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Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Milan, Italy
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • somatostatin receptors • neuropeptide Y receptors • epilepsy • neuropeptide Y • somatostatin • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Functional evidence suggests that
some neuropeptides act by inhibiting or facilitating classical
neurotransmitter functions (Hökfelt 1980
). In
particular, recent evidence suggests that somatostatin (SRIF) and
neuropeptide Y (NPY) affect synaptic transmission and neuronal
excitability and that these effects are mediated significantly by their
interaction with glutamatergic neurotransmission (Epelbaum 1986
, Mancillas et al. 1986
, Olpe et al. 1980
, Vezzani et al. 1999
).
Because an increase in glutamatergic function is involved in both
seizure initiation and propagation in the CNS (Schwarcz and Meldrum 1985
), it is of interest to investigate the
functional role of SRIF and NPY in epilepsy and whether pharmacologic
modifications of peptidergic function may control seizures by reducing
glutamate action.
Recent electrophysiologic and biochemical findings have indeed shown
that SRIF may act presynaptically by reducing glutamate release at
hippocampal synapses (Boehm and Betz 1997
), as well as
postsynaptically by depressing glutamate responses and baseline firing
(Mancillas et al. 1986
). These inhibitory effects appear
to be mediated by the SRIF1 family of receptors
(Hoyer et al. 1995
). Similarly, NPY reduces glutamate
release acting on presynaptic Y2 receptors
(Klapstein and Colmers 1993
), whereas an excitatory
component of NPY appears to be mediated by postsynaptic Y1
receptor subtypes (Brooks et al. 1987
, Gariboldi et al. 1998
).
Seizures in experimental models and humans profoundly affect the
functional status of neuropeptide-containing neurons particularly
those neurons that also contain 
-aminobutyric acid (GABA)
(DeLanerolle et al. 1989
, Schwarzer et al. 1996
, Sloviter, 1991
) and induce the ectopic
expression of NPY in glutamatergic granule cells of the dentate gyrus
(Schwarzer et al. 1996
, Sperk et al. 1992
).
In this report, we describe the pharmacologic evidence showing that SRIF and NPY analogs, acting on specific receptor subtypes, inhibit seizures induced by glutamate receptor stimulation. This suggests that selective ligands for neuropeptide receptors may be of value for controlling excessive glutamate function.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Sprague-Dawley rats (225–250 g, Charles River, Calco, Italy) were used. Procedures involving animals and their care conformed with the institutional guidelines, in compliance with national and international laws and policies.
Electrode implantation, EEG recording and intracerebral injection of drugs.
Surgical procedures for electrodes and injection cannula implantation
have been described elsewhere (Vezzani et al. 1991
). The
procedures for recording the EEG and the intracerebral injection of
drugs in unanesthetized rats have been described (Vezzani et al. 1991
). Kainic acid (Sigma-Aldrich, St. Louis, MO; 0.04
µg in 0.5 µL 0.1 mol/L PBS, pH 7.4)
was infused (60 s) into the dorsal hippocampus in the region of granule
cells. This was the smallest dose found to induce EEG seizures in all
of the animals (Vezzani et al. 1991
). Seizures consisted
of high frequency and/or multispike complexes and/or high voltage
synchronized spike or wave activity in cortical and hippocampal leads
and provided a sensitive measure of the anticonvulsant activity of
drugs (Vezzani et al. 1991
). EEG recordings were made
for at least 30 min to assess the spontaneous EEG pattern representing
a control baseline period, then continuously for at least 180 min after
kainic acid. Seizures were quantified by calculating the
latency to the first seizure, the total number of
seizures and the total time spent in seizures
(the duration of all ictal episodes) during the EEG recording period.
Systemic injection of kainic acid and evaluation of seizures.
Kainic acid or saline was injected subcutaneously into rats at a dose
of 12 mg/kg. Rats showing repeated episodes of severe generalized
limbic seizures with rearing and/or loss of postural control during the
3 h after drug injection were selected for further studies. These
rats were tested for their increase in seizure susceptibility 30 d
after kainic acid injection. A normally subconvulsive dose of
pentylentetrazol (30 mg/kg, intraperitoneal) was injected into rats and
their behavior observed for 30 min. Myoclonic (all body twitch)
convulsions were counted separately from generalized tonic-clonic
seizures (tonic-clonichindlimb extension with loss of posture). The
onset time to the first seizure episode was also measured
(Vezzani et al. 1994
).
Schedule of treatment with peptide analogs.
N-2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl-D-arginamide]
(BIBP 3226) (Rudolf et al. 1994
; Dr. Karl Thomae GmbH,
Biberach an der Riss, Germany) (5 and 10 µg in 1
µL) was dissolved in 25% polyethyleneglycol and
infused intrahippocampally at the same site as kainic acid, 10 min
before the convulsant. Octreotide (SMS 201–995; Novartis Pharma,
Basel, Switzerland; 2.5–20 µg/L) was dissolved in PBS
and infused intrahippocampally at the same site as kainic acid (in 0.5
µL) or in the ipsilateral entorhinal cortex (2
µL) 10 min before the convulsant. Seglitide (RC-160;
RBI, Natick; 6 µg in 1 µL) and NPY
13–36 (Sigma-Aldrich; 50 µg in 10
µL) were dissolved in saline and infused into the
hippocampus or given intracerebroventricularly, respectively, 15
min before pentylentetrazol. Controls received corresponding amounts of
vehicles before the convulsants.
After the experiments, the position of the electrodes and the track of the injection needle were checked visually in cryostat brain sections from each rat.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows the anticonvulsant effects of BIBP 3226, a selective NPY
Y1 receptor antagonist (Rudolf et al. 1994
) and of SMS 201–995, an agonist at
SRIF1 receptors (Reubi 1985
), on
EEG seizures induced by intrahippocampal kainic acid in rats. EEG
seizures were measured in this study because they have been found to be
the most significant, reproducible and quantifiable of the
epilepsy-like sequelae after kainic acid administration in the rat
hippocampus. EEG seizures provide a sensitive measure of the
anticonvulsant activity of drugs (Vezzani et al. 1991
).
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). The onset time
to seizures was delayed significantly by BIBP 3226 (kainic acid, 12
± 0.3 min; BIBP 3226, 33.4 ± 5.4 min, P < 0.01) and by 20 µg, but not 10 µg, SMS 201
995 in the entorhinal cortex (SMS 201–995, 39.8 ± 6.9 min,
P < 0.01).
Table 2
shows the effects of NPY 13–36 and RC 160, which act as agonists at
NPY Y2/Y5 and
SRIF1 (Reubi 1985
) receptors
(Michel et al. 1998
), respectively, on the enhanced
susceptibility to pentylenetetrazol seizures ensuing in rats 30 d
after a systemic administration of kainic acid (Vezzani et al. 1994
). Each compound reduced seizure susceptibility
significantly by decreasing the number of rats showing tonic-clonic
seizures (i.e., RC 160) or by increasing the number of rats without
seizures (i.e., NPY 13–36). NPY 13–36 also significantly delayed the
time to onset of seizures (P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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SRIF has been reported to depress glutamate responses as well as
baseline firing in both rat cortex and hippocampus (Mancillas et al. 1986
) and to inhibit glutamate release acting on
SRIF1 presynaptic receptors in the hippocampus
(Boehm and Betz 1997
). Similarly, NPY, acting on
presynaptic Y2 receptors on glutamatergic
terminals, reduces glutamate release in a highly selective manner
(Greber et al. 1994
, Klapstein and Colmers 1993
). These effects appear to be mediated by changes in
Ca2+ entry through voltage-dependent
Ca2+ channels (Boehm and Betz 1997
, Klapstein and Colmers, 1993
) and by
modifications in K+ currents (Moore et al. 1988
, Vezzani et al. 1999
). Seizure protection
by NPY Y1 antagonists may be achieved by blockade
of an excitatory component mediated by the endogenous peptide, [i.e.,
a reduction of Ca2+-dependent
K+ currents by NPY acting on postsynaptic
Y1 receptors on glutamatergic neurons
(Gobbi et al. 1996
, McQuiston 1996
) would
enhance neuronal excitability].
Recent evidence has shown that glutamate, in turn, affects
neuropeptide-expressing neurons and their receptors. Thus, the
synthesis of SRIF and NPY in cortical and hippocampal neurons and the
release of these peptides are enhanced by stimulation of metabotropic
and/or ionotropic glutamate receptors using selective agonists
(Fontana et al. 1996
, Gemignani et al. 1997
, Greber et al. 1994
). Changes in
NPY receptor subtypes, similar to those occurring after seizures
(Vezzani et al. 1999
), are induced by glutamate analogs
acting at metabotropic receptors (Schwarzer et al. 1998
). These findings suggest that the plastic changes in NPY
and SRIF receptors (Peréz et al. 1995
,
Piwko et al. 1996
, Vezzani et al. 1999
)
and the functional and morphological alteration in peptidergic neurons
found in epileptic brain tissue of experimental models and humans
(DeLanerolle et al. 1989
, Schwarzer et al. 1996
, Sperk et al. 1992
) are mediated by
glutamate released during seizures.
Because SRIF, NPY and glutamate appear to be linked functionally, changes in peptidergic transmission likely play a significant role in modulating neuronal excitability that is altered in epilepsy. This suggests that SRIF and NPY analogs may provide novel pharmacologic tools for controlling excessive glutamate function.
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FOOTNOTES |
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3 Abbreviations used: BIBP 3226,
N-2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl-D-arginamide];
CNS, central nervous system; EEG, electroencephalographic; GABA,
-aminobutyric acid; NPY, neuropeptide Y; RC-160, seglitide; SMS
201–995, octreotide; SRIF, somatostatin.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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