AJP - Regu Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 286: R927-R934, 2004. First published January 8, 2004; doi:10.1152/ajpregu.00600.2003
0363-6119/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
286/5/R927    most recent
00600.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (12)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yang, B.
Right arrow Articles by Ferguson, A. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, B.
Right arrow Articles by Ferguson, A. V.

COMPLEX FUNCTION OF THE CENTRAL NERVOUS SYSTEM, SLEEP AND LOCOMOTION

Secretin depolarizes nucleus tractus solitarius neurons through activation of a nonselective cationic conductance

Bo Yang,1 Martin Goulet,2 Richard Boismenu,2 and Alastair V. Ferguson1

1Department of Physiology, Queen's University, Kingston, Ontario K7L 3N6, Canada; and 2Repligen, Waltham, Massachusetts 02453

Submitted 15 October 2003 ; accepted in final form 30 December 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
The recent suggestion that secretin may be useful in treating autism and schizophrenia has begun to focus attention on the mechanisms underlying this gut-brain peptide's actions in the central nervous system (CNS). In vitro autoradiographic localization of 125I-secretin binding sites in rat brain shows the highest binding density in the nucleus tractus solitarius (NTS). Recent evidence suggests that intravenous infusion of secretin causes fos activation in NTS, a relay station playing important roles in the central regulation of autonomic functions. In this study, whole cell patch-clamp recordings were obtained from 127 NTS neurons in rat medullary slices. The mean resting membrane potential of these neurons was -54.7 ± 0.3 mV, the mean input resistance was 3.7 ± 0.2 G{Omega}, and the action potential amplitude of these neurons was always >70 mV. Current-clamp studies showed that bath application of secretin depolarized the majority (80.8%; 42/52) of NTS neurons tested, whereas the remaining cells were either unaffected (17.3%; 9/52) or hyperpolarized (1.9%; 1/52). These depolarizing effects were maintained in the presence of 5 µM TTX and found to be concentration dependent from 10-12 to 10-7 M. Using voltage-clamp techniques, we also identified modulatory actions of secretin on specific ion channels. Our results demonstrate that while secretin is without effect on net whole cell potassium currents, it activates a nonselective cationic conductance (NSCC). These results show that NTS neurons are activated by secretin as a consequence of activation of a NSCC and support the emerging view that secretin can act as a neuropeptide within the CNS.

gut-brain peptide; central nervous system; electrophysiology; patch clamp

SECRETIN, a 27-amino acid peptide, was the first hormone identified. In a series of experiments conducted in early 1900s, Bayliss and Starling (3, 4) showed that intravenous injection of a mucosal extract from the duodenum induced release of bicarbonate and water from the pancreas. Therefore this chemical messenger was named "secretin." Secretin actions in the digestive system have been well documented (17, 19, 27, 28, 33, 38, 47). Recent reports suggesting a potential therapeutic application for secretin in treating autistic children (15, 24) and schizophrenia patients (40) have begun to focus attention on the mechanisms underlying this gut-brain peptide's actions in the central nervous system (CNS).

The native peptide secretin, its receptors (8, 16, 21, 30, 48), secretin-like immunoreactivity, and bioactivity (29, 32) have all been detected in the brain. Intracerebroventricular administration of secretin upregulates dopamine turnover and tyrosine hydroxylase activity in the hypothalamus and inhibits prolactin release (1, 10, 39). In addition, recent evidence has suggested that after intravenous infusion, secretin can cross the blood-brain barrier to reach many brain regions (2). Intravenous secretin activates fos expression in several brain regions, including the nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMNV) (11). Stimulation of cAMP production by secretin was observed in cultured brain cells and in brain slice preparations (9, 37, 44). Moreover, secretin was found to facilitate evoked, spontaneous, and miniature inhibitory postsynaptic currents (IPSCs) in Purkinje cells in rat cerebellum (53). While highly suggestive of secretin actions in NTS, this literature does not definitively identify the source of secretin as peripheral or central.

In vitro autoradiographic localization of 125I-secretin receptor binding sites in rat brain shows the highest binding in the NTS (31). Located in the dorsomedial medulla oblongata, NTS is widely accepted as a pivotal brain region involved in the integration of cardiovascular, respiratory, gustatory, hepatic, and renal control mechanisms (25). NTS receives afferent input from and sends efferent output to many CNS areas, including essential autonomic control centers in the hypothalamus, midbrain, and spinal cord (52).

Collectively these observations support the hypothesis that NTS represents an important CNS site where endogenous and/or infused secretin could act to influence central autonomic regulation as well as other brain-mediated activities. The present electrophysiological study was designed to test the hypothesis that secretin exerts direct effects on the excitability of NTS neurons. Having identified such effects, our studies were extended to describe the modulatory roles of secretin on specific ion channels of NTS neurons.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Medullary slice preparation. Male Sprague-Dawley rats (110-140 g, Charles River, PQ, Canada) were decapitated, and the brain stem was quickly removed from the skull and immersed in cold (0-2°C) artificial cerebrospinal fluid (ACSF). Four-hundred-micrometer-thick medullary slices including NTS were cut using a vibratome (Leica VT1000S, Leica Microsystems, Nussloch, Germany) and incubated in oxygenated ACSF (95% O2-5% CO2) for at least 90 min at room temperature. Before recording, slices were transferred into an interface-type recording chamber and continuously perfused with oxygenated ACSF through a gravity pressure perfusion system. The ACSF flow rate was adjusted to ~1.5 ml/min and maintained constant throughout the entire recording period. All experiments were performed at room temperature (21-22°C). All procedures conformed to the standards outlined by the Canadian Council on Animal Care, and protocols were approved by the Queen's University Animal Care Committee.

Electrophysiological methods. Whole cell patch recordings were obtained using the whole cell configuration of the blind gigaseal patch-clamp technique to record from NTS neurons (50, 51), most of which were located in the commissural region of the nucleus. Electrodes of 4- to 7-M{Omega} resistance were pulled from TW150F-6 glass micropipettes (World Precision Instruments, Sarasota, FL) on a horizontal Flaming/Brown micropipette puller (model P-97, Sutter Instrument, Novato, CA) and were filled with the appropriate filling solution (see Experimental solutions). After establishment of >1-G{Omega} seal, a brief suction pulse was applied to rupture the membrane and achieve whole cell configuration. Signals were amplified and processed using an AxoClamp 2B (Axon Instruments, Union City, CA) amplifier. Series resistance (<15 M{Omega}) was not compensated. An Ag-AgCl electrode connected to the bath solution via a KCl-agar bridge served as reference. After recording from each NTS neuron, the pipette was withdrawn from the cell membrane, the remaining junction potential was measured (4-8 mV), and the appropriate correction was applied to all data. Drugs were applied by switching perfusion from ACSF to a solution containing the desired drug(s). In addition, the concentration of EGTA in the pipette solution was increased from 1.1 to 10 mM to decrease the Ca2+ concentration in the internal solution in one set of experiments. EGTA is a rather slow Ca2+ buffer; consequently, a faster buffer such as BAPTA might reveal a calcium contribution that EGTA fails to show. All signals were filtered at 3 kHz, digitized using the CED 1401 plus interface [Cambridge Electronic Design (CED), Cambridge, UK] at 5 kHz, and stored on computer for offline analysis. Data were collected using the Signal (episode-based capture) or Spike2 (continuous recording) packages (CED). Leak current was routinely subtracted using the option offered by the Signal program (CED).

Cells were defined as neurons by the presence of at least 70-mV action potentials (current-clamp recordings) or by the presence of large rapid voltage-activated inward currents, which were blocked by TTX (voltage-clamp recordings).

Experimental solutions. The standard internal pipette solution contained (in mM) 140 K-gluconate, 0.1 CaCl2, 2 MgCl2, 1.1 EGTA, 10 HEPES, and 2 Na2ATP and was adjusted to pH 7.25 with KOH. In experiments examining the role of Ca2+ in activating the NSCC, the concentration of EGTA in the pipette solution was increased from 1.1 to 10 mM. The control bath solution consisted of ACSF (in mM): 124 NaCl, 2 KCl, 1.25 KH2PO4, 2.0 CaCl2, 1.3 MgSO4, 20 NaHCO3, and 10 glucose. Osmolarity was maintained between 285 and 300 mosM and pH between 7.3 and 7.4.

Peptides and drugs. Secretin and [{beta}-Asp3]secretin (Repligen, Waltham, MA) were prepared on the day of experiment by diluting 50-µl aliquots of 10-5 M stock solution stored at -70°C to concentrations ranging from 10-12 to 10-7 M in ACSF. In experiments where synaptic transmission was blocked, TTX (5 µM) was added to external solutions, and blockade of Na+ channels was confirmed when either depolarizing current pulses to 0 mV failed to elicit fast spikes (current-clamp recordings) and/or the large rapid voltage-activated inward currents were abolished (voltage-clamp recordings). In voltage-clamp experiments where K+ channel activities were examined, TTX (5 µM) was added to external solutions to block the Na+ channels. All of these drugs were prepared on the day of experiment by diluting stock solutions stored at appropriate temperatures into ACSF. All chemicals, unless otherwise stated, were obtained from Sigma Chemical (St. Louis, MO).

Definition of response. A series of hyperpolarizing current pulses were applied to determine the identity of each neuron as a delayed excitation (DE), postinhibitory rebound (PIR), or neither DE nor PIR (NON) cell based on its electrophysiological fingerprint (45). Neurons were required to maintain a stable baseline for at least 2 min before application of test agents. A response to secretin was arbitrarily defined as a sustained change in membrane potential of >3 mV. In those spontaneously firing neurons, we evaluated this in time-expanded traces by looking at membrane potential between spikes, which still made up most of the recording time as peak frequency even during excitatory effects seldom exceeded 10 Hz.

Statistical analysis. For statistical analysis of effects of secretin on NTS neurons under various conditions, means were calculated from cells that were determined to have been affected using the above criteria. Changes in membrane potentials and net whole cell K+ conductances in response to secretin were compared using the Student's t-test. A minimum probability value of P < 0.05 was selected to determine significance. All values are plotted as means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Whole cell recordings were obtained from a total of 127 NTS neurons. All of these cells demonstrated action potentials with amplitude of >70 mV (arbitrary minimum cutoff for inclusion), and they had a mean resting membrane potential of -54.7 ± 0.3 mV and a mean input resistance (IR) of 3.7 ± 0.2 G{Omega}.

Secretin depolarizes NTS neurons. Current-clamp recordings from a total of 52 NTS cells showed that 82.7% (43 of 52) of this population responded to bath perfusion of secretin (see the criteria established in MATERIALS AND METHODS), whereas the remainder of neurons tested did not respond in a sustained manner and were therefore classified as nonresponders. Depolarization was the predominant effect caused by secretin exposure (42 of 52 cells, 80.8%). Similar proportions of DE (77.8%; 14 of 18), PIR (82.4%; 14 of 17), and NON (82.4%; 14 of 17) cells were found to be responsive to 10-8 M secretin. Responses were also quantitatively similar [mean depolarization for each group was 9.7 ± 0.9 mV (DE cells), 9.8 ± 0.8 mV (PIR cells), and 10.5 ± 3.8 mV (NON cells)], and therefore these cell types were grouped together for all subsequent analysis.

Depolarizations usually occurred within 2 min of secretin reaching the slice and were normally accompanied by a rapid increase in firing frequency of action potentials. Effects of secretin lasted for 7-12 min, and after washout of secretin, membrane potential and action potential frequency returned to/toward control levels as shown in Fig. 1A. Figure 1A, bottom, shows expanded time scales from the same recording (before, during, and after bath application of secretin) illustrating action potentials (truncated) and postsynaptic potentials (of up to 10 mV) with baseline noise <1 mV. In 12 cells excited by 10-8 M secretin, the mean depolarization was 9.9 ± 0.9 mV. Secretin-induced depolarizations were accompanied by a significant decrease in IR as measured by the voltage responses to hyperpolarizing current pulses [control (4.0 ± 0.3 G{Omega}) vs. 10-8 M secretin (3.0 ± 0.4 G{Omega}), P < 0.05, n = 8; Fig. 1, B and C], effects that were still observed when membrane potential was returned to baseline with injection of hyperpolarizing current before assessment of input resistance.



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 1. Secretin depolarizes nucleus tractus solitarius (NTS) neurons and decreases their input resistance. A, top: whole cell current-clamp recording from an NTS neuron demonstrates that bath administration of secretin (represented in this and other figures by the horizontal bar above each trace) resulted in rapid sustained depolarization accompanied in most cases by a rapid increase in firing frequency of action potentials. After washout of secretin, the membrane potential and action potential frequency returned to control levels. Scale bars represent 60 s (horizontal) and 10 mV (vertical). The dashed line indicates baseline membrane potential. A, bottom: expanded time scales from the same recording (before, during, and after bath application of secretin). These traces illustrate action potentials (truncated) and postsynaptic potentials (of up to 10 mV) with baseline noise <1 mV. Scale bars, 0.5 s (horizontal) and 10 mV (vertical). B: secretin also caused consistent decreases in input resistance as shown in the current-voltage relationships obtained from this NTS neuron during application of control artificial cerebrospinal fluid (ACSF; {blacksquare}) and ACSF containing 10-8 M secretin ({circ}). Successive hyperpolarizing pulses (-1 to -6 pA) were delivered and the peak changes in membrane potential were measured. C: bar chart shows summary data illustrating statistically significant effects of secretin on input resistance in NTS cells: control (4.0 ± 0.3 G{Omega}) vs. 10-8 M secretin (3.0 ± 0.4 G{Omega}), *P < 0.05, n = 8.

 

To determine if the observed actions of secretin were due to direct effects on NTS neurons, six neurons that responded to 10-8 M secretin were tested with a second application of secretin during the blockade of action potentials by bath administration of TTX (5 µM) (Fig. 2, A and B). After treatment with TTX, bath administration of secretin elicited a similar depolarizing response in all six cells tested (8.9 ± 0.7 vs. 9.9 ± 0.9 mV without TTX, n = 12, P = 0.43) (Fig. 2C).



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 2. Secretin depolarizes NTS neurons in the presence of TTX. A and B: current-clamp recordings illustrate the responses of a single NTS neuron to bath application of 10-8 M secretin in normal ACSF (A) and in ACSF containing 5 µM TTX (B). As illustrated in this example, all cells tested in this way showed maintained depolarizations in response to secretin in the presence of TTX. Scale bars represent 60 s (horizontal) and 10 mV (vertical), while the dashed horizontal lines indicate baseline membrane potentials. C: after treatment with TTX, bath administration of secretin elicited a similar depolarizing response in all 6 cells tested (8.9 ± 0.7 vs. 9.9 ± 0.9 mV without TTX, n = 12, P = 0.43).

 

Similar reversible depolarizing responses, normally accompanied by increases in spike frequency, were also recorded from NTS neurons in response to exposure to 10-7, 10-9, and 10-11 M secretin as illustrated in Fig. 3A. These effects of secretin were repeatable as a second bath application of the peptide resulted in similar changes in membrane potential. Analysis of group mean depolarization recorded from NTS neurons in response to secretin concentrations ranging from 10-12 to 10-7 M demonstrated these effects were concentration dependent as illustrated in Fig. 3B (EC50 = 8.2 x 10-10 M). Although NTS neurons did not depolarize significantly (<3 mV, see the criteria established in MATERIALS AND METHODS) in response to 10-12 M secretin, all neurons tested with this concentration were included as a group to complete the concentration-response curve.



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 3. Secretin depolarizes NTS neurons dose dependently. A: these current-clamp recordings from 3 separate NTS neurons show examples of the depolarizing effects of secretin administered at varying concentrations. B: graph summarizes the depolarizing effects of secretin on NTS neurons and shows them to be concentration dependent. Changes in membrane potential in response to 10-12 (n = 7), 10-11 (n = 7), 10-10 (n = 7), 10-9 (n = 5), 10-8 (n = 12), and 10-7 M secretin (n = 4) are plotted against bath secretin concentrations. The effect of each concentration is presented as mean ± SE. These data were then fitted to a sigmoid concentration-response function, and the corresponding curve of best fit is overlaid allowing estimation of EC50 = 8.2 x 10-10 M.

 

In view of the present lack of high-affinity specific secretin receptor antagonists (54), we used the nonfunctional analog [{beta}-Asp3]secretin (5, 46) as a control to look at the interaction between secretin and its receptor. Similar current-clamp recordings from a total of eight NTS neurons showed that only two cells were slightly depolarized (<3 mV, see the criteria established in MATERIALS AND METHODS) by bath application of [{beta}-Asp3]secretin (10-8 M), while the remaining six cells did not respond. Summary data show a statistically significant difference between effects of 10-8 M secretin and 10-8 M [{beta}-Asp3]secretin on membrane potential of NTS cells [9.9 ± 0.9 mV (n = 12) vs. 1.3 ± 0.6 mV (n = 8), P < 0.05].

Secretin is without effect on whole cell K+ currents in NTS neurons. Multiple ion channels are known to be involved in the regulation of neuronal excitability (13, 26). Our previous work demonstrated that orexin-A inhibits a specific potassium conductance (the sustained K+ current, IK) in NTS neurons (50, 51). We therefore used voltage-clamp techniques (49-51) to examine the effects of secretin on net whole cell K+ currents (measured at both the peak and the sustained values) evoked in response to 20-mV depolarizing voltage steps (0.5 s) applied from holding potentials of -100 mV (with 5 µM TTX in ACSF) before and after bath application of secretin. A typical response of an NTS neuron to secretin (10-8 M) illustrated in Fig. 4A shows no change of the net whole cell K+ currents induced by secretin. The summary data presented in Fig. 4B support the conclusion that NTS neurons (n = 13) exhibit no change in whole cell K+ currents during exposure to secretin (10-8 M). Figure 4B, inset, shows summary data of peak and sustained K+ currents evoked by the +40-mV voltage step from a holding potential of -100 mV before and during bath application of 10-8 M secretin (n = 13), illustrating secretin does not affect these currents.



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 4. Secretin does not affect K+ currents in NTS neurons. A: net whole cell K+ currents were recorded from NTS neurons in response to voltage steps (0.5 s) in 20-mV increments from a holding potential of -100 mV (ACSF contains 5 µM TTX to block Na+ currents). Left and right panels show the whole cell currents before (control) and during secretin (10-8 M) exposure, respectively. Whole cell K+ currents were not affected by secretin exposure. Scale bars represent 250 ms (horizontal) and 100 pA (vertical). B: summary data of whole cell K+ conductances (measured at the peak as well as the sustained values) recorded before ({blacksquare}, ) and during ({square}, {circ}) bath application of 10-8 M secretin (n = 13), illustrating secretin does not affect these currents. Inset: bar chart shows summary data of peak and sustained K+ currents evoked by the +40-mV voltage step from a holding potential of -100 mV before and during bath application of 10-8 M secretin (n = 13), illustrating secretin does not affect these currents.

 

Secretin activates NSCC of NTS neurons. In view of the considerable literature demonstrating peptidergic effects on neuronal excitability occurring as a consequence of the modulation of nonselective cationic conductances (14, 18) as well as our own recent studies identifying NSCC in area postrema (49) and NTS (50, 51) neurons as mediators of orexin-A actions, we next used slow voltage ramps (-100 to 0 mV in 10 s) after a prepulse to -100 mV (0.5 s) to determine if secretin influenced NTS neurons as a consequence of activation of such conductances. The data presented in Fig. 5A show average currents recorded from an NTS neuron in response to such ramps (each trace is the mean of 5 ramps) recorded before, during, and after bath administration of secretin (10-8 M). Figure 5A, inset, illustrates the difference current (i.e., secretin-induced current) obtained by subtracting control ramps from those obtained during secretin. Application of secretin (10-8 M) caused a clear change in this ramp-evoked current, and ~6-11 min after replacement of secretin with ACSF, the current recovered toward control levels. Similar effects of secretin (10-8 M) were observed in 13 of 16 (81.3%) cells tested, a proportion that closely matches the proportion (80.8%) of NTS neurons depolarized by secretin in our current-clamp experiments. The mean secretin-evoked current for this group of responsive neurons is shown in Fig. 5B (closed squares) and was found to be linear throughout the voltage range tested (r2 = 0.98), indicating a lack of voltage dependence. This conductance is voltage independent across the voltage scale of the slow ramp, which indicates that it is a NSCC (6, 14, 18). The mean reversal potential of the secretin (10-8 M)-sensitive current was -45.6 ± 2.1 mV (such values were obtained after subtraction of junction potentials), and the mean conductance of this NSCC is 0.33 ± 0.02 nS (n = 13).



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 5. Secretin activates a nonselective cationic conductance in NTS neurons. A: mean whole cell currents (each trace is the mean of 5 ramps) evoked from slow depolarizing (10 mV/s) voltage ramps before (Control), during (Secretin; dashed line), and after exposure (Washout) to secretin (10-8 M). A, inset: the difference current obtained by subtracting the control current from the current recorded during secretin application. This represents the secretin-evoked current. B: summary graph illustrates the mean ± SE secretin (10-8 M)-evoked current for responsive neurons (n = 13) ({blacksquare}). The mean reversal potential of the secretin (10-8 M)-sensitive current is -45.6 ± 2.1 mV, and the mean conductance of this nonselective cation conductance (NSCC) is 0.33 ± 0.02 nS (n = 13). It also shows that [{beta}-Asp3]secretin (10-8 M) does not activate this NSCC in NTS neurons (n = 8) ({square}).

 

We also examined the role of intracellular Ca2+ in activating this NSCC in experiments where we decreased the theoretical concentration of free intracellular Ca2+ to 10% of its normal values by increasing EGTA in the pipette solution from 1.1 mM (standard) to 10 mM. Of 13 NTS neurons recorded with this high EGTA pipette solution, 10 (76.9%) showed activation of the NSCC in response to secretin (r2 = 0.98, mean reversal potential = -44.9 ± 1.6 mV and the mean conductance = 0.37 ± 0.02 nS), which was quite similar to that observed in cells recorded with standard internal pipette solution.

We usually held NTS neurons between -52 and -53 mV before secretin (10-8 M) administration in our current-clamp recordings. At these baseline membrane potentials, secretin would be expected to activate the NSCC as a 2.3- to 2.7-pA inward current (see Fig. 5B), which we calculate to evoke an 8.5- to 10.0-mV depolarization (average input resistance of NTS neurons is 3.7 G{Omega}). This predicted depolarization is fairly close to the average depolarization (9.9 ± 0.9 mV) caused by 10-8 M secretin application that we recorded in current-clamp experiments. Additional experiments were performed while the baseline membrane potentials of NTS neurons were held at -45 mV (close to the reversal potential of this NSCC) and approximately -60 mV, before bath application of secretin. None of four neurons held at -45 mV was depolarized by secretin (10-8 M), and the depolarization of cells held at approximately -60 mV was potentiated (13.4 ± 2.3 mV, n = 4), observations further supporting the conclusion that such effects were the result of activation of this NSCC. Finally, Fig. 5B (open squares) demonstrates that bath application of [{beta}-Asp3]secretin (10-8 M) did not activate this NSCC in eight of eight NTS neurons, supporting the observations of considerable loss of potency with modification of secretin NH2 terminus (5, 12, 46).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
In vitro autoradiographic localization of 125I-secretin binding sites in rat brain shows the highest binding density in the NTS (31). Secretin appears to be widely expressed within the CNS (8, 16, 21, 30, 48) where it may act as an endogenous neuropeptide. Recent evidence showed that intravenous infusion of secretin to rats leads to fos activation in several brain regions, including the NTS and DMNV (11). Secretin was reported to cross the blood-brain barrier as an intact peptide (2). Therefore, it is a possibility that peripherally administered secretin activates secretin-responsive neurons in the NTS and/or other regions. The NTS is a relay station that plays important roles in the central regulation of autonomic functions (25, 52), which were reported to be altered in autism (20, 35, 36).

Previous studies reporting electrophysiological properties and subtypes of NTS neurons in slice preparations have suggested lower input resistances for these cells (45) than we have recorded in the current study (3.7 ± 0.2 G{Omega}, n = 127) and previous reports (50, 51). These differences are most likely the result of differences in the techniques for slice preparation [we recorded in room temperature (21-22°C) vs. 31-32°C] or the exceptionally high resistance seals obtained in the present studies (often >4 G{Omega}).

Our results are the first to demonstrate that secretin influences the excitability of NTS neurons. The only previous electrophysiological study investigating secretin's actions on CNS neurons reported that secretin facilitated GABA release from presynaptic terminals contacting Purkinje cells but was without effect on membrane potential of these neurons (53). To determine if the observed depolarizing actions of secretin were due to similar presynaptic effects (53), six secretin-responsive neurons were tested with a second application of secretin during the blockade of action potentials by bath administration of TTX. The fact that these depolarizing effects were observed in the presence of TTX suggests that they are the result of direct actions on each recorded NTS neuron. The present lack of high-affinity specific secretin receptor antagonists (54) precluded a definitive identification of the secretin receptor as the mediator of these effects. However, the clear reversibility and concentration dependence of these effects and the lack of similar effects of the nonfunctional analog [{beta}-Asp3]secretin argue strongly that they are receptor mediated. The EC50 (8.2 x 10-10 M) obtained in this study is also consistent with the literature describing secretin actions (6, 8, 12, 23, 34, 41, 42).

Multiple ion channels are known to be involved in the regulation of neuronal excitability (13, 26). Our previous work demonstrated that orexin-A inhibits a specific potassium conductance (the sustained K+ current IK) in NTS neurons (50, 51). We therefore examined the effects of secretin on voltage-gated K+ currents of NTS neurons using voltage-clamp techniques. However, our data demonstrated that secretin did not affect whole cell voltage-dependent K+ currents. In view of the clear lack of effects of the peptide on these mixed K+ currents, they were not further dissected for the current analysis, although separate experiments have shown that IA, ID, and IK all likely contribute to the total current we examined, and we would thus conclude that secretin is without effect on any of these current.

NSCCs are voltage-independent membrane channels, which allow passage of cations (Na+, K+, or Ca2+) in varying proportions (22). These channels have been shown to participate in controlling neuronal excitability in many systems, including generation of the depolarizing phase of bursting pacemaker activity in Aplysia burst-firing neurons (22) and in the intrinsic activation of rat supraoptic neurons by hyperosmotic stimuli (6), neurotensin (18), and P2 purinoceptor agonists (14). In addition, our previous work has demonstrated that orexin-A depolarizes rat area postrema and NTS neurons through activation of NSCC (49, 50, 51). The results from the current study illustrate direct reversible effects of secretin on a NSCC (voltage-clamp experiments) in a proportion of NTS neurons similar to that depolarized by the peptide (current-clamp experiments). Such effects of secretin on this NSCC likely explain the depolarization of NTS neurons in response to the peptide, especially in view of the close correlation between the predicted (obtained by calculation using biophysical features of cells and conductance) and recorded potential changes. Our data suggest that this NSCC is not activated by cytoplasmic Ca2+. These data suggest the involvement of alternative second messenger system(s) such as activation of adenylyl cyclase and a rise in intracellular cAMP in mediating secretin effects as already demonstrated in nonneuronal target tissues (27, 43) as well as neuronal tissues (9, 44, 53). However, given the small size of these NTS neurons, it seems likely that small molecules like cAMP might be completely dialyzed thru the patch pipette. Therefore, it is quite possible that other signal transduction mechanism(s) might also be involved. In fact, our own recent work (50, 51) identified that orexin-A depolarizes NTS neurons through effects on nonselective cationic and K+ conductances, and these excitatory effects are mediated by phospholipase C and protein kinase C pathways. This study and our previous papers demonstrating orexin-A effects on a NSCC in rat NTS neurons (50, 51) suggest that the modulation of this conductance by different neuropeptides may represent a common mechanism through which they exert control over neuronal excitability in NTS. The signal transduction mechanisms underlying secretin's modulation of the NSCC in NTS neurons have not been examined in the present study.

Although the electrophysiological consequences of secretin actions on NTS neurons in increasing their excitability are clear, the question still arises as to the physiological implications and the potential therapeutic benefits of such effects in autism and schizophrenia. Studies have demonstrated important roles for these neurons in cardiovascular, respiratory, neuroendocrine, and gastrointestinal control, although in our slice recordings we are unable to identify the specific output of individual neurons from which we record. The homogeneity of the observed responses of NTS neurons to secretin suggests it unlikely that the physiological consequences of this peptide's action in NTS would be limited to one or another of these specific autonomic outputs. These broad excitatory actions of secretin characterized here for NTS neurons may possibly contribute to the reported beneficial therapeutic effects of this peptide in autism and schizophrenia (15, 24, 40) by modifying autonomic and/or other responses.

It should be noted that our results presented in this paper do not exclude other possible influences of secretin in the NTS. It remains possible that in addition to the direct effects on the NSCC that we have observed here, secretin may also influence other ionic conductances in NTS neurons and/or synaptic transmission in this nucleus (similar to Ref. 53). Future studies are needed to further explore these possibilities.

In conclusion, this study provides the first evidence that secretin exerts direct effects on the excitability of NTS neurons. In addition, our studies provide description of secretin's ability to directly modulate specific ion channels in NTS neurons, supporting potential neuroregulatory roles for this gut-brain peptide. Thus the findings presented here add to an emerging electrophysiological framework for understanding the effect of endogenous secretin on neuronal activity. This knowledge may help to clarify the role of this peptide when used in the clinic to treat neuropsychiatric disease states.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
DISCLOSURES
 REFERENCES
 
This work was funded by a grant to A. V. Ferguson from the Canadian Institutes of Health Research and from Repligen. A Doctoral Research Award from the Heart and Stroke Foundation of Canada supports B. Yang.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
REFERENCES
 
R. Boismenu and M. Goulet are employed by Repligen Corp., a for-profit pharmaceutical company.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. V. Ferguson, Dept. of Physiology, Queen's Univ., Kingston, Ontario, Canada K7L 3N6 (E-mail: avf{at}post.queensu.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 

  1. Babu GN and Vijayan E. Plasma gonadotropin, prolactin levels and hypothalamic tyrosine hydroxylase activity following intraventricular bombesin and secretin in ovariectomized conscious rats. Brain Res Bull 11: 25-29, 1983.[CrossRef][ISI][Medline]
  2. Banks WA, Goulet M, Rusche JR, Niehoff ML, and Boismenu R. Differential transport of a secretin analog across the blood-brain and blood-cerebrospinal fluid barriers of the mouse. J Pharmacol Exp Ther 302: 1062-1069, 2002.[Abstract/Free Full Text]
  3. Bayliss WM and Starling EH. The mechanism of pancreatic secretion. J Physiol 28: 325-353, 1902.
  4. Bayliss WM and Starling EH. Croonian lecture. The chemical regulation of the secretory process. Proc R Soc Lond B Biol Sci 73: 310-332, 1904.
  5. Beyerman HC, Grossman MI, Scratcherd T, Solomon TE, and Voskamp D. On the instability of secretin. Life Sci 29: 885-894, 1981.[CrossRef][ISI][Medline]
  6. Bourque CW. Ionic basis for the intrinsic activation of rat supraoptic neurones by hyperosmotic stimuli. J Physiol 417: 263-277, 1989.[Abstract/Free Full Text]
  7. Chow BK. Molecular cloning and functional characterization of a human secretin receptor. Biochem Biophys Res Commun 212: 204-211, 1995.[CrossRef][ISI][Medline]
  8. Fremeau RT Jr, Jensen RT, Charlton CG, Miller RL, O'Donohue TL, and Moody TW. Secretin: specific binding to rat brain membranes. J Neurosci 3: 1620-1625, 1983.[Abstract]
  9. Fremeau RT Jr, Korman LY, and Moody TW. Secretin stimulates cyclic AMP formation in the rat brain. J Neurochem 46: 1947-1955, 1986.[ISI][Medline]
  10. Fuxe K, Andersson K, Hokfelt T, Mutt V, Ferland L, Agnati LF, Ganten D, Said S, Eneroth P, and Gustafsson JA. 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.[ISI][Medline]
  11. Goulet M, Ware C, Strong R, Tay J, Shiromani P, Boismenu R, and Rusche J. Secretin activates the central amygdale in juvenile rats: treatment for autism. Soc Neurosci Abstr 134.4, 2002.
  12. Haffar BM, Hocart SJ, Coy DH, Mantey S, Chiang HC, and Jensen RT. Reduced peptide bond pseudopeptide analogues of secretin. A new class of secretin receptor antagonists. J Biol Chem 266: 316-322, 1991.[Abstract/Free Full Text]
  13. Hille B. Ionic Channels of Excitable Membranes. Sunderland, MA: Sinauer, 1992.
  14. Hiruma H and Bourque CW. P2 purinoceptor-mediated depolarization of rat supraoptic neurosecretory cells in vitro. J Physiol 489: 805-811, 1995.[ISI]
  15. Horvath K, Stefanatos G, Sokolski KN, Wachtel R, Nabors L, and Tildon JT. Improved social and language skills after secretin administration in patients with autistic spectrum disorders. J Assoc Acad Minor Phys 9: 9-15, 1998.[Medline]
  16. Itoh N, Furuya T, Ozaki K, Ohta M, and Kawasaki T. The secretin precursor gene. Structure of the coding region and expression in the brain. J Biol Chem 266: 12595-12598, 1991.[Abstract/Free Full Text]
  17. Johnson LR. The trophic action of gastrointestinal hormones. Gastroenterology 70: 278-288, 1976.[ISI][Medline]
  18. Kirkpatrick K and Bourque CW. Effects of neurotensin on rat supraoptic nucleus neurones in vitro. J Physiol 482: 373-381, 1995.[ISI]
  19. Kleibeuker JH, Eysselein VE, Maxwell VE, and Walsh JH. Role of endogenous secretin in acid-induced inhibition of human gastric function. J Clin Invest 73: 526-532, 1984.[ISI][Medline]
  20. Kootz JP and Cohen DJ. Modulation of sensory intake in autistic children: cardiovascular and behavioral indices. J Am Acad Child Psychiatry 20: 692-701, 1981.[ISI][Medline]
  21. Kopin AS, Wheeler MB, Nishitani J, McBride EW, Chang TM, Chey WY, and Leiter AB. The secretin gene: evolutionary history, alternative splicing, and developmental regulation. Proc Natl Acad Sci USA 88: 5335-5339, 1991.[Abstract/Free Full Text]
  22. Kramer RH and Zucker RS. Calcium-dependent inward current in Aplysia bursting pace-maker neurones. J Physiol 362: 107-130, 1985.[Abstract/Free Full Text]
  23. Kwon HY, Chang TM, Lee KY, and Chey WY. Vagus nerve modulates secretin binding sites in the rat forestomach. Am J Physiol Gastrointest Liver Physiol 276: G1052-G1058, 1999.[Abstract/Free Full Text]
  24. Lamson DW and Plaza SM. Transdermal secretin for autism—a case report. Altern Med Rev 6: 311-313, 2001.[Medline]
  25. Lawrence AJ and Jarrott B. Neurochemical modulation of cardiovascular control in the nucleus tractus solitarius. Prog Neurobiol 48: 21-53, 1996.[CrossRef][ISI][Medline]
  26. Levitan IB and Kaczmarek LK. The Neuron: Cell and Molecular Biology (3rd ed.). New York: Oxford Univ. Press, 2002.
  27. McGill JM, Basavappa S, Gettys TW, and Fitz JG. Secretin activates Cl- channels in bile duct epithelial cells through a cAMP-dependent mechanism. Am J Physiol Gastrointest Liver Physiol 266: G731-G736, 1994.[Abstract/Free Full Text]
  28. Meyer JH, Way LW, and Grossman MI. Pancreatic response to acidification of various lengths of proximal intestine in the dog. Am J Physiol 219: 971-977, 1970.[Free Full Text]
  29. Mutt V, Carlquist M, and Tatemoto K. Secretin-like bioactivity in extracts of porcine brain. Life Sci 25: 1703-1707, 1979.[CrossRef][ISI][Medline]
  30. Ng SS, Yung WH, and Chow BK. Secretin as a neuropeptide. Mol Neurobiol 26: 97-107, 2002.[CrossRef][ISI][Medline]
  31. Nozaki S, Nakata R, Mizuma H, Nishimura N, Watanabe Y, Kohashi R, and Watanabe Y. In vitro autoradiographic localization of 125I-secretin receptor binding sites in rat brain. Biochem Biophys Res Commun 292: 133-137, 2002.[CrossRef][ISI][Medline]
  32. O'Donohue TL, Charlton CG, Miller RL, Boden G, and Jacobowitz DM. Identification, characterization, and distribution of secretin immunoreactivity in rat and pig brain. Proc Natl Acad Sci USA 78: 5221-5224, 1981.[Abstract/Free Full Text]
  33. Olsen PS, Kirkegaard P, Poulsen SS, and Nexo E. Effect of secretin and somatostatin on secretion of epidermal growth factor from Brunner's glands in the rat. Dig Dis Sci 39: 2186-2190, 1994.[CrossRef][ISI][Medline]
  34. Ozcelebi F, Holtmann MH, Rentsch RU, Rao R, and Miller LJ. Agonist-stimulated phosphorylation of the carboxyl-terminal tail of the secretin receptor. Mol Pharmacol 48: 818-824, 1995.[Abstract]
  35. Palkovitz RJ and Wiesenfeld AR. Differential autonomic responses of autistic and normal children. J Autism Dev Disord 10: 347-360, 1980.[CrossRef][ISI][Medline]
  36. Porges SW. Peripheral and neurochemical parallels of psychopathology: a psychophysiological model relating autonomic imbalance to hyperactivity, psychopathy, and autism. Adv Child Dev Behav 11: 35-65, 1976.[Medline]
  37. Propst F, Moroder L, Wunsch E, and Hamprecht B. The influence of secretin, glucagon and other peptides, of amino acids, prostaglandin endoperoxide analogues and diazepam on the level of adenosine 3',5'-cyclic monophosphate in neuroblastoma x glioma hybrid cells. J Neurochem 32: 1495-1500, 1979.[ISI][Medline]
  38. Rausch U, Vasiloudes P, Rudiger K, and Kern HF. In-vivo stimulation of rat pancreatic acinar cells by infusion of secretin. I. Changes in enzyme content, pancreatic fine structure and total rate of protein synthesis. Cell Tissue Res 242: 633-639, 1985.[ISI][Medline]
  39. Samson WK, Lumpkin MD, and McCann SM. Presence and possible site of action of secretin in the rat pituitary and hypothalamus. Life Sci 34: 155-163, 1984.[CrossRef][ISI][Medline]
  40. Sheitman BB, Knable MB, Jarskog LF, Chakos M, Boyce LH, Early J, and Lieberman JA. Secretin for refractory schizophrenia. Schizophrenia Res 166: 177-181, 2004.
  41. Shetzline MA, Premont RT, Walker JK, Vigna SR, and Caron MG. A role for receptor kinases in the regulation of class II G protein-coupled receptors. Phosphorylation and desensitization of the secretin receptor. J Biol Chem 273: 6756-6762, 1998.[Abstract/Free Full Text]
  42. Ulrich CD II, Holtmann M, and Miller LJ. Secretin and vasoactive intestinal peptide receptors: members of a unique family of G protein-coupled receptors. Gastroenterology 114: 382-397, 1998.[CrossRef][ISI][Medline]
  43. Ulrich CD II, Wood P, Hadac EM, Kopras E, Whitcomb DC, and Miller LJ. Cellular distribution of secretin receptor expression in rat pancreas. Am J Physiol Gastrointest Liver Physiol 275: G1437-G1444, 1998.[Abstract/Free Full Text]
  44. Van Calker D, Muller M, and Hamprecht B. 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.[Abstract/Free Full Text]
  45. Vincent A and Tell F. Postnatal changes in electrophysiological properties of rat nucleus tractus solitarii neurons. Eur J Neurosci 9: 1612-1624, 1997.[CrossRef][ISI][Medline]
  46. Voskamp D and Beyerman HC. Biological potency and chromatographic properties of [aspartoyl3]-secretin, [{beta}-Asp3]secretin, and secretin (4-27). Products of intramolecular reactions in secretin. Int J Pept Protein Res 18: 284-288, 1981.[ISI][Medline]
  47. Watanabe S, Chey WY, Lee KY, and Chang TM. Secretin is released by digestive products of fat in dogs. Gastroenterology 90: 1008-1017, 1986.[ISI][Medline]
  48. Whitmore TE, Holloway JL, Lofton-Day CE, Maurer MF, Chen L, Quinton TJ, Vincent JB, Scherer SW, and Lok S. Human secretin (SCT): gene structure, chromosome location, and distribution of mRNA. Cytogenet Cell Genet 90: 47-52, 2000.[CrossRef][ISI][Medline]
  49. Yang B and Ferguson AV. Orexin-A depolarizes dissociated rat area postrema neurons through activation of a nonselective cationic conductance. J Neurosci 22: 6303-6308, 2002.[Abstract/Free Full Text]
  50. Yang B and Ferguson AV. Orexin-A depolarizes nucleus tractus solitarius neurons through effects on nonselective cationic and K+ conductances. J Neurophysiol 89: 2167-2175, 2003.[Abstract/Free Full Text]
  51. Yang B, Samson WK, and Ferguson AV. Excitatory effects of orexin-A on nucleus tractus solitarius neurons are mediated by phospholipase C and protein kinase C. J Neurosci 23: 6215-6222, 2003.[Abstract/Free Full Text]
  52. Yates BJ, Grelot L, Kerman IA, Balaban CD, Jakus J, and Miller AD. Organization of vestibular inputs to nucleus tractus solitarius and adjacent structures in cat brain stem. Am J Physiol Regul Integr Comp Physiol 267: R974-R983, 1994.[Abstract/Free Full Text]
  53. Yung WH, Leung PS, Ng SS, Zhang J, Chan SC, and Chow BK. Secretin facilitates GABA transmission in the cerebellum. J Neurosci 21: 7063-7068, 2001.[Abstract/Free Full Text]
  54. Zhou ZC, Gardner JD, and Jensen RT. Receptors for vasoactive intestinal peptide and secretin on guinea pig pancreatic acini. Peptides 8: 633-637, 1987.[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 J. Neurophysiol.Home page
J. H. Sun, B. Yang, D. F. Donnelly, C. Ma, and R. H. LaMotte
MCP-1 Enhances Excitability of Nociceptive Neurons in Chronically Compressed Dorsal Root Ganglia
J Neurophysiol, November 1, 2006; 96(5): 2189 - 2199.
[Abstract] [Full Text] [PDF]

Home page
 Ann. N. Y. Acad. Sci.Home page
J.Y.S. CHU, W.H. YUNG, and B.K.C. CHOW
Secretin: A Pleiotrophic Hormone
Ann. N.Y. Acad. Sci., July 1, 2006; 1070(1): 27 - 50.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
K. J. Pulman, W. M. Fry, G. T. Cottrell, and A. V. Ferguson
The Subfornical Organ: A Central Target for Circulating Feeding Signals
J. Neurosci., February 15, 2006; 26(7): 2022 - 2030.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
Y. Li, X. Wu, H. Yao, and C. Owyang
Secretin activates vagal primary afferent neurons in the rat: evidence from electrophysiological and immunohistochemical studies
Am J Physiol Gastrointest Liver Physiol, October 1, 2005; 289(4): G745 - G752.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
286/5/R927    most recent
00600.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (12)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yang, B.
Right arrow Articles by Ferguson, A. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, B.
Right arrow Articles by Ferguson, A. V.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.