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Secretin modulation of behavioral and physiological functions in the rat
PeptMes, Vol. 4, pp. 73%742, 1983.©Ankho InternationalInc. Printedin the U.S.A.
Secretin Modulation of Behavioral and Physiological Functions in the Rat C L I V E L G. C H A R L T O N , * t R U S S E L L L. M I L L E R , t J A C Q U E L I N E N . C R A W L E Y , $ G A l L E. H A N D E L M A N N § A N D T H O M A S L. O ' D O N O H U E *
*Neuroendocrinology Unit, Experimental Therapeutics Branch National Institute of Neurological and Communicative Disorders and Stroke National Institutes of Health, Bethesda, MD 20205 tDivision of Clinical Pharmacology, Howard University, Washington, DC 20059 5;Unit of Behavioral Neuropharmacology, Clinical Neuroscience Branch National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20205 §Laboratory of Clinical Science, National Institute of Mental Health National Institutes o f Health, Bethesda, MD 20205
CHARLTON, C. G., R. L. MILLER, J. N. CRAWLEY, G. E. HANDELMANN AND T. L. O'DONOHUE. Secretin modulation of behavioral and physiologicalfunctions in the rat. PEPTIDES 4(5) 739-742, 1983.--The effect of secretin on behavioral and physiological functions in the rat was investigated. Secretin injected intracerebroventricularly (ICV) significantly increased defecation and decreased novel-object approaches in rats. The peptide showed no significant effects on stereotypic behavior (gnawing, grooming and rearing), open-field locomotor activity however was significantly decreased, an effect that was probably due to a decreased propensity for the rats to initiate locomotor responses. In addition, secretin showed significant effects on respiration rate in anesthetized rats. When the peptide was injected in the lateral ventricle a decrease in respiration rate occurred, but when the brain was perfused from the lateral ventricle to the cisterna magna increases in respiration rate occurred. These data, combined with the facts that secretin and secretin receptors have been identified in the brain indicate that secretin may play a neurotransmitter or neuroregulator role in the central nervous system. Secretin
Novel-object approaches
Open field locomotor activity
Respiratory
Defecation
pharmacological effects of secretin injected into the cerebral ventricles of the rat.
SECRETIN, a 27 amino acid polypeptide, was thought to be localized solely in the gut, until recently when secretin-like bioactivity was identified in extracts of porcine brain [13], and secretin immunoreactivity was identified and characterized in both pig and rat brain [4,14]. The effects of secretin on peripheral systems and its interaction with peripheral receptors are well documented [2, 5, 6, 7, 10, 11, 12, 16, 17]. However, the actions of secretin on CNS target cells were virtually unknown until recently. Secretin receptors similar to that characterized in the pancreas have recently been identified in the brain [8]. Furthermore, a secretin sensitive adenylate cyclase has been identified on neuroblastoma × glioma hybrid cells [15] and in cultured mouse brain cells [3]. The precise role of secretin in the CNS is unknown at present although intraventricular administration of the peptide appears to alter dopamine turnover rates in nucleus accumbens, olfactory tubercle and median eminence which also results in reduction of prolactin secretion from the anterior piutitary [9]. The existence of secretin and secretin receptors in the brain coupled with the observation that secretin affected CNS dopamine, cyclic AMP and prolactin, suggest that the peptide may serve as a neurotransmitter or neuromodulator in the CNS. The purpose of this study was to investigate what roles secretin in the CNS may serve by observing
METHOD
Animals Sprague-Dawley male rats weighing 250--300 g (ZivicMiller, Allison Park, PA) were used. The rats were acclimatized for at least one week, 6 to a cage, in a colony room with 12 hr light and 12 hr dark cycle. Water and food were supplied ad lib.
Surgery and Protocol Under chloropent anesthesia (Fort Dodge Labs. Inc., Fort Dodge, IA) a stainless steel guide cannula was stereotaxtically inserted into the lateral ventricle of each rat. Each cannula was affixed in place with dental cement secured to the skull with the aid of two screws. To prevent clogging, a removable 27 gauge wire was inserted into the guide cannula. The placement of the cannula with reference to bregma was 1.4 mm lateral, 0.5 mm caudal and a depth of 2.5 mm from the surface of the skull. The rats were allowed to recover for 4 days before the tests. Injections were made in the lateral ventricle 5 mm from the surface of the skull, via
739
C H A R L T O N ET AL.
740
A. DEFECATION RATE
a premeasured cannula attached by polyethylene tubing (PE20) to a 25/zl Hamilton syringe containing either phosphate buffered saline (PBS) at p H 7.4 or 5 /~g of secretin (Peninsula Lab, San Carlos, CA) dissolved in 5/xl PBS. All tests were performed between 1400 hr and 1700 hr. Each rat was used in one test only except that the rats used for determination of defecation rate were reutilized for the determination of novel object approaches and rearing without further treatments. The data are expressed as the mean-+the standard error of the mean. Differences between two means were determined using Student's two tailed t-test, utilizing a Monroe 1860, calculator (Morristown, N J).
PBS=SALINE SEC=SECRETIN
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Apparatus and Procedures Defecation rate determination. To measure defecation rate a total of 6 rats from each group (PBS and secretin) were placed in individual cages (12× 12×9") immediately after injection. The number of fecal boluses deposited by each rat for 1 hour post-injection was counted. Novel-object approaches and rearing. Immediately after the 1 hour defecation test, the rats were placed individually in an arena for the assessment of rearing and the number of approaches made to a novel object placed in the center of the arena's floor. The test time at 1 hour post-injection was selected because a pilot study showed that the decrease in novel-object approaches by secretin treated rats stabilized at about 1 hour after injection. Each rat was observed for 10 minutes. The arena consisted of a card-board box measured 21x 18x 18". The deep box was used to enhance vertical motor-activity in the rats. The novel-object was a small (21/2x3×5 ") perforated metal container. Each time a rat stood erect on its hind legs or when it sniffed or touched the novel object with its snout an incident of rearing or novelobject approaches was scored, respectively. Open-field activity. Another group of 10 surgically prepared rat (5 injected with secretin and 5 with PBS) was studied in an open field apparatus. The apparatus consisted of 3 ' × 3'×6" wooden box, painted black and covered with a steel grill. The floor was divided into 16 squares, 9"x9" each. Tests were done in a quiet room with an ambient temperature of 23+--I°C and approximately 60 foot-candles of cool white fluorescent light. The rats were tested at 1 hour post-injection as for the novel object approaches test. The number o f squares entered during a 5 minute observation period and the number of one-minute periods that the animals remained immobile were determined. The latter procedure was necessary to determine whether changes in activity were associated with a propensity to remain immobile or to changes in the rate of movement. Respiration. Each rat was anesthetized with chloropent and put in a stereotaxic apparatus for the duration of the procedure. A hole was drilled into the skull, using the cordinate described above and after incision and retraction of the overlying tissues. Injections of PBS or secretin in 6 rats each were made stereotaxically into the lateral ventricle using a 25 /xl Hamilton syringe with a 27 gauge needle. Another group of 5 aneshtetized rats was injected intravenously (femoral vein) with 5/zl PBS or 5 ~g secretin in 5/xl PBS. Respiration was counted immediately after and at 2, 5, 10, 20, 30 and 40 minutes post-injection. Since secretin injected in a 5/zl volume may localize at the point of injection, a brain perfusion technique was adopted so that the effects of secretin could be observed
PBS
SEC
FIG. 1. The effect of secretin on defecation rate in rats. Rats were injected intracerebro-ventricularly with phosphate buffered saline (PBS) or secretin (SEC) dissolved in PBS (5 ~g in 5 txt). Animals were placed in individual cages to determine defecation rate (boluses of feces deposited in 1 hour). Open column=PBS. Stippled column=SEC. N=6 *p<0.05.
following perfusion of the cerebral ventricles with the peptide. Artificial C S F (NaCI 8.89 g; KCi 0.25 g; CaC12 2HeO 0.11 g; NaH2PO4H20 0.07 g; urea 0.13 g; glucose 0.61 g per liter) was delivered into the lateral ventricle by gravity, utilizing a 27 gauge stainless steel cannula attached by polyethylene tubing (PE20) to a dispensing vessel (usually a 1.5 ml pipette tip). A 25 gauge cannula attached to a Sage Model 351 syringe pump (Sage Instruments Div. Orion Research Inc., Cambridge, MA) was used to withdraw the peffusate, via the cisterna magna at a rate of 25/xl per minute. Respiration rate was counted with the aid of a stop watch, before perfusion, during perfusion for a 20 minute period with artificial C S F and with secretin (20 ng//xl) in artificial CSF. The pH of the perfusate was also measured. RESULTS
Defecation The injection of secretin (5 /xg in 5 /xl PBS) caused a statistically significant (.o<0.05) increase in defecation rate during the first post-injection hour (Fig. 1). An average of 7.0-+1.31 boluses per rat was deposited by the secretin treated group whereas the PBS controls deposited an average of 3.0-+0.81 boluses each during the same period.
Novel-Object Appraoches and Rearing The number of novel-object approaches made by the secretin-treated rats was significantly less (p <0.05) than the number of approaches made by the PBS group. A score of 20.0+ 1.4 approaches was made by each rat treated with secretin while each rat injected with PBS approached the novel-object 34.8-+0.9 times (Fig. 2A). Rearing was essentially identical in both groups with 56.7-+6.6 for each secretin-treated rat and 51.2-+ 3.8 for each PBS control (Fig. 2B). Other stereotypic behaviors e.g., gnawing, sniffing, licking and grooming appeared to be of low incidence and preliminary results showed that secretin injections had no significant effects on these behaviors.
CNS EFFECTS OF SECRETIN
A. NOVEL-OBJECT APPROACHES PBS=SALINE SEC=SECRETIN
741 OPEN-FIELD LOCOMOTOR ACTIVITY
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FIG. 2. The effects of secretin on novel-object approaches (A), and the incidence of rearing (B) in rats. Rats were injected (ICV) with PBS or SEC (5/~g) and were tested at 1 hour post-injection. Open column=PBS. Stippled column=SEC. N=6. *p<0.05.
PBS
SEC
FIG. 3. The effect of SEC on open-field locomotor activity in rats. Rats were injected with PBS or 5 ~g secretin. The data represent the average number (mean_ SE) of squares entered by each rat during 5 minutes observation at 1 hour post-injection. Open column=PBS. Stippled column=SEC. N=5, *p<0.05.
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FIG. 4. The effects of secretin and PBS on respiration rate in anesthetized rats. Rats were anesthetized with Chloropent. When a steady respiration rate was achieved PBS or secretin (5/~g ICV) was injected and the respiration rate determined. *p<0.05 for secretin (ICV) vs. PBS (ICV) or secretin (IV).
Open-field Activity As s h o w n in Fig. 3, injection of secretin into the lateral ventricle resulted in a significant d e c r e a s e in o p e n field loc o m o t o r activity, c o m p a r e d to controls. The n u m b e r of squares entered by e a c h secretin treated rat during a 5 minute o b s e r v a t i o n period was 65.0+ 15.3 c o m p a r e d to 116.2+_8 for e a c h PBS treated rat. Additionally, when the average n u m b e r of one-minute periods of immobility was d e t e r m i n e d during the test session the group treated with secretin remained immobile for an average of 4.33+_0.95 periods as
60 MINUTES EXP. NO. 1
120
0
60 120 MINUTES EXP. NO. 2
FIG. 5. Shown are the respiration rates during perfusion of the rat brain. The open columns represent the respiration rate before perfusion, the cross-hatched columns during artifical CSF perfusion (26 p,l/min) and the stippled columns during perfusion with secretin (20 ng/p,l in artificial CSF). The upper curves represent the pH values for the corresponding perfusate. The first value is the pH of the unperfused CSF.
c o m p a r e d with 2.0---0.91 periods for the PBS control group (p <0.05---not significant).
Respiration Secretin (5 /zg) injected into the lateral ventricle of anesthetized rats gradually d e c r e a s e d respiration rate, w h e r e a s intravenous injection of secretin did not show any effect (Fig. 4). Perfusion of the brain of anesthetized rats with 20 ng//zl of secretin, h o w e v e r , markedly increased the respiration rate (Fig. 5). This effect of secretin was promptly
742
CHARLTON E T A L .
reversed when the perfusion of secretin was substituted with artificial CSF without secretin. The changes in respiration rate did not show any relationship with the pH of the perfusate. The respiratory and behavioral effects of secretin were only observed after intraventricular administration of the peptide. Intraperitoneal injections of identical doses of secretin did not affect behavior or respiration rate. DISCUSSION The results of this study demonstrated that the ICV injection of secretin significantly decreased open-field activity and novel-object approaches and increased defecation rate in freely moving rats. In addition, an ICV injection of secretin in anesthetized rats decreased respiration rate. The change in open-field locomotor activity elicited by secretin is probably not indicative of the effect of a nonspecific depressant. Rearing was not altered, there was no abnormality in gait, no apparent changes in stereotypic behavior (e.g., sniffing, licking, grooming and gnawing). In addition, the ICV injection of secretin did not alter the ability of rats to balance on a horizontal bar, climb an inclined plane nor did it change the time required for rats to regain the righting reflex following barbiturate anesthesia (unpublished data). By determining the number of one-minute periods that the rats remained immobile it was shown that the decreased locomotion was probably due to a decreased propensity to
initiate locomotor responses. The combination of decreased open field activity, decreased novel-object approaches and increased defecation rate might be interpreted to indicate fear or changes in emotional state (see [1]) of the secretin injected animals, but since the test environments may initiate exploratory activity as well as evoke fear in the rats the precise behavioral substrate affected by secretin administration is unclear. The secretin induced decrease in respiration rate in anesthetized rats seems to be independent from the effect on open-field activity, in that respiration rate returned to control values within one hour, while effects on open field activity can last for days (in preparation). Although more difficult to quantitate, decreased respiration rate also appeared in freely moving rats following ICV secretin administration. When the anesthetized rat brain was perfused between the lateral ventricle and the cisterna magna with secretin (20 ng//xl) in artificial cerebrospinal fluid, there was an increase in respiration rate as opposed to the decrease in respiration rate seen after ICV secretin injections. These data indicate that secretin at the doses studied may have opposite respiratory actions on forebrain and hind brain regions. Secretin [4,14], secretin receptors [8] and secretin sensitive adenylate cyclase [3,15] are present in neuronal tissues. These observations coupled with the fact that intraventricular administration of secretin caused behavioral and physiological changes in rats indicate that secretin may act as a neurotransmitter or neuroregulator in the CNS.
REFERENCES
I. Boradhurst, P. L. Determination of emotionality in the rat: t. Situational factors. Gen Psychol 48: 1-12, 1957. 2. Butcher, R. W. and L. A. Carlson. Effects of secretin on fat mobilizing lipolysis and cyclic AMP levels in rat adipose tissue. Acta Physiol Scand 79: 559--563, 1970. 3. Calker, D. V., M. Muller and B. Homprecht. Regulation by secretin, vasoactive intestinal peptide and somatostatin of cyclic AMP accumulation in cultured brain cells. Proc Natl Acad Sci USA 77: 6907-6911, 1980. 4. Charlton, C. G., T. L. O'Donohue, R. L. Miller and D. M. Jacobowitz. Secretin immunoreactivity in rat and pig brain. Peptides 2" Suppl 1, 45-49, 1981. 5. Chey, W. Y., S. Hitanant, J. Hendricks and S. H. Lorber. Effect of secretin and cholecystokinin on gastric emptying and gastric secretin in man. Gastroenterology 58: 820--827, 1970. 6. Chey, W. Y., S. H. Lorber, O. Kusakcioglu and J. Hendricks. Effect of secretin and pancreozymin-cholecystokinin and motor function of the duodenum. Fed Proc 26: 383, 1967. 7. Delaney, J. P. and E. Grim. Influence of hormones and drugs on canine pancreatic blood flow. Am J Physiol 211: 1398-1402, 1966. 8. Fremeau, R. T., R. T. Jensen, C. G. Charlton, R. L. Miller, T. L. O'Donohue and T. W. Moody. Secretin: Specific binding to rat brain membranes. J Neurosci, in press, 1983.
9. Fuxe, K., K. Anderson, T. Hokfelt, V. Mutt, L. Ferland, F. F. Agnati, D. Ganten, S. Said, P. Eneroth and J. A. Gustafsson. Localization and possible function of peptidergic neurons and their interactions with central catecholamine neurons and the central actions of gut hormones. Fed Proc 38: 2333-2340, 1979. 10. Goodhead, B., H. S. Himal and J. Zanbilowicz. Relationship between pancreatic secretion and pancreatic blood flow. Gut 11: 62-68, 1970. 11. Hubel, K. A. Secretin: A long progress note. Gastroenterology 62: 318-341, 1972. 12. Lazarus, R. N., N. R. Voyes, S. Devrim, T. Tanese and L. Recant. Extragastrointestinal effects of secretin, gastrin and pancreozymha. Lancet 2: 248-250, 1968. 13. Mutt, V., M. Cariquist and K. Tatemoto. Secretin-like bioactivity in extracts of porcine brain. Lift, Sci 25: 1703-1708, 1979. 14. O'Donohue, T. L., C. G. Charlton, R. L. Miller, G. Boden and D. M. Jacobowitz. Identification, characterization and distribution of secretin immunoreactivity in rat and pig brain. Proc Natl Acad Sci USA 78: 5221-5224, 1981. 15. Propst, F., L. Moroder, F. Wunsch and B. Hamprecht. The influence of secretin, glucagon and other peptides, of amino acids, prostaglandin, endoperoxide analogues and diazepam on the level of adenosine 3' 5'-cyclic mono phosphate in neuroblastoma x glioma hybrid cells. J Neurochern 32: 1495-1500, 1979. 16. Ross, G. Cardiovascular effects of secretin. Am J Physiol 218: 1116-1170, 1970. 17. Rudman, R. and A. E. Del Rio. Lipolytic activity of synthetic porcine secretin. Endocrinology 85: 214-217, 1969.
Secretin Modulation of Behavioral and Physiological Functions in the Rat C L I V E L G. C H A R L T O N , * t R U S S E L L L. M I L L E R , t J A C Q U E L I N E N . C R A W L E Y , $ G A l L E. H A N D E L M A N N § A N D T H O M A S L. O ' D O N O H U E *
*Neuroendocrinology Unit, Experimental Therapeutics Branch National Institute of Neurological and Communicative Disorders and Stroke National Institutes of Health, Bethesda, MD 20205 tDivision of Clinical Pharmacology, Howard University, Washington, DC 20059 5;Unit of Behavioral Neuropharmacology, Clinical Neuroscience Branch National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20205 §Laboratory of Clinical Science, National Institute of Mental Health National Institutes o f Health, Bethesda, MD 20205
CHARLTON, C. G., R. L. MILLER, J. N. CRAWLEY, G. E. HANDELMANN AND T. L. O'DONOHUE. Secretin modulation of behavioral and physiologicalfunctions in the rat. PEPTIDES 4(5) 739-742, 1983.--The effect of secretin on behavioral and physiological functions in the rat was investigated. Secretin injected intracerebroventricularly (ICV) significantly increased defecation and decreased novel-object approaches in rats. The peptide showed no significant effects on stereotypic behavior (gnawing, grooming and rearing), open-field locomotor activity however was significantly decreased, an effect that was probably due to a decreased propensity for the rats to initiate locomotor responses. In addition, secretin showed significant effects on respiration rate in anesthetized rats. When the peptide was injected in the lateral ventricle a decrease in respiration rate occurred, but when the brain was perfused from the lateral ventricle to the cisterna magna increases in respiration rate occurred. These data, combined with the facts that secretin and secretin receptors have been identified in the brain indicate that secretin may play a neurotransmitter or neuroregulator role in the central nervous system. Secretin
Novel-object approaches
Open field locomotor activity
Respiratory
Defecation
pharmacological effects of secretin injected into the cerebral ventricles of the rat.
SECRETIN, a 27 amino acid polypeptide, was thought to be localized solely in the gut, until recently when secretin-like bioactivity was identified in extracts of porcine brain [13], and secretin immunoreactivity was identified and characterized in both pig and rat brain [4,14]. The effects of secretin on peripheral systems and its interaction with peripheral receptors are well documented [2, 5, 6, 7, 10, 11, 12, 16, 17]. However, the actions of secretin on CNS target cells were virtually unknown until recently. Secretin receptors similar to that characterized in the pancreas have recently been identified in the brain [8]. Furthermore, a secretin sensitive adenylate cyclase has been identified on neuroblastoma × glioma hybrid cells [15] and in cultured mouse brain cells [3]. The precise role of secretin in the CNS is unknown at present although intraventricular administration of the peptide appears to alter dopamine turnover rates in nucleus accumbens, olfactory tubercle and median eminence which also results in reduction of prolactin secretion from the anterior piutitary [9]. The existence of secretin and secretin receptors in the brain coupled with the observation that secretin affected CNS dopamine, cyclic AMP and prolactin, suggest that the peptide may serve as a neurotransmitter or neuromodulator in the CNS. The purpose of this study was to investigate what roles secretin in the CNS may serve by observing
METHOD
Animals Sprague-Dawley male rats weighing 250--300 g (ZivicMiller, Allison Park, PA) were used. The rats were acclimatized for at least one week, 6 to a cage, in a colony room with 12 hr light and 12 hr dark cycle. Water and food were supplied ad lib.
Surgery and Protocol Under chloropent anesthesia (Fort Dodge Labs. Inc., Fort Dodge, IA) a stainless steel guide cannula was stereotaxtically inserted into the lateral ventricle of each rat. Each cannula was affixed in place with dental cement secured to the skull with the aid of two screws. To prevent clogging, a removable 27 gauge wire was inserted into the guide cannula. The placement of the cannula with reference to bregma was 1.4 mm lateral, 0.5 mm caudal and a depth of 2.5 mm from the surface of the skull. The rats were allowed to recover for 4 days before the tests. Injections were made in the lateral ventricle 5 mm from the surface of the skull, via
739
C H A R L T O N ET AL.
740
A. DEFECATION RATE
a premeasured cannula attached by polyethylene tubing (PE20) to a 25/zl Hamilton syringe containing either phosphate buffered saline (PBS) at p H 7.4 or 5 /~g of secretin (Peninsula Lab, San Carlos, CA) dissolved in 5/xl PBS. All tests were performed between 1400 hr and 1700 hr. Each rat was used in one test only except that the rats used for determination of defecation rate were reutilized for the determination of novel object approaches and rearing without further treatments. The data are expressed as the mean-+the standard error of the mean. Differences between two means were determined using Student's two tailed t-test, utilizing a Monroe 1860, calculator (Morristown, N J).
PBS=SALINE SEC=SECRETIN
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Apparatus and Procedures Defecation rate determination. To measure defecation rate a total of 6 rats from each group (PBS and secretin) were placed in individual cages (12× 12×9") immediately after injection. The number of fecal boluses deposited by each rat for 1 hour post-injection was counted. Novel-object approaches and rearing. Immediately after the 1 hour defecation test, the rats were placed individually in an arena for the assessment of rearing and the number of approaches made to a novel object placed in the center of the arena's floor. The test time at 1 hour post-injection was selected because a pilot study showed that the decrease in novel-object approaches by secretin treated rats stabilized at about 1 hour after injection. Each rat was observed for 10 minutes. The arena consisted of a card-board box measured 21x 18x 18". The deep box was used to enhance vertical motor-activity in the rats. The novel-object was a small (21/2x3×5 ") perforated metal container. Each time a rat stood erect on its hind legs or when it sniffed or touched the novel object with its snout an incident of rearing or novelobject approaches was scored, respectively. Open-field activity. Another group of 10 surgically prepared rat (5 injected with secretin and 5 with PBS) was studied in an open field apparatus. The apparatus consisted of 3 ' × 3'×6" wooden box, painted black and covered with a steel grill. The floor was divided into 16 squares, 9"x9" each. Tests were done in a quiet room with an ambient temperature of 23+--I°C and approximately 60 foot-candles of cool white fluorescent light. The rats were tested at 1 hour post-injection as for the novel object approaches test. The number o f squares entered during a 5 minute observation period and the number of one-minute periods that the animals remained immobile were determined. The latter procedure was necessary to determine whether changes in activity were associated with a propensity to remain immobile or to changes in the rate of movement. Respiration. Each rat was anesthetized with chloropent and put in a stereotaxic apparatus for the duration of the procedure. A hole was drilled into the skull, using the cordinate described above and after incision and retraction of the overlying tissues. Injections of PBS or secretin in 6 rats each were made stereotaxically into the lateral ventricle using a 25 /xl Hamilton syringe with a 27 gauge needle. Another group of 5 aneshtetized rats was injected intravenously (femoral vein) with 5/zl PBS or 5 ~g secretin in 5/xl PBS. Respiration was counted immediately after and at 2, 5, 10, 20, 30 and 40 minutes post-injection. Since secretin injected in a 5/zl volume may localize at the point of injection, a brain perfusion technique was adopted so that the effects of secretin could be observed
PBS
SEC
FIG. 1. The effect of secretin on defecation rate in rats. Rats were injected intracerebro-ventricularly with phosphate buffered saline (PBS) or secretin (SEC) dissolved in PBS (5 ~g in 5 txt). Animals were placed in individual cages to determine defecation rate (boluses of feces deposited in 1 hour). Open column=PBS. Stippled column=SEC. N=6 *p<0.05.
following perfusion of the cerebral ventricles with the peptide. Artificial C S F (NaCI 8.89 g; KCi 0.25 g; CaC12 2HeO 0.11 g; NaH2PO4H20 0.07 g; urea 0.13 g; glucose 0.61 g per liter) was delivered into the lateral ventricle by gravity, utilizing a 27 gauge stainless steel cannula attached by polyethylene tubing (PE20) to a dispensing vessel (usually a 1.5 ml pipette tip). A 25 gauge cannula attached to a Sage Model 351 syringe pump (Sage Instruments Div. Orion Research Inc., Cambridge, MA) was used to withdraw the peffusate, via the cisterna magna at a rate of 25/xl per minute. Respiration rate was counted with the aid of a stop watch, before perfusion, during perfusion for a 20 minute period with artificial C S F and with secretin (20 ng//xl) in artificial CSF. The pH of the perfusate was also measured. RESULTS
Defecation The injection of secretin (5 /xg in 5 /xl PBS) caused a statistically significant (.o<0.05) increase in defecation rate during the first post-injection hour (Fig. 1). An average of 7.0-+1.31 boluses per rat was deposited by the secretin treated group whereas the PBS controls deposited an average of 3.0-+0.81 boluses each during the same period.
Novel-Object Appraoches and Rearing The number of novel-object approaches made by the secretin-treated rats was significantly less (p <0.05) than the number of approaches made by the PBS group. A score of 20.0+ 1.4 approaches was made by each rat treated with secretin while each rat injected with PBS approached the novel-object 34.8-+0.9 times (Fig. 2A). Rearing was essentially identical in both groups with 56.7-+6.6 for each secretin-treated rat and 51.2-+ 3.8 for each PBS control (Fig. 2B). Other stereotypic behaviors e.g., gnawing, sniffing, licking and grooming appeared to be of low incidence and preliminary results showed that secretin injections had no significant effects on these behaviors.
CNS EFFECTS OF SECRETIN
A. NOVEL-OBJECT APPROACHES PBS=SALINE SEC=SECRETIN
741 OPEN-FIELD LOCOMOTOR ACTIVITY
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FIG. 2. The effects of secretin on novel-object approaches (A), and the incidence of rearing (B) in rats. Rats were injected (ICV) with PBS or SEC (5/~g) and were tested at 1 hour post-injection. Open column=PBS. Stippled column=SEC. N=6. *p<0.05.
PBS
SEC
FIG. 3. The effect of SEC on open-field locomotor activity in rats. Rats were injected with PBS or 5 ~g secretin. The data represent the average number (mean_ SE) of squares entered by each rat during 5 minutes observation at 1 hour post-injection. Open column=PBS. Stippled column=SEC. N=5, *p<0.05.
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MINUTES
FIG. 4. The effects of secretin and PBS on respiration rate in anesthetized rats. Rats were anesthetized with Chloropent. When a steady respiration rate was achieved PBS or secretin (5/~g ICV) was injected and the respiration rate determined. *p<0.05 for secretin (ICV) vs. PBS (ICV) or secretin (IV).
Open-field Activity As s h o w n in Fig. 3, injection of secretin into the lateral ventricle resulted in a significant d e c r e a s e in o p e n field loc o m o t o r activity, c o m p a r e d to controls. The n u m b e r of squares entered by e a c h secretin treated rat during a 5 minute o b s e r v a t i o n period was 65.0+ 15.3 c o m p a r e d to 116.2+_8 for e a c h PBS treated rat. Additionally, when the average n u m b e r of one-minute periods of immobility was d e t e r m i n e d during the test session the group treated with secretin remained immobile for an average of 4.33+_0.95 periods as
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0
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FIG. 5. Shown are the respiration rates during perfusion of the rat brain. The open columns represent the respiration rate before perfusion, the cross-hatched columns during artifical CSF perfusion (26 p,l/min) and the stippled columns during perfusion with secretin (20 ng/p,l in artificial CSF). The upper curves represent the pH values for the corresponding perfusate. The first value is the pH of the unperfused CSF.
c o m p a r e d with 2.0---0.91 periods for the PBS control group (p <0.05---not significant).
Respiration Secretin (5 /zg) injected into the lateral ventricle of anesthetized rats gradually d e c r e a s e d respiration rate, w h e r e a s intravenous injection of secretin did not show any effect (Fig. 4). Perfusion of the brain of anesthetized rats with 20 ng//zl of secretin, h o w e v e r , markedly increased the respiration rate (Fig. 5). This effect of secretin was promptly
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CHARLTON E T A L .
reversed when the perfusion of secretin was substituted with artificial CSF without secretin. The changes in respiration rate did not show any relationship with the pH of the perfusate. The respiratory and behavioral effects of secretin were only observed after intraventricular administration of the peptide. Intraperitoneal injections of identical doses of secretin did not affect behavior or respiration rate. DISCUSSION The results of this study demonstrated that the ICV injection of secretin significantly decreased open-field activity and novel-object approaches and increased defecation rate in freely moving rats. In addition, an ICV injection of secretin in anesthetized rats decreased respiration rate. The change in open-field locomotor activity elicited by secretin is probably not indicative of the effect of a nonspecific depressant. Rearing was not altered, there was no abnormality in gait, no apparent changes in stereotypic behavior (e.g., sniffing, licking, grooming and gnawing). In addition, the ICV injection of secretin did not alter the ability of rats to balance on a horizontal bar, climb an inclined plane nor did it change the time required for rats to regain the righting reflex following barbiturate anesthesia (unpublished data). By determining the number of one-minute periods that the rats remained immobile it was shown that the decreased locomotion was probably due to a decreased propensity to
initiate locomotor responses. The combination of decreased open field activity, decreased novel-object approaches and increased defecation rate might be interpreted to indicate fear or changes in emotional state (see [1]) of the secretin injected animals, but since the test environments may initiate exploratory activity as well as evoke fear in the rats the precise behavioral substrate affected by secretin administration is unclear. The secretin induced decrease in respiration rate in anesthetized rats seems to be independent from the effect on open-field activity, in that respiration rate returned to control values within one hour, while effects on open field activity can last for days (in preparation). Although more difficult to quantitate, decreased respiration rate also appeared in freely moving rats following ICV secretin administration. When the anesthetized rat brain was perfused between the lateral ventricle and the cisterna magna with secretin (20 ng//xl) in artificial cerebrospinal fluid, there was an increase in respiration rate as opposed to the decrease in respiration rate seen after ICV secretin injections. These data indicate that secretin at the doses studied may have opposite respiratory actions on forebrain and hind brain regions. Secretin [4,14], secretin receptors [8] and secretin sensitive adenylate cyclase [3,15] are present in neuronal tissues. These observations coupled with the fact that intraventricular administration of secretin caused behavioral and physiological changes in rats indicate that secretin may act as a neurotransmitter or neuroregulator in the CNS.
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9. Fuxe, K., K. Anderson, T. Hokfelt, V. Mutt, L. Ferland, F. F. Agnati, D. Ganten, S. Said, P. Eneroth and J. A. Gustafsson. Localization and possible function of peptidergic neurons and their interactions with central catecholamine neurons and the central actions of gut hormones. Fed Proc 38: 2333-2340, 1979. 10. Goodhead, B., H. S. Himal and J. Zanbilowicz. Relationship between pancreatic secretion and pancreatic blood flow. Gut 11: 62-68, 1970. 11. Hubel, K. A. Secretin: A long progress note. Gastroenterology 62: 318-341, 1972. 12. Lazarus, R. N., N. R. Voyes, S. Devrim, T. Tanese and L. Recant. Extragastrointestinal effects of secretin, gastrin and pancreozymha. Lancet 2: 248-250, 1968. 13. Mutt, V., M. Cariquist and K. Tatemoto. Secretin-like bioactivity in extracts of porcine brain. Lift, Sci 25: 1703-1708, 1979. 14. O'Donohue, T. L., C. G. Charlton, R. L. Miller, G. Boden and D. M. Jacobowitz. Identification, characterization and distribution of secretin immunoreactivity in rat and pig brain. Proc Natl Acad Sci USA 78: 5221-5224, 1981. 15. Propst, F., L. Moroder, F. Wunsch and B. Hamprecht. The influence of secretin, glucagon and other peptides, of amino acids, prostaglandin, endoperoxide analogues and diazepam on the level of adenosine 3' 5'-cyclic mono phosphate in neuroblastoma x glioma hybrid cells. J Neurochern 32: 1495-1500, 1979. 16. Ross, G. Cardiovascular effects of secretin. Am J Physiol 218: 1116-1170, 1970. 17. Rudman, R. and A. E. Del Rio. Lipolytic activity of synthetic porcine secretin. Endocrinology 85: 214-217, 1969.