animals. *, P < 0.001 compared with other groups [by ANOVA, F(2 22 ) = 104.93; P < 0.0001].

 
In sham-operated rats, cold exposure for 90 min increased gastric transit to 94.3 ± 1.8% compared with 70.9 ± 5.8%, in rats maintained at room temperature (n = 6 in each group; P < 0.05). Subdiaphragmatic vagotomy 48 h before the experiments reduced the rate of gastric emptying in animals maintained at room temperature to 44.2 ± 9.6% (n = 6; P < 0.05 vs. sham vagotomy at room temperature; Fig. 2Go). Vagotomy completely prevented the stimulation of gastric emptying induced by cold exposure, and values (41.4 ± 5.5%) were similar to those observed in vagotomized rats maintained at room temperature (Fig. 2Go).



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  		Figure 2. Effects of vagotomy and atropine on basal and cold-induced stimulation of gastric emptying in conscious rats. Sham operation or subdiaphramatic vagotomy was performed 48 h before the experiments; atropine (0.1 mg/kg, ip) was administered 30 min before the onset of cold exposure. The 20-min rate of gastric emptying of a nonnutrient viscous solution was determined during the 70–90 min after the onset of cold exposure. Data represent the mean ± SEM of six animals per group. *, P < 0.05 vs. sham vagotomy at room temperature; #, P < 0.05 vs. sham vagotomy and cold exposure [by ANOVA, F(3 20 ) = 15.557; P < 0.0001]. *, P < 0.05 vs. vehicle at room temperature; #, P < 0.05 vs. vehicle and cold exposure [by ANOVA, F(3 20 ) = 17.816; P < 0.0001].

 
Cholinergic blockade with atropine reduced the basal rate of gastric emptying to 26.1 ± 7.9%, compared with 63.7 ± 5.7% in vehicle-treated animals maintained at room temperature (n = 6 for each group; P < 0.05; Fig. 2Go). Cold exposure increased the rate of gastric emptying to 91.8 ± 2.8% in vehicle-pretreated rats (n = 6; P < 0.05 vs. room temperature). In atropine-treated rats, cold exposure no longer stimulated gastric emptying, and values (41.5 ± 8.9%, n = 6) were decreased compared with those in the vehicle-injected group and were not significantly different from those in the atropine-treated group maintained at room temperature (Fig. 2Go).

Effect of TRHr antisense oligonucleotides on intracisternal TRH-, BIM-23052-, and cold exposure-induced stimulation of gastric emptying
In rats maintained at room temperature, intracisternal TRH (0.1 µg in 10 µl) increased gastric emptying to 82.9 ± 1.7% compared with 61.8 ± 4.1% in rats injected intracisternally with vehicle (n = 4 for each group; P < 0.05; Fig. 3Go). Intracisternal injections of either saline or the mismatch oligodeoxynucleotides against the TRHr did not modify the stimulatory effect of intracisternal TRH on gastric emptying [saline, 80.7 ± 2.9% (n = 5); mismatch, 77.9 ± 3.5% (n = 6); Fig. 3Go]. However, pretreatments with the TRHr antisense completely prevented the stimulatory effect of TRH injected intracisternally (50.0 ± 3.2%; n = 5; Fig. 3Go). None of the intracisternal pretreatments, including saline, TRHr mismatch, or antisense oligodeoxynucleotides, modified the basal rate of gastric emptying in animals injected intracisternally with vehicle (Fig. 3Go).



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  		Figure 3. Prevention of intracisternal TRH-induced stimulation of gastric emptying in conscious rats by pretreatment with TRHr antisense oligodeoxynucleotides. Animals were injected intracisternally (ic) with saline or TRHr mismatch (100 µg) or TRHr antisense (100 µg) oligodeoxynucleotides at 48 and 24 h before the experiment. The 20-min rate of gastric emptying of a nonnutrient viscous solution was determined during the 10- to 30-min period after intracisternal injection of vehicle or TRH (0.1 µg). Data represent the mean ± SEM of four to six animals per group. *, P < 0.05 vs. respective vehicle-treated group; #, P < 0.05 vs. TRH-treated groups [by ANOVA, F(7 32 ) = 25.86; P < 0.0001].

 
In saline- or TRHr mismatch-pretreated animals, cold exposure for 90 min significantly increased the rate of gastric emptying to 88.3 ± 1.8% and 86.7 ± 5.8%, respectively (n = 5 for each group; Fig. 4Go). The enhanced basal gastric emptying rate elicited by cold exposure was no longer observed in animals pretreated intracisternally with the TRHr antisense [56.3 ± 4.0% (n = 6); P > 0.05 vs. antisense at room temperature: 49.0 ± 3.1% (n = 5 in each group); Fig. 4Go].



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  		Figure 4. Prevention of acute exposure to cold-induced stimulation of gastric emptying by intracisternal injections of TRHr antisense oligodeoxynucleotides. Animals were injected intracisternally (ic) with saline or TRHr mismatch (100 µg) or TRHr antisense (100 µg) oligodeoxynucleotides 48 and 24 h before the experiment. The 20-min rate of gastric emptying of a nonnutrient viscous solution was determined during the 70- to 90-min period after the onset of cold exposure. Data represent the mean ± SEM of four to six animals per group. *, P < 0.05 vs. respective room temperature group; #, P < 0.05 vs. cold exposure [by ANOVA, F(5 26 ) = 21.402; P < 0.0001].

 
The somatostatin subtype 5 receptor-preferring analog, BIM-23052, injected intracisternally (1 µg/rat, 10 µl) increased the rate of gastric emptying to 72.2 ± 6.2% compared with that produced by saline injection (Table 1Go). The stimulatory effect of BIM-23052 was not modified in animals pretreated with either the TRHr mismatch or antisense oligodeoxynucleotides (Table 1Go).


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  		Table 1. Effect of intracisternal injections of TRHr antisense oligodeoxynucleotides on intracisternal somatostatin analog BIM-23052-induced stimulation of gastric emptying in conscious rats

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present results show that cold exposure for 90 min increased the basal rate of gastric emptying of a nonnutrient solution by 64% in rats maintained singly in semirestraining cages. The stimulation of gastric transit resulted from the specific effect of cold exposure, as similar semirestraint at room temperature did not influence basal gastric emptying compared with that of rats in the home cage. This observation provides an additional functional parameter consistent with other markers indicative of lack of stress (absence of Fos expression in the brain, defecation, and gastric erosions) in rats semirestrained at room temperature (23, 24, 34). Cold-induced stimulation of gastric transit and motility is mediated through vagal cholinergic pathways, as shown by the blockade of the gastric motor changes by subdiaphragmatic vagotomy and atropine in conscious or anesthetized rats (Ref. 35 and present observations).

The alteration of gastric emptying is a sensitive visceral response to exposure to various stressors (36). However, different challenges, including surgical, immunological (interleukin-1), chemical (anesthesia), and physical (immobilization, forced swimming), all delay gastric emptying through the activation of brain CRF receptors influencing autonomic output to the stomach in rats (36, 37). Therefore, the marked vagal stimulation of gastric motor function induced by acute cold exposure is unlikely to be mediated by brain CRF-related mechanisms and represents a specific response to cold exposure.

One characteristic pathway activated by cold exposure is the stimulation of TRH gene expression in neurons of the paraventricular nucleus of the hypothalamus and medullary raphe nuclei (obscurus and pallidus) (2, 7). Convergent evidence supports a role for medullary TRH in mediating the stimulation of gastric transit induced by acute cold exposure. Intracisternal injection of TRH mimicked the vagal cholinergic dependent stimulation of gastric emptying and motility induced by cold (Refs. 6, 8, 30, 38 and present observation). Moreover, pretreatments with antisense oligodeoxynucleotides, that are complementary to the TRHr mRNA, blocked the gastric emptying response to cold. The suppressive effect of TRHr antisense reflects a sequence-specific mechanism of action. First, similar administration of the TRHr antisense was equally effective in blocking intracisternal injection of TRH (0.1 µg)-induced increase in gastric emptying. Second, the mismatch oligodeoxynucleotides with similar composition to the TRHr antisense did not modify the stimulatory response elicited by either intracisternal TRH or cold exposure. Under basal conditions neither the sense nor the antisense oligodeoxynucleotides influenced the rate of gastric emptying, which, as above mentioned, is a sensitive visceral index of stress response (36). Furthermore, the TRHr antisense oligodeoxynucleotides were unable to alter the stimulation of gastric emptying elicited by the intracisternal injection of the somatostatin receptor subtype 5 agonist, BIM-23052. Taken together, these observations rule out a nonsequence-specific action of the TRHr antisense pretreatments (26, 39). We recently showed that intracisternal BIM-23052-induced gastric emptying is prevented by vagotomy and atropine in conscious rats (31). The lack of blockade of the BIM-23052 effect by intracisternal TRHr antisense pretreatments shows also that the central vagal cholinergic dependent action of the somatostatin subtype 5-preferring analog is not secondary to the activation of medullary TRH pathways.

The TRHr antisense oligodeoxynucleotides may act by preventing TRHr-mediated activation of DMN neurons. Consistent with such a possibility, cold increases TRH gene expression in raphe nuclei projecting to the dorsal vagal complex (7). Abundant TRH-binding sites and TRHr mRNA are present on DMN neurons (12, 40), and TRH increases the spontaneous firing rate in DMN neurons by a direct postsynaptic effect (13, 14, 15). Phosphorothioate oligodeoxynucleotides have been shown to remain stable upon injection into the rat cerebrospinal fluid (39, 41) and to be rapidly taken up into cells close to the injection site (25). The TRHr antisense oligodeoxynucleotides were delivered into the cisterna magna located in a position immediately dorsal to the dorsal vagal complex (42), and this route of administration blocks the increases in gastric intraluminal pressure and pyloric motility induced by TRH microinjected in the dorsal vagal complex (21).

The existence of a tonic cholinergic vagal outflow has been proposed as a mechanism regulating gastric functions under basal conditions in rats. For instance, the high interdigestive basal gastric acid secretion and emptying seem to depend upon vagal cholinergic mechanisms, as atropine and vagotomy reduce basal rates of gastric function (Refs. 30, 43 and present observation). The lack of changes in basal gastric emptying by the TRHr antisense pretreatments ruled out a role of tonic TRHr activation in the modulation of vagal outflow to the stomach. Similarly, the TRHr antisense did not modify basal gastric acid secretion, but modulated the acid response to sham feeding (44). Together, these observations suggest that medullary TRH does not participate in the control of basal gastric vagal tone in conscious fasted rats.

In summary, intracisternal pretreatments with TRHr antisense oligodeoxynucleotides induced a sequence-specific blockade of intracisternal injection of TRH- and cold-induced vagal cholinergic dependent stimulation of gastric emptying while not influencing basal gastric emptying. These results provide evidence that the activation of medullary TRH receptors plays a key role in cold exposure-induced vagal dependent stimulation of gastric motor function. By contrast, these receptors are not involved in maintaining basal gastric emptying or the vagus-dependent increase in gastric emptying induced by intracisternal injection of the somatostatin receptor subtype 5-preferring compound, BIM-23052. As intracerebroventricular injection of TRH antibody was reported to attenuate the vagus-dependent stimulation of gastric acid secretion and lesion formation induced by hypothermia in anesthetized rats (35, 45), these results indicate that brain TRH may have implications in autonomic adaptive visceral responses to acute cold exposure.


   Acknowledgments
 
The authors thank Dr. David H. Coy (Tulane University Medical Center, New Orleans, LA) for the generous supply of BIM-23052. Mr. Paul Kirsch is acknowledged for his help in the preparation of the manuscript.


   Footnotes
 
1 This work was supported by NIMH Grant MH-00663 and NIAMDD Grant MK-30110. Back

2 Present address: CEU San Pablo, Department of Physiology, Veterinary School, 46113 Moncada, Valancia, Spain. Back

Received March 4, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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