Neuropeptide Y5 Receptors Reduce Synaptic Excitation in Proximal Subiculum, But Not Epileptiform Activity in Rat Hippocampal Slices

Melisa W. Y. Ho,1 Annette G. Beck-Sickinger,2 and William F. Colmers1

 1Department of Pharmacology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada; and  2Department of Pharmazie, Swiss Federal Institute, CH 8057 Zurich, Switzerland


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega ) 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 GOmega ) 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 MOmega ) 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. Binding of neuropeptide Y (NPY) and [ahx8-20]Pro34NPY to membranes containing Y1 (A) and Y5 (B) receptors. IC50 values are given in the bottom left of each panel. The binding affinities for these and the Y2 receptor are given in the text.

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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Fig. 2. Y1/Y5 agonist, [ahx8-20]Pro34NPY (1 µM) inhibits synaptic excitation in proximal subiculum. In this and most subsequent figures, current traces recorded under different conditions are shown superimposed for comparison. A: excitatory postsynaptic current (EPSC) evoked in control is reduced by application of [ahx8-20]Pro34NPY (- - -). Wash out reverses agonist action. B: response of neuronal membrane to a voltage step (-20 mV) is unaffected by the agonist (- - -). C: membrane response to slow (2.5 s) voltage ramp is unaffected by the agonist. Cell was obtained from an 11-day-old rat.

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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Fig. 3. Different NPY agonists inhibit EPCS in subicular neurons. Left: whole cell synaptic currents evoked with paired stimuli (40 ms interstimulus interval). Right: membrane current response to a voltage ramp in the corresponding cell to the left. A: Y1/Y5 preferring agonist [ahx8-20]Pro34NPY (3 µM) inhibits the EPSC in this neuron obtained from a 21-day-old rat, which reversed almost entirely after 5 min wash out. There was no significant change in the response to the voltage ramp in the presence of the agonist. B: Y5-selective agonist D-Trp32NPY (1 µM) also inhibited the EPSC in the same cell as in A, but to a lesser extent. The D-Trp32NPY effect recovered more slowly (25 min). As in A, D-Trp32NPY did not significantly alter the membrane current response. C: in a different subicular neuron obtained from a 13-day-old rat, the nonselective NPY agonist, [ahx8-20]NPY (1 µM) profoundly suppressed the evoked EPSC, also without affecting the neuron's postsynaptic properties. Note the underlying outward (upward) synaptic current, presumably representing a GABAA-mediated inhibitory postsynaptic current (IPSC), which is revealed in the presence of the agonist. The EPSC recovered to >90% of control amplitude within 12 min wash.

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.



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Fig. 4. Y5-mediated presynaptic responses in subicular neurons from rats of different ages. Each point represents the effect of a single application of different NPY agonists on the intracellularly recorded EPSCs of subicular pyramidal cells [, D-Trp32NPY (1 µM); , [ahx8-20]Pro34NPY (3 µM); black-triangle, [ahx8-20]NPY (1 µM)], plotted against the age of animals. A: D-Trp32NPY inhibition of the EPSC was observed until age 22 days. Linear regression of the data yielded a significant negative slope (R = -0.521, P < 0.0002). B: [ahx8-20]Pro34NPY effect on the EPSC also declined with age (R = -0.538, P < 0.0002). C: effects of the poorly selective agonist, [ahx8-20]NPY, were not significantly affected by the animal's age (R = 0.136, P > 0.5).

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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Fig. 5. Y1 receptors do not mediate synaptic inhibition in the subiculum. A: the Y1 antagonist, BIBP3226 (1 µM), did not affect the EPSC, whereas co-application of [ahx8-20]Pro34NPY (3 µM) in the presence of BIBP3226 reduced the ESPC to 40% of control values. B: neither antagonist nor agonist affected the membrane response to a voltage ramp. C: average data from 6 neurons. Pro34 = [ahx8-20]Pro34NPY (3 µM).

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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Fig. 6. Effects of different NPY agonists on stimulus train-induced bursting (STIB) induced afterdischarge recorded in area CA3. Stable 1°AD was established in this slice after 25 min incubation in modified artificial cerebrospinal fluid (ACSF). Stimulus trains (13 × 4 stimuli) were delivered every 8 min (see METHODS for details). A: typical 1°AD recorded in CA3, with single (tonic) spikes developing shortly after the end of stimulus train, then gradually developed into multiple (clonic) discharges. B: D-Trp32NPY (1 µM) had no significant effect on the duration of 1°AD. C: [ahx8-20]Pro34NPY (3 µM) also had no effect on 1°AD. D: the Y2 selective agonist, [ahx5-24]NPY (1 µM), suppressed 1°AD but had no effect on the interictal discharge. E: the effect of [ahx5-24]NPY reversed within one cycle (i.e., 8 min) of wash. F: NPY itself (1 µM) strongly suppressed the 1°AD. Some recovery was observed after 70 min wash (not illustrated). All traces were recorded from a single slice from a 15-day-old animal.

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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Fig. 7. Effects of different NPY agonists on STIB-induced afterdischarges recorded in the subiculum. Stable 1°AD was established in this slice after 40 min incubation in modified ACSF. Stimulus trains (10 × 4) were delivered every 5 min. A: typical 1°AD recorded in the subiculum. Unlike in CA3, the 1°AD in the subiculum usually consisted of single spikes throughout its duration. B and C: neither [ahx8-20]Pro34NPY (3 µM; B) nor D-Trp32NPY (1 µM; C) had any significant effect on the duration of the subicular 1°AD. D: as in area CA3, the Y2-selective agonist, [ahx5-24]NPY (1 µM) was also effective in inhibiting the development of the 1°AD recorded in the subiculum, and its effects also reversed rapidly (~5 min) on wash out. E: [ahx8-20]NPY (1 µM) was effective in suppressing the 1°AD, which usually returned within a few minutes of wash out. F: NPY (1 µM) completely suppressed the 1°AD, but did not wash out even after 30 min. The response began to recover after 55 min wash (not illustrated). All traces were recorded from a single slice from an 18-day-old animal.



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Fig. 8. Effect of different NPY agonist peptides on the duration of STIB-induced 1° afterdischarges recorded in area CA3 or the subiculum. Neither of the Y5 agonists, D-Trp32NPY (1 µM) nor [ahx8-20]Pro34NPY (3 µM) was effective in suppressing 1°AD duration recorded in area CA3 (; 2/10 slices responding for D-Trp32NPY and 1/10 for [ahx8-20]Pro34NPY) or in the subiculum (; 0/4 for both agonists). The Y2 agonist, [ahx5-24]NPY, showed higher sensitivity in CA3 (5/7 slices responding) than in the subiculum (1/4 slices), whereas nonselective NPY agonist [ahx8-20]NPY (1 µM) was more or less equally effective in the subiculum (4/4 slices responding) and in CA3 (4/5 slices). NPY itself was equally effective in both area CA3 (4/4 slices) and the subiculum (3/3 slices). An effective response was defined as at least a 50% reduction in the 1°AD duration after treatment of agonist. All average reductions of 1°AD duration by various NPY agonists were reported as means ± SE. Average (±SE) age of animals used for CA3 area and the subiculum was 19.08 ± 0.69 and 17.0 ± 0.41 days, respectively.


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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.


    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.


    FOOTNOTES

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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ABSTRACT
INTRODUCTION
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