Address correspondence and reprint requests to Dr. S. C. Baraban at Box 0520, Department of Neurological Surgery, 513 Parnassus Avenue, UCSF, San Francisco, CA 94143, U.S.A. E-mail: email@example.com
Summary: Purpose: Recent evidence suggests an antiepileptic role for neuropeptide Y (NPY) in the central nervous system. The precise receptor subtypes mediating the inhibitory actions of NPY in the hippocampal formation, however, remain unclear. In vitro studies suggest a role for Y2 receptors in modulating excitatory hippocampal synaptic transmission and epileptiform discharge. In vivo studies implicate Y5 receptors. Here we used pharmacologic tools and Y5-receptor knockout mice to examine the role of Y5 receptors in mediating the antiexcitatory and antiepileptic actions of NPY in the hippocampal formation.
Methods: Hippocampal slices were obtained from age-matched wild-type (WT; 129 s3/svimj) and Y5-receptor knockout (Y5R KO) mice generated on the same background strain. Extracellular or whole-cell voltage-clamp recordings were obtained in area CA3 pyramidale. Evoked population spikes or excitatory postsynaptic currents were monitored during bath application of NPY, NPY13-36, or human pancreatic polypeptide (hPP). In some slices, zero-magnesium cerebrospinal fluid (CSF) was used to evoke spontaneous epileptiform discharges.
Results: NPY and NPY agonists with preference for either Y2 (NPY13-36) or Y5 (hPP) receptor subtypes caused a significant reduction in population spike and excitatory postsynaptic current (EPSC) amplitudes in slices from WT mice. NPY (and NPY agonists) also suppressed zero-magnesium epileptiform burst discharge in slices from WT mice. In contrast, bath application of NPY, NPY13-36, or hPP had no effect in slices from Y5R KO mice.
Conclusions: NPY modulates excitatory synaptic transmission and inhibits limbic seizure activity in the mouse hippocampus. The antiepileptic actions of NPY, in the mouse, appear to require activation of hippocampal Y5 receptors.
Neuropeptide Y (NPY) is one of several brain peptides capable of inhibiting epileptiform discharge (1–4). Within the hippocampus, a region long implicated in epileptogenesis, NPY has been shown to inhibit excitatory synaptic transmission at Schaffer collateral-to-CA1 and mossy fiber-to-CA3 synapses (5). Hippocampal NPY content is increased after an acute seizure (6,7), and plastic changes in NPY-receptor subtype expression have been noted in various experimental models of limbic seizure activity (8,9). Furthermore, mice lacking NPY are unable to terminate limbic seizures initiated by the glutamate analogue kainic acid (10). Taken together, these findings have led to the hypothesis that NPY, released from γ-aminobutyric acid (GABA)ergic interneurons during abnormal electrical discharge, reduces presynaptic glutamate release, resulting in a powerful inhibition of limbic seizure activity. The precise receptor mechanisms responsible for the antiepileptic actions of NPY, however, remain uncertain.
In the absence of selective NPY-receptor antagonists, in vitro studies using peptide fragments with receptor subtype “preference” suggest that the antiepileptic actions of NPY are mediated through Y2 receptors (11). In contrast, using similar peptide fragments, in vivo studies suggest a role for Y5 receptors (12). Because peptidergic modulation of presynaptic glutamate release may represent a novel avenue for anticonvulsant drug (AED) design, it is important to understand the precise mechanisms through which NPY exerts its antiepileptic actions. To address this issue, we examined NPYergic modulation of excitatory synaptic transmission in the hippocampus of age-matched WT and Y5R KO mice. Using NPY and NPY peptide fragments with preference for either Y2 or Y5 receptor subtypes, we studied modulation of population spike activity, excitatory postsynaptic currents, and spontaneous epileptiform discharge in the CA3 region of acute hippocampal slices. Our results are consistent with the hypothesis that activation of a hippocampal Y5 receptor is necessary to produce the antiepileptic actions of NPY in the mouse.
Mice were housed and handled according to guidelines approved by the UCSF Committee on Animal Care. The methods for preparing acute hippocampal slices have been described in detail (10). Slices were stored in artificial cerebrospinal fluid (aCSF) containing (in mM) 124 NaCl, 3 KCl, 26 NaHCO3, 1.24 NaH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, and 10 mM dextrose bubbled with 95% O2 and 5% CO2. For acute studies, a slice was then transferred to a recording chamber and continuously perfused with oxygenated aCSF at 32–34°C. The internal solution for field recordings contained 2 M NaCl, and a cesium gluconate–based internal solution was used for patch-clamp recordings (13). The current and voltage were recorded with an Axopatch 1D amplifier (Axon Instruments, Foster City, CA, U.S.A). All patch-clamp recordings were obtained from visually identified CA3 pyramidal cells using an Olympus microscope (BX50, Japan) equipped with infrared differential interference contrast optics and a CCD camera (Hamamatsu C2400, Japan). In each slice, a monopolar stimulating electrode was placed in the dentate hilus. To isolate evoked excitatory postsynaptic currents (EPSCs), the bathing medium was supplemented with the GABAA-receptor antagonist bicuculline (5 μM). Evoked EPSCs were abolished by application of bathing medium supplemented with the glutamate-receptor antagonists, 10 μMd-2-amino-5-phosphonopentanoic acid (D-APV) and 50 μM 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt (CNQX) (see Fig. 2C). To elicit spontaneous epileptiform discharge, magnesium was removed from the bathing medium (i.e., zero-magnesium CSF). All NPY agonists were stored as aqueous solutions of 1 mM and kept frozen at –20°C until immediately before use. Slices were exposed to peptides via the perfusate for 1–10 min at a flow rate of ∼2 ml/min.
NPY responses in wild-type mouse hippocampus
Extracellular recordings were made from area CA3, and population spikes were elicited by stimulating electrodes placed in the mossy fiber pathway (n = 13). Application of 1 μM NPY caused a significant reduction in population spike amplitude for responses obtained in hippocampal slices from WT mice (Fig. 1). Application of 1 μM NPY13-36 (Y2R-preferring peptide agonist) or 1 μM human pancreatic polypeptide (hPP; Y5R-preferring peptide agonist) also caused a reduction in CA3 population spike amplitude in hippocampal slices from WT mice (Fig. 1A).
Whole-cell voltage-clamp recordings were made from visually identified CA3 pyramidal cells, and EPSCs were elicited by stimulating electrodes placed in the mossy fiber pathway (n = 9). Application of 1 μM NPY caused a marked reduction in evoked EPSC amplitude in hippocampal slices from WT mice (Fig. 2). Application of 1 μM NPY13-36 or 1 μM hPP also caused a significant reduction in evoked EPSCs recorded on CA3 pyramidal cells in hippocampal slices from WT mice (Fig. 2A). NPY agonists had no effect on the holding current or membrane properties of CA3 pyramidal cells (data not shown).
Spontaneous epileptiform discharge generated in zero-magnesium CSF was monitored using an extracellular recording electrode placed in area CA1 or CA3 (n = 12). Application of 1 μM NPY reversibly suppressed epileptiform discharge in hippocampal slices from WT mice (Fig. 3A). Application of 1 μM NPY13-36 or 1 μM hPP also suppressed epileptiform discharge in hippocampal slices from WT mice (Fig. 3A).
NPY responses in Y5 receptor KO mouse hippocampus
To examine the role of Y5 NPY receptors in mediating the inhibitory and antiepileptic actions of NPY, in vitro experiments were performed in slices from Y5R KO mice under identical recording conditions used for the WT control studies described earlier. Application of 1 μM NPY did not significantly alter population spike amplitudes in hippocampal slices from Y5R KO mice (n = 13; baseline, 3.9 ± 0.8 mV; NPY, 4.1 ± 0.8) Application of 1 μM NPY did not significantly alter evoked EPSC amplitudes in hippocampal slices from Y5R KO mice (n = 3; baseline, 7.9 ± 0.2 pA; NPY, 7.7 ± 0.2 pA; Fig. 2C). Application of 1 μM NPY did not suppress zero-magnesium–induced epileptiform discharge in hippocampal slices from Y5R KO mice (n = 7; baseline burst frequency, 0.1 ± 0.02 Hz; NPY burst frequency, 0.1 ± 0.01 Hz; Fig. 3C).
Here we have shown that NPY mediates excitatory synaptic transmission at mossy fiber-to-CA3 excitatory synapses in the mouse hippocampus. We also demonstrate that NPY effectively inhibits spontaneous epileptiform activity in the mouse hippocampus. On the basis of these results, we then tested the hypothesis that Y5 receptors are responsible for the actions of NPY using hippocampal slices from Y5R KO mice. Although earlier studies suggested that Y2 receptors mediate the inhibitory and antiepileptic actions of NPY in the rat hippocampus (2,11), we were unable to demonstrate a significant NPY effect in slices from Y5R KO mice. Y2 receptors are present in the hippocampal formation of rats/mice (14,15) and may participate in NPYergic modulation of inhibitory synaptic transmission (as was recently demonstrated in the thalamus) (16) or modulation of a different excitatory synapse than was studied here. Conversely, the discrepancy between our findings and previously published results may be related to species differences in the expression and/or function of NPY receptor subtypes.
Evoked synaptic activity
We first attempted to demonstrate that NPY inhibits excitatory synaptic transmission onto CA3 pyramidal cells in the mouse hippocampus. Previous work by Colmers et al. (5) showed that 1 μM NPY application reduced CA3 excitatory postsynaptic potentials by ∼44% in hippocampal slices from adult rats. The inhibitory effect of NPY in rat hippocampus was also observed at Schaffer collateral-to-CA1 and Schaffer collateral-to-subiculum synapses (5,11) and was mimicked by application of putative Y2R-preferring ligands (e.g., NPY13-36,[ahx8-20]NPY, PYY3-36). At the single-cell level, NPY and Y2R-preferring ligands had no effect on the membrane properties of CA3 pyramidal neurons but produced a significant reduction in the amplitude of evoked excitatory postsynaptic potentials or currents (17,18). These findings suggest that NPY, binding to presynaptic Y2 receptors (14), mediates excitatory synaptic transmission in the rat hippocampus. In hippocampal slices from WT mice, we observed a significant reduction in field excitatory postsynaptic potential and evoked excitatory postsynaptic current amplitude after bath application of 1 μM NPY. Data in the mouse hippocampus are qualitatively similar to that reported for the rat hippocampus (e.g., 30–45% inhibition of evoked excitatory field activity in both species). Interestingly, the inhibitory effect of NPY in mouse hippocampus was mimicked by application of 1 μM NPY13-36 (a Y2R-preferring ligand) or hPP (a Y5R-preferring ligand). These latter findings raise the issue of peptide-agonist specificity, as it is possible that micromolar concentrations of NPY analogues activate multiple receptor subtypes in vitro. This hypothesis is supported by our subsequent observation that NPY has no such inhibitory actions at excitatory CA3 synapses in hippocampal slices from Y5R KO mice. We conclude that in the absence of selective NPY receptor agonists or antagonists, genetic approaches appear to be the most accurate means of assessing NPY receptor function in vitro.
Spontaneous epileptiform activity
We next attempted to demonstrate that NPY inhibits epileptiform activity in the mouse hippocampus. Previous work showed that 1 μM NPY application abolished stimulus train-induced bursting in area CA3 of rat hippocampal slices (11). Epileptiform activity produced by the STIB protocol was also suppressed by application of [ahx5-24]NPY and [ahx8-20]NPY, putative Y2R-preferring NPY agonists. In addition, Klapstein and Colmers (2) demonstrated that NPYergic suppression of epileptiform bursting could not be mimicked by the Y1 receptor–preferring agonist [Leu31,Pro34]NPY. Y1R expression increases immediately after an acute seizure, and recent studies suggest that Y1R activation may enhance the early stages of epileptogenesis (19), suggesting a permissive (but not necessarily anticonvulsant) role for this receptor subtype in limbic seizures. In hippocampal slices from WT mice, we observed suppression of zero-magnesium bursting with application of NPY, NPY13-36, and hPP. However, NPY agonists had no effect on epileptiform activity recorded in hippocampal slices from Y5R KO mice, and these animals showed no overt signs of altered hippocampal excitability owing to inactivation of the Y5 receptor (20). Thus, we conclude that a Y5R plays a key role in suppression of limbic seizure activity. Indeed, our hypothesis that Y5Rs mediate the antiepileptic actions of NPY is supported by in vivo experiments performed by Woldbye et al. in rats (12). In their studies, intracerebral injection of various NPY agonists suggests an antiepileptic profile that is consistent with activation of Y5 but not Y2 or Y1 receptors. Receptor-expression studies by Kopp et al. (21) demonstrated a significant elevation of Y5R mRNA levels in the hippocampus after an acute seizure and in vivo peptide-infusion studies by Reibel et al. (22) indicated suppression of focal hippocampal seizures with Y5R agonists, lending further support to the hypothesis that Y5 receptors play a critical antiepileptic role.
These results clearly show that exogenous NPY is capable of inhibiting excitatory synaptic transmission and epileptiform discharge in the mouse hippocampus. These effects appear to represent a biologic role for NPY in modulation of seizure activity, consistent with the following observations: (a) NPY is found at release sites near the glutamatergic presynaptic terminal in the hippocampus (23), (b) NPY is upregulated after acute seizure activity (6,7), (c) NPY receptor expression is altered during epileptogenesis (21), and (d) mice lacking NPY are unable to terminate limbic seizure activity (10). In the absence of any experimental evidence for developmental compensation in Y5R KO mice (20,24,25), we conclude that the antiepileptic actions of NPYare mediated by a hippocampal Y5 receptor.