1Department of Pharmacology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada; and 2Department of Pharmazie, Swiss Federal Institute, CH 8057 Zurich, Switzerland
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ABSTRACT |
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Ho, Melisa W. Y., Annette G. Beck-Sickinger, and William F. Colmers. Neuropeptide Y5 Receptors Reduce Synaptic Excitation in Proximal Subiculum, But Not Epileptiform Activity in Rat Hippocampal Slices. J. Neurophysiol. 83: 723-734, 2000. Neuropeptide Y (NPY) potently inhibits excitatory synaptic transmission in the hippocampus, acting predominantly via a presynaptic Y2 receptor. Recent reports that the Y5 receptor may mediate the anticonvulsant actions of NPY in vivo prompted us to test the hypothesis that Y5 receptors inhibit synaptic excitation in the hippocampal slice and, furthermore, that they are effective in an in vitro model of anticonvulsant action. Two putative Y5 receptor-preferring agonists inhibited excitatory postsynaptic currents (EPSCs) evoked by stimulation of stratum radiatum in pyramidal cells. We recorded initially from area CA1 pyramidal cells, but subsequently switched to cells from the subiculum, where a much greater frequency of response was observed to Y5 agonist application. Both D-Trp32NPY (1 µM) and [ahx8-20]Pro34NPY (3 µM), a centrally truncated, Y1/Y5 agonist we synthesized, inhibited stimulus-evoked EPSCs in subicular pyramidal cells by 44.0 ± 5.7% and 51.3 ± 3.5% (mean ± SE), in 37 and 58% of cells, respectively. By contrast, the less selective centrally truncated agonist, [ahx8-20] NPY (1 µM), was more potent (66.4 ± 4.1% inhibition) and more widely effective, suppressing the EPSC in 86% of subicular neurons. The site of action of all NPY agonists tested was most probably presynaptic, because agonist application caused no changes in postsynaptic membrane properties. The selective Y1 antagonist, BIBP3226 (1 µM), did not reduce the effect of either more selective agonist, indicating that they activated presynaptic Y5 receptors. Y5 receptor-mediated synaptic inhibition was more frequently observed in slices from younger animals, whereas the nonselective agonist appeared equally effective at all ages tested. Because of the similarity with the previously reported actions of Y2 receptors, we tested the ability of Y5 receptor agonists to suppress stimulus train-induced bursting (STIB), an in vitro model of ictaform activity, in both area CA3 and the subiculum. Neither [ahx8-20]Pro34NPY nor D-Trp32NPY were significantly effective in suppressing or shortening STIB-induced afterdischarge, with <20% of slices responding to these agonists in recordings from CA3 and none in subiculum. By contrast, 1 µM each of [ahx8-20]NPY, the Y2 agonist, [ahx5-24]NPY, and particularly NPY itself suppressed the afterdischarge in area CA3 and the subiculum, as reported earlier. We conclude that Y5 receptors appear to regulate excitability to some degree in the subiculum of young rats, but their contribution is relatively small compared with those of Y2 receptors, declines with age, and is insufficient to block or significantly attenuate STIB-induced afterdischarges.
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INTRODUCTION |
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Neuropeptide Y (NPY) has profound actions on excitatory
synaptic transmission in rat and human hippocampus (Colmers and
Bleakman 1994; Colmers et al. 1987
;
Vezzani et al. 1999
). NPY inhibits glutamate release
presynaptically (McQuiston and Colmers 1996
), mediated
by an inhibition of presynaptic Ca2+ influx (Qian et
al. 1997
). In the absence of selective antagonists, experiments
with selective agonists strongly suggest that this action of NPY is
mediated via Y2 receptors (Klapstein and Colmers 1997
; McQuiston and Colmers 1996
). The
presynaptic NPY receptor appears important in the control of
excitability in the hippocampus. NPY knockout mice are more prone to
spontaneous seizures, but are profoundly sensitive to kainate-induced
status epilepticus, failing to recover and dying from this procedure,
whereas their heterozygous littermates recovered completely. NPY
knockout animals were rescued from kainate-induced status epilepticus
by intracerebroventricular (icv) pretreatment with NPY (Baraban
et al. 1997
). In vitro, NPY Y2 receptors are
capable of interrupting interictal events caused by removal of
Mg2+ from the extracellular solution or by addition of the
GABAA receptor blocker, picrotoxin. Furthermore, NPY and
Y2 receptor-preferring or -selective agonists, but not
the Y1 agonist, Leu31,
Pro34 NPY were shown to effectively prevent ictaform bursts
induced in hippocampal slices by a series of stimulus trains
[(stimulus train-induced bursting (STIB)] (Colmers and
Bleakman 1994
; Klapstein and Colmers 1997
).
These and data from other laboratories (Smialowska et al.
1996
) suggested that Y2 receptors are important in
the control of excitation in the hippocampus.
An in vivo study has suggested that a newly cloned NPY receptor,
designated Y5 (Gerald et al. 1996), may
mediate the anticonvulsant actions of NPY in adult rats in vivo
(Woldbye et al. 1997
). Specifically, seizures induced by
kainate injection in adult rats were reduced or abolished by NPY or
related agonists injected into the lateral cerebral ventricle.
Interestingly, in these experiments, the Y2 receptor
agonist, NPY13-36, had little or no effect on the seizures
induced by kainate in these animals, but NPY3-36 (which
binds Y2 and Y5 receptors), Leu31,
Pro34NPY (which binds Y1, Y4, and
Y5 receptors) (Gerald et al. 1996
) and human
pancreatic polypeptide (hPP, which binds Y4/PP1
and Y5 receptors) were effective (Woldbye et al.
1997
), consistent with the agonist binding profile of the
Y5 receptor (Gerald et al. 1996
;
Michel et al. 1998
). These results were surprising, given the response to Y2 agonists in the in vitro
preparations. However, recent binding studies, using Leu31,
Pro34PYY (which binds to Y1, Y4,
and Y5 receptors) in the presence of saturating
concentrations of the selective Y1 antagonist, BIBP3226 (Doods et al. 1995
) suggested the presence of
Y5 receptors in the rat hippocampus (Dumont et al.
1998
; Widdowson et al. 1997
).
Although we earlier failed to observe effects of the
Y1/Y5 agonist,
Leu31Pro34NPY on STIB ictaform events
(Klapstein and Colmers 1997), it did reduce the
frequency of spontaneous bursts (SB) that occurred between stimulus
train-induced afterdischarges. This action of Leu31Pro34NPY was insensitive to the
Y1 antagonist 1229U91. We hypothesized that this might
represent a Y5 receptor-mediated action of
Leu31Pro34NPY (Klapstein and Colmers
1997
).
Here we tested the hypothesis that Y5 receptors could
inhibit excitatory synaptic transmission in rat hippocampus. Because we
earlier failed to see a significant effect on STIB, which is initiated
and sustained in area CA3 (Bindokas et al. 1998;
Stasheff et al. 1985
), we concentrated our whole cell
patch-clamp recordings in areas CA1 and the subiculum. We synthesized a
novel, centrally truncated NPY analogue with a preference for
Y1 and Y5 receptors and compared its actions
with those of other agonists. Our results suggest that Y5
receptors do mediate some of the actions of NPY in the hippocampus of
young rats, but their contribution is relatively small compared with
those of Y2 receptors, declines with age, and is
insufficient to block or significantly attenuate STIB-induced afterdischarges.
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METHODS |
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Synthesis and preparation of a centrally truncated peptide analogue
The peptide was synthesized by automated multiple solid phase
peptide synthesis using Fmoc/tert. butyl strategy as described previously (Rist et al. 1995, 1998
). The
peptides were analyzed and purified to homogeneity >94% by
reversed-phase HPLC. Correct mass was identified by ion-spray mass
spectrometry (API III, Sciex, Toronto).
Membrane preparation
SMS-KAN cells (Y2 receptor-expressing)
were grown in 50% nutrient mixture Ham's F12/50% Dulbecco's
modified Eagle medium with 15% fetal calf serum, 2 mM glutamine, and
nonessential amino acids at 37°C and 5% CO2
until they were confluent. SK-N-MC cells (Y1 receptor-expressing) were grown under the same conditions using MEM
Earle's salts medium with 10% fetal calf serum, while BHKrY5 cells
(Y5 receptor-expressing) were raised in DMEM (4.5 g/l glucose) with 10% fetal calf serum, 1% PENStrep, and 1 mg/ml
Geneticin. Membrane preparation was performed as described
before (Beck-Sickinger et al. 1994; Ingenhoven
and Beck-Sickinger 1997
). After determination of the protein
concentration, inhibitors were added accordingly. Aliquots of the
membrane suspension of 1 ml were stored at
80°C.
All cell culture media and supplements were purchased from GIBCO, Pefabloc SC from Serva, and Bacitracin from Sigma.
Receptor binding
Membrane preparations of SK-N-MC, SMS-KAN, or BHKrY5 cells were
diluted in incubation buffer (MEM/25 mM HEPES, 1% bovine serum albumin, 50 µM Pefabloc SC, 0.1% bacitracin, 3.75 mM
CaCl2). Two hundred microliters of the suspension
containing 20 µg protein were incubated with 25 µl 1.2 nM
3H-propionyl-NPY (3.18 TBq/mmol; Amersham) and 25 µl of solutions of the analogues in increasing concentrations to give
a total volume of 250 µl. After 1.5 h at room temperature the
incubation was terminated by centrifugation of the samples for 10 min
at 3,000 × g and 4°C. The pellets were washed with
PBS, resuspended in PBS, and mixed with scintillation cocktail, and
radioactivity was determined (Ingenhoven and Beck-Sickinger
1997). Nonspecific binding was defined in the presence of 1 µM NPY. All experiments were performed in triplicate, and data are
provided as means ± SE.
Brain slice preparation
Male Sprague-Dawley rats were used for both whole cell intracellular recording (ages 7-35 days old) and extracellular recording experiments (ages 15-22 days) in conjunction with stimulus train-induced bursting (STIB). Animals were decapitated without prior anesthesia according to Canadian Council on Animal Care guidelines in a protocol approved by the University of Alberta Health Sciences Laboratory Animal Care Committee. Brains were rapidly removed and placed in cold (2-4°C) artificial cerebrospinal fluid (ACSF) bubbled continuously with 95% O2-5% CO2 (carbogen). A block of tissue containing a hippocampus and overlying cortex, including the entorhinal cortex, was mounted in a chamber and sliced transversely with a Vibratome (TPI, St. Louis, MO) to a thickness of 350 and 600 µm for whole cell and STIB experiments, respectively. Slices containing both hippocampus and entorhinal cortex were equilibrated at 32°C or room temperature for 30-60 min for whole cell and STIB experiments, respectively. Slices were then transferred to a perfusion chamber on the stage of an upright microscope (Zeiss Axioskop FS) and perfused, submerged, with warm (34 ± 0.5°C), carbogenated ACSF (flow rate ~ 2-2.5 ml/min), and equilibrated for a minimum of 10 min before the beginning of experiments.
Whole cell recordings
Composition of the ACSF used for dissection and storage of
hippocampal slices was (in mM) 124 NaCl, 3 KCl, 1.3 MgSO4, 4 MgCl2, 2 CaCl2, 1.4 NaH2PO4, 26 NaHCO3, and 10 glucose. The ACSF used for
recording was identical to the one used for dissection with the
exception that MgCl2 was reduced to 2 mM and 0.5 mM CaCl2 was added. Whole cell recordings were
performed with fiber-fill borosilicate patch pipettes (4-6 M)
filled with saline containing (in mM) 130 potassium gluconate, 2 KCl, 5 HEPES, 5 MgATP, 1 NaGTP, and 1.1 BAPTA tetrapotassium salt. Potassium
hydroxide was used to adjust the pH of the pipette saline to
7.22-7.25, and the final osmolarity for the pipette saline was
adjusted to 270-285 mOsm.
All whole cell experiments were performed on neurons in either CA1 or
the subicular regions of the hippocampal slice. Neurons in the cell
body layer of area CA1 or the subiculum having typical pyramidal cell
morphology were identified using a water immersion objective (Zeiss
×40) with an infrared filter and differential interference contrast
(DIC) optics. The patch pipette was guided to the identified cell under
visual control using infrared illumination and a televison monitor
(DAGE-MTI, Michigan City, IN), a gigohm seal (>2 G) was established
and the patch ruptured to gain access to the cell. Membrane potential
and action potential waveform were initally monitored to confirm
pyramidal cell properties, then the cell in voltage clamp was held near
their resting potentials (
58 to
70 mV). Synaptic currents were
evoked via a sharpened tungsten, monopolar stimulating electrode placed
in stratum radiatum of area CA1 or the subiculum, using a paired pulse
protocol (5-40 V, 100-200 µs, 40-ms interstimulus interval),
delivered from a stimulus isolation unit (IsoFlex, AMPI, Jerusalem).
The intensity of the stimuli was adjusted until a submaximal and stable
synaptic current was induced. Such paired stimuli were used to
demonstrate that stimulus amplitudes were not close to saturating the
synaptic responses and to emphasize the presynaptic nature of the
receptor actions. All whole cell currents were recorded using a
single-electrode voltage-clamp amplifier (NPI SEC 05 l, NPI,
Tamm, Germany) with a switching frequency of 35-38 kHz. Care
was taken with clamp gain and capacity compensation settings to ensure
proper square-wave headstage voltage throughout the duration of a
voltage-clamp experiment. Whole cell synaptic currents were filtered at
0.7-1.3 kHz, and each current trace was the digital average of three
successive responses, evoked at 0.1 Hz. In most cases, a voltage step
(50 ms, 10-20 mV negative to rest) was performed during the protocol, after the synaptic stimulus, to monitor for changes in access resistance. Passive postsynaptic membrane properties near rest were
also tested with a slow (2 s), 35 mV, positive-going voltage ramp
starting 20 mV negative to rest. Data were acquired and membrane potential controlled using pClamp (Axon Instruments, Foster City, CA).
Results from experiments in which whole cell access resistance had
changed significantly (>10%) were discarded.
All NPY agonists were stored as aqueous solutions of 100 µM to
1 mM kept frozen (20°C) in small aliquots until immediately before
use, then diluted to their final concentration with 10 ml ACSF and
perfused through the recording chamber in 4-5 min. In antagonist
experiments, slices were first perfused with ACSF containing a blocking
concentration of the antagonist for 4-5 min before either
[ahx8-20]Pro34NPY or
D-Trp32NPY was added to the
antagonist-containing ACSF. All reported effects of different NPY
agonists were calculated as the percent inhibition of the peak EPSC
amplitude of the first of the paired synaptic responses. Statistical
comparisons were made using paired t-tests in most
cases, with neurons serving as their own controls.
STIB recordings
Composition of the ACSF used for dissection, storage of slices,
and testing for field potential was (in mM) 120 NaCl, 3.3 KCl, 1.2 MgSO4, 1.8 CaCl2, 1.23 NaH2PO4, 25 NaHCO3, and 10 glucose. All experiments were
recorded with glass pipette (5-10 M) filled with ACSF, connected to
the headstage of the voltage-clamp amplifier used in the bridge
current-clamp mode. Field potentials were evoked from a monopolar
stimulating electrode placed in stratum radiatum of CA2/CA3a. A 30-V,
100-µs pulse was used to optimize placement of recording and
stimulating electrodes. Field potentials were recorded from stratum
pyramidale of CA3a, CA3b, or CA3c, or the subiculum. Recordings in area
CA3 were made for comparison with previous results (Klapstein
and Colmers 1997
), and electrophysiological (Bragdon et
al. 1992
) and imaging experiments (Bindokas et al. 1998
) suggested that in STIB, activity in area CA1 follows that in area CA3 consistently. Once the evoked field potential response was
stable, slices were incubated without synaptic stimulation in the
modified ACSF (identical with the above with the exception of
MgCl2 being reduced to 0.9 mM and
CaCl2 being reduced to 1.6 mM) for 20-40 min
before the beginning of the STIB experiments.
Stimulus trains were delivered every 8 min and 5-6 min to CA3 and the
subiculum, respectively. Each stimulus train contained four stimuli (30 V, 100-200 µs) at 100 Hz, repeated 20-25 times at 5 Hz. Once a
stable primary ictal afterdischarge (1°AD) was recorded, meaning the
duration remained constant within ~10% (Klapstein and Colmers
1997), the number of stimulus trains were reduced until the
1°AD failed to occur, and was then raised gradually until at least
three consecutive, stable 1°ADs were elicited. The average number of
stimulus trains at threshold needed to elicit stable 1°AD was 13 ± 2.5 (mean ± SE, n = 14). The field
afterdischarge potential was routinely amplified with an AC-coupled
amplifier (×10 to ×100), filtered at 1-3 kHz, and recorded with a
rectilinear chart recorder (Gould RS3200). Different NPY agonists were
applied to slices via the perfusate during the last 4 min of a stimulus train cycle. A positive effect of an agonist on AD was determined as at
least 50% reduction in 1°AD duration. All data were taken from
slices in which the effect of an agonist reversed completely, i.e., the
1°AD duration remained stable for three successive stimulus train
cycles on wash out. All STIB experiments were performed at 32 ± 0.5°C.
Materials
[ahx8-20] NPY,
[ahx5-24]NPY (Rist et al. 1995)
and
[ahx8-20]Pro34NPY
were all synthesized in Zürich as detailed above,
D-Trp32NPY was purchased from Bachem
California (Torrance, CA). BIBP3226 was purchased from Penninsula
Laboratories (Belmont, CA). Porcine sequence NPY was purchased from Dr.
S. St-Pierre, Peptidec Technologies, St-Laurent, Quebec.
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RESULTS |
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Binding of [ahx8-20]Pro34 NPY to Y1, Y2, and Y5 receptors
Binding curves were constructed for NPY and
[ahx8-20]Pro34NPY in
membrane preparations containing and Y1,
Y2, and Y5 receptors. Figure 1 illustrates competitive binding
profiles for Y1 and Y5 receptors for NPY and
[ahx8-20]Pro34NPY.
Although NPY has approximately the same affinity for both receptors in
this assay,
[ahx8-20]Pro34NPY had
about 10-fold less affinity for Y1 receptors, and
about 200-fold less affinity for Y5
receptors than the native peptide. Although previous experiments show
NPY binding to the Y2 receptor with an affinity
of 0.04 nM (Rist et al. 1996),
[ahx8-20]Pro34NPY had
essentially no affinity for Y2 receptors
(IC50 > 10,000 nM, not illustrated).
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Whole cell recordings
To investigate the possible involvement of
Y5 receptors in the regulation of synaptic
transmission, we first tested the action of the
Y5-preferring agonists,
D-Trp32NPY, or
[ahx8-20]Pro34 NPY
on excitatory synaptic responses evoked in pyramidal neurons of area
CA1. In whole cell patch-clamp recordings from 33 neurons in area CA1,
only 7 cells exhibited significant responses to applications of either
Y5-preferring agonist (not illustrated). We
noticed that the responsive cells of area CA1 lay distal, close to the subiculum. We next hypothesized that synaptic inputs to neurons of the
subiculum might respond to Y5-preferring
agonists, and we made whole cell recordings of neurons within the
pyramidal cell layer of the subiculum. For these experiments, to best
make comparisons with pyramidal cells in area CA1, we chose neurons (mostly in the proximal subiculum) that did not respond to stimulation with bursting (Greene and Totterdell 1997). Figure
2 illustrates typical excitatory
postsynaptic currents (EPSCs) recorded under control conditions in
such a subicular neuron; paired-pulse stimulation demonstrated
facilitation of the second response. Bath application of the
Y5 receptor-preferring agonist,
[ahx8-20]Pro34 NPY
(1 µM) caused a 67.4% reduction in the amplitude of the first EPSC
response, with an accompanying increase in the facilitation ratio of
the first to the second EPSC response (1.6 vs. 2.4). At the same time,
the response to a
20-mV voltage step (Fig. 2B) or a 35-mV
voltage ramp (Fig. 2C) was not significantly affected by the
application of the peptide agonist. The response reversed on agonist
wash out (Fig. 2A).
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Synaptic responses of subicular neurons were sensitive to both
Y5-preferring agonists tested, in addition to
less selective agonists. Figure 3,
A and B, illustrates the response of one
subicular pyramidal cell to both
[ahx8-20]Pro34NPY (3 µM) and D-Trp32NPY (1 µM). Both
agonists were effective in reducing EPSC in this neuron.
[ahx8-20]Pro34NPY
inhibited the EPSC by 41.8%, and
D-Trp32NPY inhibited it by 23.0%.
The synaptic response reversed almost completely on wash out of the
agonists. In this cell, the response recovered stably to 80% of
control values within 5 min wash out of
[ahx8-20]Pro34NPY,
and to 100% of control within 25 min wash out of
D-Trp32NPY (Figs. 3A and
2B). Effects of the centrally truncated agonist, [ahx8-20]Pro34NPY
usually recovered within 5-10 min wash, whereas recovery from the
effects of the full-length agonist,
D-Trp32NPY required between 20 and 40 min. The difference in wash out rates between centrally truncated and
full-length NPY agonists is similar to results reported earlier in the
hippocampal slice (Klapstein and Colmers 1997;
McQuiston and Colmers 1996
).
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To compare the relative potency of these
Y5-preferring agonists with that of a full
agonist at this synapse, we tested the effect of
[ahx8-20] NPY, a relatively nonselective,
centrally truncated agonist on which the more selective agonist
examined here was based (Rist et al. 1995), on subicular
neurons. Figure 3C shows a typical response of the synaptic
input to a subicular neuron to 1 µM
[ahx8-20] NPY. In this cell,
[ahx8-20] NPY caused an 88.5% reduction of
the EPSC amplitude, revealing an inhibitory postsynaptic current (IPSC)
underlying the first synaptic response. The inhibition of the EPSC
recovered within 12 min of wash. Recovery from the effects of this
concentration of [ahx8-20] NPY generally
occurred within 10-20 min.
In all three experiments in this figure, voltage ramps were applied to
the cell in the absence of synaptic stimulation in the absence or
presence of the agonist, after peak effects on the EPSC were observed
(insets in Fig. 3, A-C). As can be
seen, there were no significant changes in somatic conductance that accompanied the significant changes in synaptic response amplitudes, consistent with previous observations of NPY action in hippocampal pyramidal cells (McQuiston and Colmers 1992,
1996
). These results are consistent with an entirely
presynaptic action of the NPY agonists in the subiculum.
At the respective concentrations tested, [ahx8-20]Pro34NPY and D-Trp32NPY were roughly equipotent at inhibiting the EPSC in pyramidal cells of the subiculum. The average effect of 1 µM D-Trp32NPY was an inhibition of 44 ± 5.7%, whereas 3 µM [ahx8-20]Pro34NPY inhibited the EPSC on average by 51.3 ± 3.5%. However, [ahx8-20]Pro34NPY was more frequently effective at inhibiting the EPSC in subicular pyramidal cells (26/45 cells tested) than was D-Trp32NPY (17/46 cells tested. By contrast, the nonselective NPY agonist, [ahx8-20]NPY (1 µM), induced an even greater inhibition in the subiculum (66.4 ± 4%), in a far greater proportion of neurons (18/21 cells tested). The average ages for the animals from which neurons showing responses to [ahx8-20]Pro34NPY and D-Trp32NPY were observed were 15.3 ± 0.78 (n = 26) and 14.1 ± 0.96 days (n = 18), respectively (n.s., P = 0.314). The average age for animals from which neurons showing responses to [ahx8-20]NPY were observed was 19.1 ± 0.79 days (n = 21) and was significantly greater than either [ahx8-20]Pro34NPY- (P = 0.001) or D-Trp32NPY-responsive cells (P < 0.001).
We tested the hypothesis that the expression of presynaptic responses to Y5 agonists was a function of the age of the animal (Fig. 4). In general, we more commonly observed a Y5 agonist-induced inhibition of the subicular EPSC in slices prepared from younger animals, whereas the effect of the nonselective agonist, [ahx8-20]NPY, was equally potent among each age group (Fig. 4). Although the degree of inhibition by either [ahx8-20]Pro34NPY or D-Trp32NPY within each age group was highly variable (Fig. 4), regression analysis showed that the reductions with age in Y5-agonist responses were statistically significant (R2 = 0.277, P < 0.0005 for D-Trp32 and R2 = 0.271, P < 0.0003 for [ahx8-20]Pro34NPY), but there was no significant relationship with age for nonselective agonist, [ahx8-20]NPY.
|
Because the centrally truncated Y5 agonist we
tested also has a high affinity for Y1 receptors,
we next tested the hypothesis that the response in the subiculum was
mediated by a Y1 receptor. We thus compared the
action of
[ahx8-20]Pro34NPY alone
and in the presence of a blocking concentration of the Y1-specific antagonist BIBP3226 (Doods et
al. 1995) on the EPSC recorded in subicular neurons. In Fig.
5A, BIBP3226 alone had no
effect on the EPSC in this neuron, whereas co-application of [ahx8-20]Pro34NPY with
BIBP3226 caused a reduction in EPSC as observed with [ahx8-20]Pro34NPY alone.
Neither BIBP3226 alone nor
[ahx8-20]Pro34NPY in the
presence of BIBP3226 affected the membrane response to a voltage ramp
(Fig. 5B). Results of these experiments are summarized in
Fig. 5C. Similar results were also observed with D-Trp32NPY and BIBP3226 (not
illustrated).
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STIB recordings
To assess the anticonvulsant effects of the
Y5 agonists
[ahx8-20]Pro34NPY and
D-Trp32NPY in the in vitro slice
preparation, we performed extracelluar field potential recording in
either area CA3 or the subiculum, using the STIB model for epilepsy
(Klapstein and Colmers 1997; Stasheff et al.
1985
). Figure 6A shows
a typical field potential recording from stratum pyramidale of CA3
after stable 1°AD was obtained. Once a 1°AD developed in response
to stimulation, it remained stable for as long as 5 h under our
experimental conditions. Afterdischarges generally occurred after a
short delay from the end of a stimulus train and were generally clonic
or tonic-clonic in nature (Klapstein and Colmers 1997
).
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AREA CA3.
Neither
[ahx8-20]Pro34NPY(3
µM) nor D-Trp32NPY(1 µM) were
significantly effective in suppressing or shortening the 1°AD in area
CA3 (Fig. 6, B and C). Of 10 slices tested,
1°ADs recorded in area CA3 were totally suppressed by
D-Trp32NPY in only two, whereas only
one slice responded strongly to [ahx8-20]Pro34NPY (Fig.
8). By contrast, the Y2-selective agonist,
[ahx5-24]NPY (1 µM), totally abolished
1°AD in most slices tested, but in some cases did not abolish the
spontaneous bursts that followed after the stimulus train (Fig.
6D). The effect of [ahx5-24]NPY on
1°AD usually reversed within one stimulus cycle after washing (Fig.
6E). Unlike the Y5 agonists,
[ahx5-24]NPY was effective in most (5/7
slices), but not all slices (Fig. 8). Similarly, the nonselective
agonist, [ahx8-20]NPY (1 µM), was also
effective in inhibiting 1°AD duration (not illustrated), and its
effect was observed in most slices (4/5 slices, Fig. 8). Finally, NPY
(1 µM) induced a profound, long-lasting suppression of
1°AD duration (Fig. 6F), its effect often persisting even
after 60 min of washing, as was reported earlier (Klapstein and
Colmers 1997).
SUBICULUM. We next examined the effects of various NPY agonists on bursting behavior in the subiculum (Fig. 7). Unlike in area CA3, the 1°AD recorded in subiculum in response to stimulation of stratum radiatum in area CA2/3 was generally briefer and smaller in amplitude and rarely exhibited multiple spike waveforms (Fig. 7A). It was also more difficult to obtain stable 1°AD in the subiculum. Often, spontaneous bursts occurred between two train stimuli. Nonetheless, our experience showed that stable 1°AD could be achieved with an interval of between 5 and 6 min between two stimulus trains. Neither [ahx8-20]Pro34NPY nor D-Trp32NPY had any significant inhibitory effect on 1°AD in the subiculum among the four tested slices (Figs. 7, B and C, and 8). Contrary to our observations in area CA3 [ahx5-24]NPY was less commonly effective in the subiculum (1/4 slices; Fig. 8). The response of the one slice to [ahx5-24]NPY recovered within one cycle of washing (Fig. 7E). [ahx8-20]NPY (1 µM) effect on 1°AD was more frequently observed in the subiculum (4/4; Fig. 8), and its effect also recovered within one cycle of washing (Fig. 7D). Finally, NPY was a potent inhibitor, and its effect on 1°AD was consistently observed in the subiculum (3/3; Fig. 8). However, NPY's effect in the subiculum was not as readily reversed as in CA3 (Fig. 7F). Differences in response to various NPY agonists in these two areas could not be simply explained by the ages of animals used, because the mean ages were 19.08 ± 0.69 days (area CA3) and 17.0 ± 0.41 days (subiculum; P = 0.14).
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DISCUSSION |
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We have shown here that Y5 receptors can
mediate presynaptic inhibition of stratum radiatum-evoked
glutamatergic responses in some pyramidal neurons of area CA1 and the
proximal subiculum, in vitro, although this response declines rapidly
with age. On the basis of these results, we then tested the hypothesis
that Y5 receptors can control hyperexcitability
in the hippocampal STIB model of epileptiform activity, using slices
from animals in the age range where Y5 inhibitory
responses were significant. Although earlier results demonstrated that
NPY receptors, predominantly of the Y2 subtype,
can inhibit epileptiform activity in this model (Klapstein and
Colmers 1997), we were unable to demonstrate a significant
action of Y5 receptors in this model, compared
with the potent and prolonged effects of NPY itself. We conclude that, although Y5 receptors can inhibit the synaptic
excitation of some neurons in area CA1 and the proximal subiculum in
young rats, they do not appear to contribute significantly to the
overall action of NPY in regulating hyperexcitability in the
hippocampal formation, at any age tested.
Whole cell recordings
We first attempted to demonstrate that Y5
receptor agonists affected hippocampal synaptic responses already known
to be sensitive to NPY and Y2 agonists. In the
absence of consistent, Y5-mediated responses in
area CA1, we examined the sensitivity of excitatory synaptic responses
in neurons of the proximal subiculum to Y5 agonists. These cells exhibited typical pyramidal morphology and did
not fire bursts on stimulation, in agreement with observations of
Greene and Totterdell (1997) that nonbursting neurons
were most often found in this area. However, such typical morphology was not often observed in rats younger than 12 days of age. In these
young rats, the subicular neurons usually appeared round using infrared
DIC videomicroscopy, with a less prominent or invisible apical
dendrite. Nonetheless, we could stably evoke EPSCs in these neurons on
stimulation of stratum radiatum, and their action potentials appeared
like those of pyramidal cells from older animals.
In many of these experiments, we utilized a centrally truncated
analogue of NPY, [ahx8-20]Pro34NPY. We had
originally designed this molecule to be a Y1- selective agonist, but as we demonstrate here, it also shows a reasonably high-affinity binding for Y5 (but not Y2)
receptors. The central truncation conferred the advantage of rapid wash
out for this peptide in the hippocampal slice (Klapstein and
Colmers 1997; McQuiston and Colmers 1996
). The
specificity of [ahx8-20]Pro34NPY for
Y1 and Y5 receptors allowed us to attribute the
contribution of these two receptors to the effects of this agonist.
Both the centrally truncated peptide
[ahx8-20]Pro34NPY (3 µM), and
D-Trp32NPY (1 µM), a full-length, specific,
but weak Y5 agonist (Gerald et al. 1996
)
were effective in inhibiting to a greater or lesser degree the
evoked EPSCs in most subicular neurons. However, we did not observe any
synaptic inhibition by these Y5 agonists at concentrations below those used throughout our experiments. As expected, the effect of
[ahx8-20]Pro34NPY was short lived in
comparison with D-Trp32NPY.
Before attributing the actions of
[ahx8-20]Pro34NPY to the activation of
Y5 receptors, we had to ensure that the effects we observed were not mediated by Y1 receptors, by comparing its action
in the absence and presence of the specific Y1 receptor
antagonist, BIBP3226 (Rudolf et al. 1994). BIBP3226 has
no significant affinity for Y2 (Jacques et al.
1995
; Rudolf et al. 1994
), Y4
(Bard et al. 1995
), or Y5 (Gerald et
al. 1996
) receptor subtypes. A blocking concentration of the
antagonist (1 µM) had no effect on the action of
[ahx8-20]Pro34NPY, ruling out a
Y1 receptor as mediating the effect we observed. Although
the expression of moderate levels of Y1 receptor mRNA has
been reported in the pyramidal layer of area CA1 and the subiculum (Larsen et al. 1993
), based on the present results there
is no evidence for a Y1 response in this region. We
conclude that this centrally truncated agonist acts predominantly at
Y5 receptors in this preparation.
Regardless of their relative potency, the effects of both Y5 agonists were more often seen in younger animals, suggesting a waning role for the receptor in this brain region with maturity. NPY itself remains effective regardless of age, as does the nonselective agonist, [ahx8-20]NPY. We must, however, caution that the present results are based on animals between 1 to 4 wk of age. Immunocytochemical studies of Y5 receptor distribution in the hippocampus with age would be of significant interest and complement existing pharmacological studies.
We did not investigate the exact mechanism(s) by which NPY receptors
reduced synaptic excitation in subicular neurons. However, our
voltage-ramp and -step data indicated that there was no change in
postsynaptic membrane properties with application of any agonist tested. This is consistent with previous findings of NPY actions in the
hippocampus that demonstrated that they resulted entirely from a
presynaptic mechanism (Colmers et al. 1987;
McQuiston and Colmers 1996
; Qian et al.
1997
). Although it is possible that Y5 receptors
may also suppress Ca2+ currents in subicular neurons, as
has been observed in dentate granule cells (McQuiston et al.
1996
), such a mechanism is unlikely to contribute to the
suppression of synaptic input observed here, because it did not alter
synaptic excitation of rat granule cells (Klapstein and Colmers
1993
).
STIB recordings
The STIB model has been frequently used to study epileptogenesis
in vitro (Klapstein and Colmers 1997;
Stasheff et al. 1985
). The 1°AD that follows
the stimulus trains resembles the electrographic activities that occur
during seizures both in human epilepsy patients and in animal models
(McNamara 1994
) and has been validated to be sensitive
to several anticonvulsants when applied at clinically relevant
concentrations (Clark and Wilson 1992
). Our previous STIB experiments suggested that, although Y2 receptors
played a significant role in suppressing 1°AD in CA3, spontaneous
bursting was also reduced by the Y1 agonist,
Leu31-Pro34NPY,
even in the presence of a Y1 antagonist (Klapstein
and Colmers 1997
). This agonist also has a very high affinity
for Y5 receptors (Gerald et al. 1996
). Given
the recent in vivo studies reporting that Y5 receptors are
anticonvulsant in the kainate model (Woldbye et al.
1997
) and the present observations in subicular neurons, we
hypothesized that Y5 receptors might also play a role in controlling neuronal excitability in the STIB model. Surprisingly, our data do not
support this hypothesis.
The present results from area CA3 were consistent with our
previous observations that Y2 receptors were mostly
responsible for the suppression of the 1°AD in area CA3
(Klapstein and Colmers 1997). The nonselective agonist,
[ahx8-20]NPY (1 µM) was as effective as
Y2 agonist, [ahx5-24]NPY (1 µM), in
suppressing 1°AD. However, neither of the two Y5
agonists, [ahx8-20]Pro34NPY (3 µM) nor
D-Trp32NPY (1 µM) was clearly effective in
preventing 1°AD expression. Of the 10 slices tested, only two had a
measurable response to D-Trp32NPY, whereas only
one responded to [ahx8-20]Pro34NPY.
In the subiculum, the Y5-selective agonists,
D-Trp32NPY and
[ahx8-20]Pro34NPY were not significantly
effective in reducing 1°AD duration in the four slices tested. In the
same slices, the Y2-selective agonist,
[ahx5-24]NPY (1 µM), was unexpectedly not very
effective in suppressing 1°AD, whereas the nonselective agonist
[ahx8-20]NPY (1 µM) was as potent as NPY (1 µM) in inhibiting 1°AD expression in this region. Such
differences in distribution of Y2 receptors in these two
areas were not a result of any developmental factors because there was
no significant difference (P = 0.14) in the age of
animals used for either set of experiments. This may instead reflect
the relative density of Y2 receptors in this region.
Gustafson et al. (1997) reported a concentration of
Y2 receptor mRNA in area CA3, presumably reflecting the
high concentration of Y2 receptors we have observed
functionally in the Schaffer collaterals of area CA1 (Klapstein
and Colmers 1993
; McQuiston and Colmers 1996
). However, lower levels of Y2 receptor mRNA expression in CA1
pyramidal cells might be reflected in lower concentrations of
functional receptors in their projections to the subiculum.
The experiments thus suggest that there are Y5 receptors that can inhibit synaptic excitation, particularly in distal area CA1 and the proximal subiculum of young rats. This action is not universal, because the inputs to a majority of neurons tested failed to express a response to the Y5 agonists. Furthermore, although these agonists share a site (and presumably a mechanism) of action with the nonspecific NPY agonists, they were essentially incapable of mimicking the anticonvulsant actions observed with the Y2-selective or nonselective agonists in the STIB model.
Experiments using rhodamine 123 imaging to study patterns of activity
and the actions of NPY in the STIB model suggest that subthreshold
activity persists even with concentrations of NPY sufficient to prevent
1°AD expression (Bindokas et al. 1998). It is thus
tempting to speculate that, if the scarcity of whole cell responses to
Y5 agonists encountered here reflects the actual receptor
distribution, then the poor response of the STIB preparation to
Y5 agonists may result from an insufficient density of
Y5 receptors to suppress this regenerative network event.
Alternatively, or in addition, the concentration of Y5
receptors in the subiculum may not be adequate to prevent the
regenerative activity of STIB, which relies heavily on reciprocal
excitatory connections within area CA3 for its expression (Wong
and Traub 1983
).
This might explain why our current results contradict an earlier in
vivo study of the anticonvulsant action of NPY (Woldbye et al.
1997). These authors used intracerebroventricular preinjections of NPY and related agonists to suppress seizures induced by acute peripheral kainate injection, and concluded that NPY acted via a
Y5 receptor to suppress these seizures. It is possible
that, in the in vivo kainate model as used by these investigators,
sufficient Y5 receptors are located somewhere in the
pathways mediating the seizures, but are located outside the
hippocampal circuitry included in our in vitro preparation.
Alternatively, the mechanism by which putative Y5 receptors
suppress picrotoxin-induced interictaform bursts (Klapstein and
Colmers 1997
) may have greater significance in vivo.
Nonetheless, based on the present data, the Y5 receptors in
young rats appear incapable of significantly contributing to the
actions of NPY receptors in suppressing epileptiform activity in
hippocampal circuitry even when they are present.
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ACKNOWLEDGMENTS |
---|
We are grateful to the University of Alberta Hospitals Foundation for the purchase of the infrared videomicroscope used in the experiments. W. F. Colmers is a Medical Scientist of the Alberta Heritage Foundation for Medical Research.
This work was supported by the Medical Research Council of Canada.
Present addresses: M.W.Y. Ho, Inositide Signaling Section, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27713; A. G. Beck-Sickinger, Institut of Biochemistry, University of Leipzig, Talstr. 33, D 04103 Leipzig, Germany.
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FOOTNOTES |
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Address for reprint requests: W. F. Colmers, Dept. of Pharmacology, University of Alberta, 9-36 MSB, Edmonton, Alberta T6G 2H7, Canada.
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.
Received 2 April 1999; accepted in final form 18 October 1999.
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REFERENCES |
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