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Neuroscience Vol. 53, No. 1, pp. 239-250, 1993 Printed in Great Britain
0306-4522/93 $6.00 + 0.00 Pergamon Press Ltd
0 1993 IBRO
THE ACTIONS OF THE IC, OPIOID AGONIST U-50,488 ON PRESYNAPTIC NERVE TERMINALS OF THE CHICK
CILIARY GANGLION
G. H. FLETCHER and V. A. CHIAPPINELLI*
Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, MO 63104, U.S.A.
Abstract-The actions of the K, opioid receptor agonist U-50,488 (rrunr-( + )-3,4-dichloro-N-methyl-N-[2- (I-pyrrolidinyl)cyclohexyll_benzene-acetamide methane sulfonate) on the membrane properties of presyn- aptic calyciform nerve terminals of the chick ciliary ganglion were examined using intracellular recordings obtained from intact ganglion preparations maintained in vifro. U-50,488 produced a concentration- dependent (3&lOOO~M) hyperpolarization with an apparent increase in input resistance. This hyper- polarization resulted from inhibition of the Na+-K+ inward rectifier, since it was blocked by 3 mM Cs+ and was not observed when terminals were depolarized beyond resting potential where inward rectification was voltage inactivated. A depolarizing effect on membrane potential with a further rise in input resistance was commonly observed at the highest perfused U-50,488 concentration (1 mM). The depolarizing event appears to result from a decrease in membrane potassium conductance, as the reversal Potential for the response was estimated to he between - 70 and - 90 mV and the potassium channel blocker Ba* + (1 mM) abolished the response. The K, opioid receptor agonist also blocked spontaneously occurring miniature hyperpolarizations in the terminals, which are considered to be due to a Ca2 + -dependent K+ conductance. Most of the responses to U-50,488 were abolished in the presence of the K, receptor antagonist norbinaltorphimine.
In conclusion, the excitability of presynaptic nerve terminals in the chick ciliary ganglion can be modulated by the inhibition of at least three separate ion conductances following activation of K, opioid receptor sites in the nerve terminal region.
Opioid receptors are currently classified according to three major subtypes, designated as p (mu), 6 (delta) and K (kappa),with the enkephalins (for p and 6) and dynorphins (for K) being regarded as the endogenous ligands. “J* When presynaptic opioid receptors are activated, they may function to decrease the amount of transmitter released by the neuron following inva- sion of the terminal region by an action poten- tial.4~‘1J6~‘9~23~27~29~39 Pharmacological assays performed in neuronal preparations, such as brain slices from anatomically distinct regions or in isolated tissues, suggest that this prejunctional modulation of endo- genous transmitter release can occur by both p and K receptors co-existing on the same nerve termi- nal.4v’6,19,23 The ionic mechanisms that govern the inhibition of neurotransmission seem to be different for the various opioid receptor subtypes. For in- stance, activation of ,u receptors in myenteric plexus neurons reduces acetylcholine release by increasing an outward membrane potassium conductance, which effectively shortens the duration of the presyn- aptic action potential. 4,27 By contrast, agonists selec-
*To whom correspondence should be addressed. Abbreviations: DC, depolarizing current; HEPES, N-2-hy-
droxyethylpiperazine-N’-2-ethanesulfonic acid; nor-BNI, norbinaltorphimine; R,, input resistance; U-50,488, trans-( f )-3,4-dichloro-N-methyl-N-[2-( l-pyrrolidinyl)- cyclohexyl]-benzene-a&amide methane sulfonate.
tive at K-opioid receptors inhibit neurotransmission in the same neurons by decreasing voltage-regulated inward Ca* + conductance and cytosolic free Ca*+ levels in presynaptic terminals.4*27 For some drugs, such as the classical p agonist morphine and the K agonist U-50,488 (tram-( + )-3,4-dichloro-N-methyl- N-[2-( l-pyrrolidinyl)cyclohexyl]-benzene-acetamide methane sulfonate), relatively high concentrations (2 100 PM) are required to observe these effects.‘5s23
The information summarized above regarding the ionic mechanisms that govern p and K opioid modu- lation of acetylcholine release is based largely on indirect methods of assessment. To better understand the electrical events mediated by p and K receptors which may be involved in the process of neuro- secretion, it is necessary to record directly from the presynaptic terminal. Although this is not possible in most situations, the chick ciliary ganglion is an ideal preparation in which to examine the physiological actions of opioids at the nerve ending. In the ciliary ganglion of the chick, preganglionic fibers emanating from the nucleus of Edinger-Westphal, which is situ- ated in the avian midbrain, terminate and envelop the postsynaptic ciliary neurons in a cap or calyx-like ending.sJ8 Studies have shown that presynaptic fibers to these ciliary neurons contain a variety of neuro- peptides, including substance P, the enkephalins and vasoactive intestinal peptide, all of which co-occur with the conventional transmitter acetylcholine in
239
this parasympathetic ganglion.” ‘(‘,” As presynaptic calyces in the ciliary ganglion are large enough fat stable intracellular impalements, it has been possible to show that these peptides mediate physiological responses at the terminal membrane.‘.“.’ For example. both substance P and the enkephalins depolarize these nerve terminals.“.“.” The mechanism of action of leu-enkephalin is at least in part an activation of a Nat conductance, an etrect which is scnsitivc to blockade by the ,u-preferring antagonist naloxone.” This p receptor-mediated response seems to agree with preliminary data suggesting the existence of h (and K) opioid binding sites in the chick ciliary ganglion. Ih Although the exact location of these binding sites was not determined, it is tempting to speculate that p and K opioid receptors are present together on the terminal membranes of the calyces, as they can be on other neurons.4.“.‘9.‘3 Therefore, the present study sought to examine whether ti opioid receptors are involved in excitation or inhibition of presynaptic calyciform nerve terminals by using the classical agonist U-50,488.‘* Previous functional stud- ies, performed further along the neural axis, have shown that both K and p opioid agonists modulate release of acetylcholine at intact neuromuscular junc- tions between ciliary ganglion neurons and choroidal smooth muscle.‘(’
EXPERIMENTAL PROCEDURES
Ciliary gangliu preparation
The preparation and acute maintenance of intact ciliary ganglia in uirro, including the description of electrophysio- logical recording and drug application techniques, have previously been described in full.” Briefly, one-day-old hatched White Leghorn chicks (SPAFAS, Peoria, IL) were killed by decapitation and the ciliary ganglion, along with pre- and postsynaptic nerve trunks, was dissected out and positioned in a recording chamber (capacity of I ml) through which perfused (3-4 ml/min) a continuous stream of normal physiological saline (composition in mM: NaCl, 150; KCl, 3; CaCl,, 5; MgCl,, 2; HF,PES, 10; glucose, 17; oH 7.4) maintained at 36-37°C under 100% 0,. Glass suction~electrodes placed on the oculomotor (presinaptic) and ciliary (postsynaptic) nerves were used to deliver ortho- dromic and antidromic stimuli (Grass S-88 stimulator and SIU-5 isolation unit), respectively, to identify the individual synaptic elements of the ciliary ganglion, i.e. presynaptic calyciform nerve terminals vs ciliary or choroid neurons.
Electrical recording
Intracellular recordings were made with glass microelec- trodes filled with 3 M KC1 (60-90 MR) using the bridge circuit mode of the Axoclamp-2A (Axon Instruments, Inc.) amplifier. All membrane potentials and current signals were displayed on a digital oscilloscope (Nicolet 3091), and simultaneously recorded on a two-channel chart recorder (Gould RS 3400) and VCR recording system (Vetter 420) - . for later analysis. Current clamp recordings were utilized in this study due to the difficulty of maintaining an adeauate space clamp during voltage-clamp recording in the sheei-like calycifonn terminals.”
Drugs and solutions
Drugs and test solutions were applied by either changing the superfusing solution via a three-way valve or by pressure
qcction lrom a micropipette positioned il few mrllm~clcr\ upstream from the ganglion. The compounds used in lhlq study included /runs-( f )-3,4-dichloro-N-methyl-,~-[3-( 1. pyrrolidinyl)cyclohexyl]-benzene-acetamide meth;tncsul- fonatc (U-50,488), naloxone. C‘sCI. t&Cl, and Cd(‘l, (all obtained from Sigma, St. Louis, MO). The x opiold ,~ntag- onist norbinaltorphine (nor-BNI) dihydrochloride HIS pur- chased from Research Biochemicals Inc. (Natick. MA).
RESULTS
Physiological characteristics qf’ presynuptic, cul~~tm
Presynaptic calyciform nerve terminals were distin- guished from postsynaptic cells (i.e. ciliary and choroid neurons) in intact chick ciliary ganglia in
vitro by known electrophysiological criteria.Y,‘“,‘~.24,2~ Specifically, orthodromic and antidromic stimulation of the oculomotor and ciliary nerves, respectively, initiates a single action potential in nerve terminals without any nicotinic excitatory postsynaptic poten- tial. When nerve stimulation is superimposed on a
hyperpolarizing pulse of > 10 mV, the action poten- tial is blocked, leaving a small depolarization known as the coupling potential. The presence of the coup- ling potential confirms the identity of the presynaptic calyx, as the nerve terminal of the calyx-type synapse is electrically coupled in the forward and backward directions. Additional distinguishing characteristics include the appearance of spontaneous miniature hyperpolarizations and the firing of single spikes (80-100 mV in amplitude) in response to prolonged (a65 ms, I Hz) depolarizing current pulses of 0.6-1.0 nA.”
Presynaptic actions of U-50,488
The micropipette ejection of U-50,488 (20~1 of 6mM in the delivery pipette) into the perfusion stream (3-4 ml/min) bathing the ganglion elicited two noticeable responses in presynaptic calyces. In most cases (25 out of 32 terminals), the pressure application of the K, agonist produced a marked hyperpolarization of 6.2 + 0.7 mV (mean + S.E.M., range 2-17 mV, n = 25) consequent with an increase in input resistance (R,,). This increase in R, is clearly seen in Fig. 1A by the larger amplitude of electrotonic potentials in response to constant- current hyperpolarizing pulses, which persisted when the membrane potential was returned to the control value (by passing depolarizing current through the microelectrode) during the response. In the remaining seven calyces tested, similar application of U-50,488 produced a predominant membrane depolarization of 7.1 + 1.5 mV (mean + S.E.M., range 4-l 5 mV, n = 7) associated with an increase in R,, (Fig. 1B).
To investigate the apparent dual nature of the response induced by U-50,488, ciliary ganglia were superfused with various known concentrations (30-1000pM) of the agonist. Up to 3OOpM, U- 50,488 elicited a dose-dependent increase in mem- brane hyperpolarization. This was accompanied by
A
U-50,488
Actions of U-50,488 in chick ciliary ganglion 241
I25mV
dlnA 8 30s
U-50,488
Fig. I. Hyperpolarization (A) and depolarization (B) produced in presynaptic calyciform nerve terminals by pressure ejection of U-50,488. Downward deflections in voltage recording (upper trace in each pair) are electrotonic potentials caused by passing hyperpolarizing current pulses (0.2 s, 0.5 Hz, lower traces) of constant amplitude. (A) Hyperpolarization recorded from a calyciform terminal in response to pressure ejection of 20 pl of 6 mM U-50,488 (during the period indicated by the arrow) into the recording chamber. At the peak of the U-50,488-induced response, the membrane potential was briefly brought back to its control value by applying depolarizing current (+DC) through the microelectrode, which indicated an apparent increase in input resistance f&J. Membrane potential = -63 mV. (3) F’resynaptic terminal showing a depolarization in response to a similar pressure-pipette application of U-50,488 as outlined above. At the height of the depolarizing response, the membrane potential was returned to its original value (by passing negative DC through the electrode), which demonstrated an increase in R,,. Membrane
potential = - 65 mV.
an increase in input resistance (Fig. 2A-C). U-50,488 evidence of de~nsiti~tion. Longer perfusion times caused hyperpolarizations of 2.3 f 0.6 mV (mean & were not examined. The response did not wash out S.E.M.,rangel-SmV,n=8)at30~M,3.8+0.6mV rapidly after removal of the drug, and a typical (range 2-8 mV, n = 12) at 100 PM and 4.5 + 0.6 mV recovery was usually of the order of 45-60 min. (range 2-12 mV, n = 24) at 300 FM. However, at the In some calyces (n = 6), repeated exposures to highest concentration of U-50,488 (1 mM), there was U-50,488 separated by one to several hours caused first a rn~br~e h~~la~tion (3.7 f 0.8 mV, responses of similar amplitude with no sign of range O-9 mV, n = 12), followed by a slow depolariz- progressive decline (not shown), thus indicating an ation (4.4 + 0.9 mV, range O-l 1 mV, n = 12) that was apparent lack of long-term receptor desensitization. associated with a further rise in I&, (Fig. 2D). The A consistent feature in some of the records examined onset of the depolarization occurred within 4-5 min at 1000 pM U-50,488 was the apparent instability of after the start of the hyperpolarization. Over the the membrane potential during the initial depolar- ~n~ntration range tested, the effects of U-50,488 izing phase of the biphasic response. We are uncertain persisted for up to 10 min of perfusion without any at this time whether this is due to inherent properties
‘47 G. tt. FI.ETCHEI~ and V. A. ~‘HIAPPINELI I
U-50,488
30pM
_-I 15 mV
30s
Fig. 2. Dose-dependent effects of U-50,488 on membrane polarization at concentrations between 30 and 1000 p M. Chart recorder traces of a presynaptic calyciform nerve terminal subjected to constant amplitude hyperpolarizing current pulses (3OOpA, 200ms, 0.5 Hz), which were injected into the terminal region through the recording microelectrode. The downward deflections observed on the trace thus give an indication of membrane input resistance (Rio). U-50,488 was applied for the period indicated by the solid bar. Application of U-50,488 from 30 to 300 pM elicited a dose-dependent hyperpolarizing response in this presynaptic calyx (A-C). Under these conditions, R,, appeared to increase. Bath administration of 1 mM U-50,488 (D) produced two distinct responses, the first of which was a membrane hyperpolariz- ation, which was followed by a depolarization and a further rise in R,,. It should be noted that the amplitudes of the hyperpolarizing and depolarizing responses are reported with reference to the resting
membrane potential (-62mV), as shown by the dashed line.
of the benzene-acetamide compound or whether it is a consequence of the competing actions of the two different membrane polarizing effects of U-50,488. It should also be noted that on six separate occasions of perfusion with 3OOpM U-50,488, duaf responses of hy~~la~zation (I .2 & 0.3 mV, mean + S.E.M., range O-2 mV) and depolarization (3.5 & 0.6 mV, range 3-6 mV) were observed, similar to those result- ing from 1 mM U-50,488.
Pharmacology of U-50,488 responses
Figure 3 shows one presynaptic calyciform termi- nal that responded with a biphasic pattern of hyper- polarization and depolarization to perfusion with 300pM U-50,488. The subsequent pretreatment of
this nerve terminal with the selective ti, antagonist nor-BNI (30 Jo M), 22,36 before the repeated application of the same concentration of the K agonist, resulted in a suppression of both the hyperpolarizing and depolarizing actions of U-50,488. However, at this concentration of nor-BNI, it was still possible to observe a response to U-50,488, as evidenced by the increase in electrotonic potential amplitude, indicat- ing an increase in R,. The inhibition of these two U-50,488-induced responses by nor-BNI was par- tially restored (only for depolarization) after a 40-min recovery period in normal saline (Fig. 3). Similar results were obtained in 30 FM nor-BNI in two other calyciform terminals with the 1 mM concentration of U-50.488.
Actions of U-50,488 in chick ciliary ganglion 243
U-50,488 (300)iM) + nor-BNI (301~IUt
. ‘..
U-50,488 (~OOJJM)
Fig. 3. No&N1 antagonizes the actions of U-50,488. The chart records show the response to 300 PM U-50,488 before (upper trace), during (middle trace) and after (lower trace) perfusion of nor-BNI (30 y M). Superfusion of U-50,488 produced a 2 mV hy~rpola~~ation followed by a 5 mV depolarization away from the normal resting membrane potential of -60 mV (dashed line). Superfusion for 20 mm with the selective K~ receptor antagonist nor-BNI had no apparent effects on its own, but attenuated the responses induced by U-50,488. After a 40.min washout in normal tyrode solution, there was a partial recovery. Downward deflections are electrotonic potentials evoked by current pulses of 250 pA for 200 ms (0.5 Hz).
Lower concentrations of nor-BNI (I-IO FM; n = 13) and a concentration of 10 FM of the g- preferring antagonist naloxone (n = 2) had no effect on U-50,488-induced responses, and also did not alter membrane potential or Rj, in the absence of agonist (not shown).
We have previously shown that under conditions similar to those of the present experiments, Cs+ is a potent blocker of a prominent Na+-K+ inward rec- tifier in the nerve terminal region of the calyx.r3 Thus, when 3 mM Cs+ was added to the bathing solution, three characte~stic changes were observed in the recordings which were due to blockade of this cat- ionic inward rectifier. These included a hyperpolariz- ation of 2-4 mV (corrected ex~~rnen~liy to resting values by injection of positive depolarizing current, +DC, an apparent increase in input resistance and the abolition of the prominent relaxations in the hype~la~~ng electrotonic potentials. When the ganglion was perfused with solution containing both
300 FM U-50,488 and 3 mM Cs+ following a 20-min exposure to 3 mM Cs+ , the hype~ola~~ng response to this opioid agonist observed in normal physiologi- cal solution was totally suppressed (n = 6; Fig. 4) Instead, the depolarizing action of U-50,488 was revealed, as well as an increase in Ri,, Thus, it seems likely that the hyperpolarizing action of U-50,488 in normal solution is due to the inactivation of the voltage-activated inward rectifier. Furthermore, this result indicates that the inward rectifier is normally responsible for a depolarizing effect at resting mem- brane potential in this presynaptic terminal region.
Although Ba2 + does not block the inward rectifier of the presynaptic calyx, I3 the addition of this K+ channel blocker at a co~~ntration of 1 mM to perfusate already containing 3 mM Cs+ depolarized the membrane of the nerve terminal by w 20 mV and further increased pi”. H~~ola~zing holding cur- rents of 100-300 pA were injected through the recording electrodes to maintain membrane poten- tials near their control values. Since Ba*+ caused a depolarization and an increase in Ri,, it appears that an outward K” fh~x across the membrane of the calyx
U-50,488 (300pM)
U-50,488 (300~M) + Cs* (3mM)
U-50,488 (300pM ) + Cs* (3 mM) + Ba’* ( 1 mM )
I l --
10 mV
o --l-_-J---
I e L-J-J3o.v
10 mV 100 ms
0
I -c ,(--- i 20mV i
_--
30s
Fig. 4. U-50,488-induced hyperpolarization and depolarization are blocked by cesium (0’) and barium (Ba’+) respectively. During superfusion with 3OOpM U-50.488 (time indicated by the bar) in control solution, a hyperpolarizing response was recorded from this terminal region. The addition of 3 mM Cs ’ to the bathing medium abolished the sag in the hyperpolarizing electrotonic potentials and produced a marked increase in input resistance. Repeating the administration of 300 PM U-50,488 in this Cs * - modified tyrode solution revealed that the hyperpolarizing response to U-50,488 was abolished and d prominent depolarization occurred instead (middle trace). The addition of Ba2+ (1 mM) to the 3 mM Cs+-containing tyrode solution depolarized the calyciform nerve terminal by _ 20 mV and produced a further increase in membrane resistance (note voltage scale was adjusted). Negative DC was therefore applied to bring the membrane potential back to control levels before further testing with 3OOpM U-50,488. U-50,488 was without effect when applied during the continued presence of Cs+ and Ba’+ In all of the above chart records, downward deflections are electrotonic potentials evoked by passing hyperpolarizing current pulses of 200 pA, 200 ms at 0.5 Hz. The traces on the right show the electrotonic potentials on an expanded time base taken before (open circles) and during (filled circles) perfusion of
U-50.488. Membrane potential = - 66 mV.
must be a major determinant of normal resting
potential. When 3OOpM U-50,488 was perfused in the presence of Cs+ and Ba’ +, the SC] opioid agonist had no effect on either membrane potential or R,, (n = 3; Fig. 4). The U-.50,488-induced depolarization thus involved a decrease in outward membrane pot- assium conductance at the presynaptic terminal re- gion. Taken together, it appears that the K opioid agonist produces its hyperpolarizing and depolarizing effects by reductions of opposing conductances which are active at or near the resting membrane potential of the presynaptic calyx.
To investigate whether the U-50,488-evoked de- polarization is the result of inhibition of an outward Ca’+-dependent K+ flux, recordings were made
during perfusion with buffer containing 100 PM CdCl, to block Ca2+ entry. Since the depoiarizing response induced by 1 mM U-50,488 persisted during superfusion with this Ca2+ channel blocker (n = 2; results not shown), it appears that the outward potassium conductance inactivated by the K, opioid agonist in the calyciform nerve terminals is not dependent on calcium entry.
Effect of membrane potential on the responses to U-50,488
To determine the effects of varying membrane potential on U-50,488-induced hyperpolarizations and depolarizations, 1 mM of this compound was applied at various holding potentials (n = 2; Fig. 5).
U-5
0.48
8 (1
OO
Op
M)
-52
mV
O
----
-4-
-.--
-d-
-72
mV
--
----
----
----
--e-
e
“v
7-f-
-J-
Fig.
5.
The
am
plitu
de
of t
he h
yper
pola
riza
tion
and
depo
lari
zatio
n in
duce
d by
U-5
0,48
8 w
as d
epen
dent
on
mem
bran
e po
tent
ial.
U-5
0,48
8 (1
mM
) w
as
appl
ied
for
the
peri
od
indi
cate
d by
the
sol
id b
ar t
o a
pres
ynap
tic
caly
x w
hose
m
embr
ane
pote
ntia
l w
as h
eld
at d
iffe
rent
le
vels
abo
ve
and
belo
w r
est
by
inje
ctio
n of
dep
olar
izin
g an
d hy
perp
olar
izin
g cu
rren
t, re
spec
tivel
y.
At
the
rest
ing
mem
bran
e po
tent
ial
of
- 62
mV
, th
e bi
phas
ic
resp
onse
pa
ttern
of
hy
perp
olar
izat
ion
follo
wed
by
a d
epol
ariz
atio
n w
as o
btai
ned
with
per
fusi
on
of
1 m
M
U-5
0,48
8. W
hen
the
mem
bran
e w
as h
yper
pola
rize
d to
-7
2mV
, th
e am
plitu
de
of t
he d
rug-
indu
ced
hype
rpol
ariz
atio
n w
as g
reat
ly i
ncre
ased
, w
hile
the
dep
olar
izin
g ac
tion
was
abs
ent.
At
a m
ore
depo
lari
zed
leve
l (-
52
mV
) fr
om
rest
ing
mem
bran
e po
tent
ial,
the
ampl
itude
of
the
U-5
0,48
8-in
duce
d de
pola
riza
tion
was
enh
ance
d,
whi
le t
he h
yper
pola
rizi
ng
resp
onse
w
as a
bsen
t. N
otic
e th
at
duri
ng
the
time
spen
t in
the
co
ntro
l so
lutio
n be
fore
dr
ug
perf
usio
n,
the
base
line
nois
e is
due
to
th
e pr
esen
ce
of
spon
tane
ous
min
iatu
re
hype
qola
riza
tions
. T
hese
mom
enta
ry
volta
ge
even
ts
subs
eque
ntly
di
sapp
kare
d up
on
appl
icat
ion
of U
-50,
488
(see
Fig
. 6
for
furt
her
deta
ils).
E
xpan
ded
elec
trot
onic
po
tent
ials
sh
own
at t
he e
xtre
me
righ
t of
the
fig
ure
wer
e ob
tain
ed
befo
re
(ope
n ci
rcle
s) a
nd
duri
ng
(clo
sed
circ
les)
dru
g ap
plic
atio
n.
746 <;. kj. 1'1k~HtK iid v. A. ~HIAPPINI~I I.1
RMP -62mV
NT{
u-50,488 CLS~
NTlyb,
J 1OmV
200 ms Fig. 6. Effects of bath-applied 1 mM U-50,488 on spontaneous hyperpolarizations of presynaptic calyces. The top trace shows spontaneous hyperpolarizing potentials commonly observed in normal saline (NT). Following a IO-min perfusion with the K, opioid agonist (middle trace), these discrete potential fluctuations were abolished. However, > IO-min -very period in standard tyrode restored these spontaneous
membrane voltage events (bottom). Membrane potential = -62 mV.
At the resting membrane potential (-62 mV) of the nerve terminal. perfusion with 1 mM U-50,488 in- duccd the characteristic biphasic response of hyper- polarization followed by depolarization. When the membrane was hyperpolarizcd IOmV beyond rest. application of U-50,488 produced a dramatic increase in the amplitude of the hyperpolarization, by up to 20 mV. without any evidence of depolarization during drug administration. This result suggested that the reversal potential for the depolarizing effect of U-50,488 was between - 70 and - 90 mV, a value consistent with the inactivation of a potassium con- ductance. AI a more depolarized level (- 52 mV) 01 membrane potential. the depolarizing response to U-50,488 was considerably enhanced. while the hyperpolarization was abolished. On this evidence, it would appear that the reversal potential for the hypcrpolarizing effect of U-50,488 lies between - 52 and -62 mV. However. close inspection of hyper- polarizing clectrotonic potentials at the more de- polarized level indicates that no obvious sagging occurs in the voltage records. compared to a pro- nounced sag at the resting potential (Fig. 5). Thus, it seems more likely that at the depolarized potential. the terminal is removed from the voltage range required for activation of the inward rectifier.
In a previous report, we described the presence in prcsynaptic calyces of small. transient hyperpolar- izing potentials. which seemed to be due to an outward K ’ flux that was dependent on the release
of Ca’ - from internal stores.‘J The discharge of thcsc spontaneous miniature hyperpolarizations can bc observed as rapid fluctuations in membrane voltage noise around the level of the resting potential (Fig. 6). When U-50,488 was applied to the bathing solution, these discrete hyperpolarizing potentials were completely abolished, only to reappear on return to drug-free saline after a period of 5 -IO min (Fig. 6). This transient disappearance was more noticeable with the highest perfused concentration (1 mM) of U-50,488. Thus, U-50,488 may cause a reduction in cytosolic free Ca’ ’ levels. Alternatively. the K, agonist may inactivate these Ca’ +-dependent K channels without altering intracellular Ca-’
DISCUSSION
Ionic mechanisms involved in I;-50,488~inducrd ucticmc
Intracellular recordings from identified presynaptic calyccs of the chick ciliary ganglion have been used to characterize the actions of the K, opioid rcceplor agonist U-50.488 on these large nerve endings. The results indicate that U-50.488 has at least three prin- cipal presynaptic actions on ionic conductanccs. lead- ing to a hyperpolarizdtion. a depolarization and the abolition of small spontaneous hyperpolarizations.
The hyperpolarization elicited by L-50.488 is due to the inactivation of an inward rectitier that is active at the resting membrane potential. Previously. we have determined that Cs’ in the millimolar concen- tration range is a potent blocker of this mixed cationic inward rectifier in the terminal membrane. since it causes a hyperpolarization of -2 4mV. an
Actions of U-50,488 in chick ciliary ganglion 247
increase in 1pi, and the abolition of the characteristic sagging of hyperpolarizing electronic potentialsI These particular changes in the properties of the caiyciform membrane are very similar to events which occur when U-50,488 is bath administered. In ad- dition, when U-50,488 is superfused in the presence of extracellular Cs+, the xi agonist-induced hyperpol- arization is totally abolished. Finally, when the mem- brane of the presynaptic calyx is depolarized by N 10 mV from resting values, inspection of the indi- vidual electrotonic potentials at this depolarized level does not reveal any obvious sagging in the voltage records, indicating that no inward rectification is present. The absence of a hy~~la~zing response to U-50,488 at this depolarized potential supports the hypothesis that the U-50,488-indn~d hyperpolariz- ation is due to inhibition of the inward rectifier.
The mechanism which governs the depolarization induced by U-50,488 may involve K+ channel clo- sure. When the membrane of the presynaptic calyx was driven to a more hyperpolarized level (e.g. -70 to - 80 mV) from rest, the depolarizing effect evoked by U-50,488 (1 mM) was lost, indicating that the reversal potential (E,,) lies close to the predicted equilibrium potential for K+ ions. A further indi- cation for a decreased K+ conductance was obtained when at the more positive potential from rest (-50mV), the augmentation of the U-50,488- induced depoia~zation was clearly associated with an increase in apparent R,. This was evidenced from the increased amplitude of the electrotonic potentials when the membrane potential was returned to the new level at the height of the response (Fletcher and Chiappinelli, unpublished observations). Since the voltage records did not encroach into the activation range of the inward rectifier, we feel confident that the increase in Ri, reflects a decreased conductance to K+. Moreover, the addition of the K+ channel blocker Ba*+ to the bath chamber not only elimi- nated the U-50,488~induced depolarization, but by itself produced effects similar (though more pro- nounced) to those observed with U-.50,488. The addition of the Ca*+ channel blocker Cd*+ to the perfusing solution did not affect the actions of U-50,488. Thus, inward CaZ + eonductances through N-type channels at the terminal membrane of the calyx do not appear to be directly involved in the it agonist-indu~d response profile.34.35,42,43
Presynaptic calyces display spontaneous miniature hyperpolarizations,14 which are random and brief fluctuations in membrane potential.‘4.26*33 These hyperpolarizing events are thought to occur through the release of CaZ+ from intracellular stores, which then activates a brief spon~neous miniature outward current carried by K+ ions.2~3,‘4~26,33 The disappear- ance of these momentary voltage events during the period of perfusion with U-50,488 may involve a reduction in cytosolic free Ca*+ levels, which sub- sequently causes a decreased capacity for gating those channels responsible for the outward K’ flux from
the presynaptic nerve ending. Evidence in favor of this proposal has recently been obtained from synap- tosomal preparations enriched with hippocampal mossy fiber terminals, where U-50,~8 depressed cytosolic Ca ‘+ levels by possibly redistributing this divalent ion to endogenous stores.ls Alternatively, it is possible that these Ca*+-dependent K+ channels are inactivated independent of changes in intracellu- lar CaZ+
P~~r~a~ofogy of responses to U-50,488
Subtypes of IC opioid receptors, designated as K,, IC? and Q, have been suggested on the basis of physio- logical and binding studies.7.20*22 These separate classes of K receptors appear to exhibit further subdi- visions; for example, in the guinea-pig cerebellum X,~ and K,,, opioid receptors have been proposed.‘* In our experiments, exposure to the IC, antagonist nor- BN122*36 at one specific concentration (30pM) was able to abolish most of the pha~acolo~cal effects which we observed in presynaptic calyces in response to U-50,488 (i.e. membrane hy~~ola~zation and depola~~tion and blockade of spontaneous minia- ture hyperpolarizations). Occupancy and subsequent activation of a single population of 1c, receptor sites by U-50,488 might therefore account for these ac- tions. Yet, it is also possible that the membrane hyperpolarization and depolarization may be distinct pha~acologica1 responses. In one calyx terminal, perfusion with 300 FM U-50,488 elicited only a 5 mV hyperpolarizing response. However, in the presence of 10 FM nor-BNI, the response to U-50,488 became a depolarizing event of similar magnitude (Fletcher and Chiappinelli, unpublished observations). In ad- dition, one of the effects of U-50,488 (increased cell impedance) persisted in the presence of 30 FM nor- BNI. These latter observations raise the possibility that several K opioid receptor subtypes may be present within the calyces. Preliminary evidence“’ demon- strates the presence of IC opioid receptor sites in the chick ciliary ganglion, but it is not known whether more than one subtype of K receptor is found there.
It is possible that non-K opioid receptor subtypes may mediate some of the actions of U-50,488 on presynaptic calyces. *’ Previous work suggests that nor-BNI at concentrations 2 20 PM can interact with p and 6 receptors. 36 However, the lack of effect of the p-preferring antagonist naloxone on U-50,488- induced responses would seem to argue against this point. Furthermore, we have previously shown that p and 6 opioid agonists such as leu-enkephalin and met-enkephalin produce depolarization accompanied by decreases in input resistance in calyciform nerve terminals.5” This effect is due in large part to an enhanced sodium conductance, and is quite distinct from the effects observed with the IC opioid agonist in the present study. The micromolar (2 100 FM) con- centrations of U-50,488 needed to elicit a response from calyciform nerve terminals are comparable to concentrations required to activate presynaptic K
opioid sites that inhibit neurotransmitter releasc.‘“~“ We therefore believe that in our study, U-50,488 retains its pharmacological specificity, acting on K, opioid receptors located in the calyciform nerve terminal region.
Agonists selective for p and ti receptor subtypes inhibit transmitter release at chohnergic (and other) synapses by different mechanisms. The presynaptic inhibition caused by p receptor activation probably results from an increased K+ conductance, which effectively shortens the action potential, thereby re- ducing the amount of Ca2+ entry.4.27 In contrast, it has been proposed that K receptor agonists depress neurotransmitter release by directly reducing Ca’ + entry into nerve terminals through N-type calcium channe,s,f~~t7~~7,4J Of course, different opioid-activated mechanisms may also suppress neurotransmission. In the ciliary ganglion, activation of presynaptic K, opioid receptors can affect at least three separate ionic mechanisms, all of which may play a role in neurotransmitter release.
Recent evidence supports an inhibitory role of ti opioid receptors in synaptic transmission in the cil- iary ganglion. Nicotinic transmission through ciliary neuron synapses was measured by recording the size of compound action potentials elicited in the ciliary nerve by orthodromic stimulation of the oculomotor nerve. We found that exposure of ganglia to either 30-100 PM U-50,488 or l-3 PM dynorphin reduced nicotinic transmission in the postsynaptic ciliary nerve by up to 30% (Feng C., Novoa-Takara K. and Chiappinelli V. A., manuscript in preparation). While leu-enkephalin and met-enkephalin have been ident- ified in calyciform nerve terminals of the avian ciliary ganglion,“.3’*40 it is not known whether dynorphin or another IC receptor-preferring peptide is also present within these terminals. If present, such peptides could be released during synaptic activity and exert effects similar to those of U-50,488. At the developmental stage used in the present experiments, we and other?” find that as much as 6t-90% of the synaptically evoked compound action potential recorded in the ciliary nerve is mediated by acetylcholine released from calyciform terminals. These chemically acti- vated ciliary neurons are more sensitive to the inhibitory actions of both ps and IC (Feng C., Novoa- Takara K. and Ch~appinelli V. A., in preparation) opioid agonists than the remaining ciliary neurons,
which exhibit supramaximal electrical coupling to their presynaptic nerve terminals.
The hyperpolarization induced by U-50.488 in the calyciform nerve terminal following the inactivation of the inward rectifier appears to be the predominant action on membrane excitability at the resting mcm- brane potential, and therefore may exert the greatest effect on neurotransmitter release. The underlying depolarizing effect of U-50,488 in the nerve terminal could provide a mechanism by which the predomi- nant K, receptor-mediated inhibition of the presyn- aptic calyx might be finely tuned.’ The absence of drug-induced tachyphylaxis from our experimental records would suggest that this regulation is reason- ably well maintained.
Ca’ t -activated K ’ channels in nerve terminals of the frog neuromuscular junction appear to shorten the duration of the presynaptic action potential, since following their blockade there is a broadening of the presynaptic spike, which results in greater Cal + entry into the nerve terminal and increased terminal release.” A similar role could be proposed for the Ca’+ -dependent K + channels responsible for the spontaneous miniature hyperpolarizations in the calyciform terminals. The blockade of these spontaneous hyperpolarizing responses may underlie at least a portion of the depolarizing response to U-50,488.
CONCLUSIONS
The present study demonstrates that the K, opioid receptor agonist U-50,488 has at least three actions on neuronal excitability of presynaptic terminals in the chick ciliary ganglion. The most pronounced effect of U-50,488 on the terminal membranes is hype~olari~tion resulting from the inactivation of an inward rectifier permeable to both Na+ and K+ ions. Depolarization is mediated by the suppression of at least two outward K” conductances, one of which is dependent on the release of intracellular Ca*+ I The modulation of this repertoire of ion channels on the presynaptic terminal by U-50,488 may have an influence on the frequency of nerve discharge and the release of neurotransmitters at this synapse.
Acknowledgements--We thank Dr Rodrigo Andrade for many helpful camments on the manuscript, Chunhua Feng for participating in some earlier experiments. and Linda Russell for typing skiifs. This work was supported by NIH grant EYO6564 to V.A.C.
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(Acc~~ptrd 23 Sqmvdwr 1992)
