Department of Neurosurgery, Yale University Medical School, New Haven, Connecticut 06520
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ABSTRACT |
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Patrylo, Peter R., Anthony N. van den Pol, Dennis D. Spencer, and Anne Williamson. NPY Inhibits Glutamatergic Excitation in the Epileptic Human Dentate Gyrus. J. Neurophysiol. 82: 478-483, 1999. Neuropeptide Y (NPY) has been shown to depress hyperexcitable activity that has been acutely induced in the normal rat brain. To test the hypothesis that NPY can also reduce excitability in the chronically epileptic human brain, we recorded intracellularly from dentate granule cells in hippocampal slices from patients with hippocampal seizure onset. NPY had a potent and long-lasting inhibitory action on perforant path-evoked excitatory responses. In comparison, the group 3 metabotropic glutamate receptor agonist L-2-amino-4-phosphonobutyric acid (L-AP4) evoked a mild and transient decrease. NPY-containing axons were found throughout the hippocampus, and in many epileptic patients were reorganized, particularly in the dentate molecular layer. NPY may therefore play a beneficial role in reducing granule cell excitability in chronically epileptic human tissue, and subsequently limit seizure severity.
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INTRODUCTION |
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Neuropeptide Y (NPY) is found within axons and
somata in the hippocampus and many other regions of the brain
(Chronwall et al. 1985). In the rat hippocampus, acting
at a presynaptic site, NPY inhibits release of the excitatory
transmitter glutamate and can thereby reduce hyperexcitability
(Bleakman et al. 1992
; Colmers et al.
1988
; Klapstein and Colmers 1993
) as has been
demonstrated in acute models of seizurelike activity (Klapstein
and Colmers 1997
; Woldbye et al. 1997
).
Neurochemical studies suggest that there are changes in the expression
of both NPY and its receptors in the hippocampus and dentate gyrus of
epileptic humans and rodent models (de Lanerolle et al.
1992
; Gruber et al. 1994
; Marksteiner et
al. 1990
; Mathern et al. 1995
; Schwarzer
et al. 1998
; Sperk et al. 1992
). In most cases
the levels of expression are increased, suggesting that NPY may serve
as an endogenous mechanism to reduce seizure activity.
Electroencephalographic (EEG) and depth electrode studies have shown
that temporal lobe epilepsy can have temporal neocortical or
hippocampal onset (Spencer et al. 1993). In hippocampal
slices from patients with temporal lobe epilepsy with cortical onset, perforant path stimulation generally leads to a single spike and a
small excitatory postsynaptic potential (EPSP). In contrast, in slices
from patients with hippocampal onset, the same stimulation leads to
multiple spikes, and a larger and generally longer polysynaptic EPSP,
indicative of dentate excitability (Williamson 1994
).
This chronic excitability may be due in part to enhanced
N-methyl-D-aspartate (NMDA) receptor-mediated
events and the presence of altered glutamatergic circuits. Therefore we
tested the hypothesis that NPY would reduce the glutamatergic synaptic
response evoked in hippocampal slices from epileptic patients with
hippocampal onset. We compared the actions of NPY with another agonist
that also acts at presynaptic receptors to reduce glutamate release,
the metabotropic group 3 receptor agonist,
L-2-amino-4-phosphonobutyric acid (L-AP4) (Gereau and Conn 1995
; Macek et al.
1996
). Although the importance of glutamatergic circuits has
been well-documented in epileptic tissue, regulation of this activity
by neuromodulators has received relatively little attention. The
actions of NPY have not been studied in chronically epileptic tissue,
or in human hippocampi.
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METHODS |
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The tissue used for the physiological experiments was obtained
from patients diagnosed with medically intractable temporal lobe
epilepsy with hippocampal onset. This determination was based on
intracranial EEG recordings and/or a presurgical evaluation suggestive
of medial temporal lobe sclerosis (Spencer et al. 1993). Subsequent neuropathological examination showed cell loss, whereas structural lesions and/or tumors were not observed. The methods for
preparation and maintenance of hippocampal slices from human tissue
have been described previously (Williamson et al. 1995
). These experiments were approved by the Yale University Human
Investigation Committee, and informed consent was obtained.
Intracellular recordings from dentate granule cells were made with
microelectrodes filled with 4 M K-acetate pulled on a Brown-Flaming electrode puller (Sutter Instruments). Perforant path fibers were activated by delivering electrical stimuli (0.1 Hz; 0.3 ms duration; 100-800 µA) to the outer molecular layer with bipolar stimulating electrodes. The stimulating current was maintained at a single intensity for any given cell. Drugs were dissolved in the bath medium
immediately before use and were applied as microdrops using a
picospritzer (General Valve). All compounds were obtained from Sigma.
We examined neuronal input resistance, membrane potential, and maximal
evoked response [number of action potentials, duration, and the area
(measured as the time-voltage integral)] before and 15 s after
the initiation of drug application. Data are presented as means ± SE, and statistical significance (P 0.05) was
determined using a two-tailed paired Student's t-test.
Histological sections (30-50 µm) were cut and then stained with
rabbit NPY antiserum, a gift of Dr. T. Gorcs. The antiserum is specific
for NPY, as described elsewhere (Csiffary et al. 1990). After overnight incubation in primary NPY antiserum, sections were
washed, immersed in biotinylated goat anti-rabbit antiserum, washed,
incubated in avidin-biotin-peroxidase complex (Vector Labs), and then
stained with diaminobenzidine and hydrogen peroxide as previously
described in greater detail (van den Pol 1997
).
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RESULTS |
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The data presented are from 32 neurons recorded from 22 patients. Intracellularly recorded cells exhibited a range of excitability that often appeared as a synaptically evoked burst of action potentials riding on complex EPSPs (Figs. 1 and 2). The amplitude and duration of these events were significantly (P < 0.05) and reversibly reduced by 2-amino-5-phosphonovaleric acid (APV; 200 µM), an NMDA receptor antagonist (Figs. 1A and 2C; n = 13 cells, 10 patients). Whether this excitability can also be attenuated by neuromodulators has not been extensively addressed in chronically epileptic tissue.
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We examined the effects of L-AP4, a group 3 metabotropic glutamate receptor agonist, on evoked responses (Fig. 1B; n = 8 cells from 6 patients). L-AP4 (100 µM) significantly and reversibly decreased the number of action potentials by 29 ± 9.3% [mean ± SE, from mean 3.1 ± 1 (range, 1-10) to 2.1 ± 0.7 (range, 1-7) spikes; P = 0.02], the EPSP duration by 14.3 ± 3.2% [mean pre-L-AP4 82 ms duration (range 71-129 ms); P = 0.006] and the time-voltage integral of the synaptic response by 18.8 ± 2.8% [from mean 4,860 ± 730 mVs (range, 1,666-8,034 mVs) to 3,908 ± 565 mVs (range, 1,481-5,885); P = 0.004]. In contrast, L-AP4 did not alter the membrane potential or input resistance. These data suggest that mGluR activation can produce a modest decrease in glutamatergic synaptic actions in this tissue (Fig. 2C).
The effects of NPY (1 µM) on evoked responses were also examined (n = 13 cells from 5 patients). Although NPY produced no consistent change in either the membrane potential or input resistance, dramatic inhibitory effects were observed on the evoked excitatory responses (Fig. 2). Specifically, NPY reduced the number of action potentials evoked by a single stimulus by 53.4 ± 2.7% [mean pre-NPY 2.7 ± 0.6 (range, 0-6), post-NPY 0.9 ± 0.3 (range, 0-3) spikes; P = 0.001], the duration of the response by 18.1 ± 3.3% mean (pre-NPY control 135 ms duration; P = 0.0002), and the time-voltage integral by 52.0 ± 6.3% [pre-NPY mean 10,108 ± 1,432 (range, 5,323-25,003), post-NPY mean 4,615 ± 779 mVs (range, 1,688-10,424); P = 0.0002; Fig. 2C].
Additionally, NPY's effect appeared to be greater in more excitable cells. Specifically, a positive correlation was found (r = 0.56) when the relative excitability of the neurons, as assessed by their control time-voltage integral, was plotted relative to the effect of NPY on their time-voltage integral (percent decrease). Furthermore, NPY had a greater effect in tissue in which an evoked inhibitory postsynaptic potential (IPSP) was not observed. In cells with a biphasic IPSP (4 of 13 cells), NPY produced a 34.5 ± 9.8% decrease in the time-voltage integral, whereas in the group of cells in which evoked IPSPs were not detected, NPY caused a more substantial (60.1 ± 6.3%) decrease (Fig. 2D; P = 0.05).
Although recovery after L-AP4 and APV was generally
complete, a comparable level of recovery was not noted after NPY wash out (Fig. 2B). This could be due to long-term actions of NPY
on synaptic activity as previously reported in slice (Colmers et al. 1988) and culture (Obrietan and van den Pol
1996
; van den Pol et al. 1996
), but could in
part also be due to the potentially sticky nature of this peptide in slices.
The electrophysiology experiments above were done on patients with
hippocampal seizure onset. The pattern of NPY immunostaining in the
dentate gyrus typical for this type of patient is shown in Fig.
3, A, B, and F. NPY
immunoreactive fibers were found in the inner and outer molecular
layer, the granule cell layer, and the hilus. Scattered NPY-positive
neuron somata were found in the hilus. In contrast, immunostaining of
the dentate gyrus from patients with cortical seizure onset, typically
lacking signs of hippocampal sclerosis, showed a relative paucity of
NPY fibers in the inner molecular layer and granule cell layer compared
with the outer molecular layer in the same section (Fig. 3,
C-E); NPY positive neurons were common in the hilus. These
data are consistent with previous data suggesting a reorganization of
the NPY fiber system in some patients with temporal lobe epilepsy
(de Lanerolle et al. 1992; Mathern et al.
1995
).
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DISCUSSION |
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NMDA receptor antagonists can reduce the excitability of responses
evoked in granule cells from epileptic hippocampi (e.g., Fig.
1A of Williamson 1994). However, the role
that neuromodulators may play in reducing this excitability has not
been extensively examined, and the actions of NPY have not previously
been reported in the human hippocampus. We report here that both
L-AP4 and NPY can significantly reduce cellular
excitability. Previous work in rodents has shown that NPY
(Colmers et al. 1988
; Klapstein and Colmers
1993
) and the group 3 metabotropic glutamate receptor (Gereau and Conn 1995
; Macek et al. 1996
)
act primarily on the presynaptic axon terminal to reduce glutamate
release, although postsynaptic actions may also occur (McQuiston
et al. 1995
). Although we did not directly address the site of
action here, our results showing a decrease in evoked excitatory
responses without an apparent change in input resistance or membrane
potential are consistent with the presynaptic receptor actions
previously reported.
In this study we show that NPY can attenuate excitatory responses
evoked by perforant path stimulation in dentate granule cells from
epileptic humans. These data are in apparent contrast to parallel work
on control rats where NPY had little effect on perforant path-evoked
activity (Klapstein and Colmers 1993). Whereas this
difference may be due to species-specific differences in receptor
expression (Dumont et al. 1998
), we cannot rule out the possibility that an NPY-mediated action on glutamate release at the
recurrent collaterals of the granule cell mossy fibers might also be
involved (Babb et al. 1991
; Dudek et al.
1994
; Houser et al. 1990
; Patrylo and
Dudek 1998
; Sutula et al. 1989
). Previous work
in the rodent has shown that NPY can alter the release of glutamate
from these fibers (Klapstein and Colmers 1993
).
NPY has been shown to reduce excitatory activity in the rat hippocampus
made acutely hyperexcitable by either reducing GABA inhibition or
enhancing glutamate excitation (Klapstein and Colmers 1997; Woldbye et al. 1997
). The relative
efficacy (this study) and long-lasting effect of NPY (Colmers et
al. 1988
; van den Pol et al. 1996
) in reducing
excitatory activity in hyperexcitable neurons and in the epileptic
human hippocampus, support the hypothesis that NPY may be particularly
effective in reducing glutamate-mediated hyperactivity. The probable
release pattern of NPY makes this peptide a prime candidate for an
endogenous anticonvulsant. NPY is located within dense core vesicles
(Pickel et al. 1995
) that are believed to be released
during high-frequency firing (Hökfelt 1991
). Thus
NPY may be released during a seizure and thereby limit its severity.
Indeed, data from transgenic NPY knockout mice have shown that the
threshold for seizure induction does not appear altered, yet the
severity and lethality of induced seizures are significantly greater
than in controls (Baraban et al. 1997
). However, whether
NPY plays a role in restricting seizure onset in chronically epileptic
tissue is unknown, especially given the extensive changes in the NPY
system seen in epileptic tissue.
Anatomic studies on the human epileptic hippocampus also suggest that
NPY can reduce glutamateric excitation. Although there appears to be a
reduction in the number of NPY neurons in the hilus of temporal lobe
epileptics with hippocampal seizure onset, there is an increase in the
distribution of NPY immunoreactive axons in the inner molecular layer.
In contrast, in control tissue and tissue from patients with a cortical
seizure onset, the inner molecular layer shows relatively fewer axons
(de Lanerolle et al. 1992; Mathern et al.
1995
). This reorganization of NPY immunoreactive fibers in the
patient population used in the present study (hippocampal seizure
onset) occurs in the same area where glutamatergic circuits may be
formed due to reorganization of the mossy fibers. NPY may therefore
play a role in limiting activity at this recurrent excitatory synapse.
In conclusion, our data support the hypothesis that NPY can act as an endogenous neuromodulator that may limit hyperexcitability in the epileptic human dentate gyrus. We postulate that one neuronal mechanism that restricts the severity and propagation of seizures may be presynaptic inhibition of glutamate release, in part mediated by NPY.
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ACKNOWLEDGMENTS |
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We thank Dr. Y. Yang for help with histology.
This research was supported by National Institutes of Health Grants NS-31573, NS-34887, NS-37788, NS-06208, NS-30619, and AG-00795.
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FOOTNOTES |
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Address for reprint requests: A. N. van den Pol, Dept. of Neurosurgery, Yale University Medical School, 333 Cedar St., PO Box 208082, New Haven, CT 06520-8082.
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 29 January 1999; accepted in final form 1 March 1999.
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REFERENCES |
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