Laboratory of Neuropsychiatry, Department of Psychiatry, University Hospital Rigshospitalet, Department of Pharmacology, University of Copenhagen, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark
Received 16 October 2001; in revised form 20 December 2001; accepted 16 January 2002
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ABSTRACT |
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INTRODUCTION |
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NPY receptors are potential pharmacological targets for treatment of several neuropsychiatric disorders. For instance, it has been shown in our laboratory (Woldbye et al., 1996, 1997
; Woldbye, 1998
; Klemp and Woldbye, 2001
) and by others (Vezzani et al., 1999
; Reibel et al., 2001
) that exogenous application of NPY inhibits epileptic and epileptiform seizures as well as opioid withdrawal (Woldbye et al., 1998
; Clausen et al., 2001
). Both seizures (Meldrum, 1994
) and opioid withdrawal (Aghajanian et al., 1994
) are associated with increased release of glutamate and neuronal hyperexcitability. Ethanol withdrawal is a serious medical condition also characterized by neuronal hyperexcitability and increased glutamate release (Tsai and Coyle, 1998
). Gamma-aminobutyric acid A receptor-modulating drugs, such as benzodiazepines and barbiturates, are efficient at reducing ethanol withdrawal symptoms in humans and rodents (Kramp and Rafaelsen, 1978
; Ulrichsen et al., 1986
, 1995
). However, these drugs may not yield sufficient protection against the neurotoxic effects of withdrawal, and glutamate antagonists have therefore been suggested as a potential alternative treatment strategy (Tsai and Coyle, 1998
). NPY inhibits presynaptically the release of glutamate in hippocampal slices of rats (Vezzani et al., 1999
) and humans (Patrylo et al., 1999
). Thus NPY receptors might be a potential target for alternative treatment of withdrawal. The purpose of the present study was therefore to test whether exogenous application of NPY can attenuate symptoms of ethanol withdrawal in rats.
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MATERIALS AND METHODS |
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At 1617 h after the last infusion of ethanol, all rats were scored for signs of withdrawal according to the following rating scale (Ulrichsen et al., 1986). Three signs were noted: irritability, rigidity, and tremor. Each sign was given a score of 03 (0 = not present; 1 = slight; 2 = moderate; 3 = severe). Withdrawal rating was repeated 15 min later to establish baseline levels for each animal. Rats with a mean total withdrawal score of
4 were excluded at this point. Assessed on the mean baseline score, the rats were divided into two groups using stratified randomization (Altman, 1991
) to ensure equal inter-group withdrawal levels. One group received an i.c.v. injection (10 µl) containing human/rat synthetic NPY (12 or 24 nmol; #N-5017, Sigma, St Louis, MO, USA) while the other group received vehicle (0.9% isotonic saline and 1% bovine serum albumin) administered for a duration of 1 min. The two different doses of NPY were tested in separate experiments. Fifteen minutes later and subsequently every 15 min, the withdrawal score was determined during the next 2 h and again at 5 h after the i.c.v. injection. Subsequently, the rats were killed. Withdrawal rating was done by an observer blinded as to whether NPY or vehicle had been injected. Rats developing seizures during the observation period were excluded because the withdrawal score, post-ictally, was not believed to represent truly the effects of withdrawal per se, considering the possible influence of post-ictal behavioural depression.
For each rat, the withdrawal score was plotted as a function of time. Visual inspection of these plots made it reasonable to assume a linear relationship between the withdrawal score and the time from 15 min to 2 h post-i.c.v. For each rat, the withdrawal slope of the line that would best fit the data was calculated by linear regression analysis (Altman, 1991). The withdrawal slopes and the withdrawal score at 5 h post-i.c.v. were statistically analysed using MannWhitney U-tests with P < 0.05 as the level of significance.
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RESULTS |
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The total withdrawal scores during the observation period are shown in Fig. 1. For both NPY doses, the withdrawal score of NPY-treated rats was lower than vehicle from 75 min post-i.c.v., reaching its lowest level at ~105 min. The withdrawal slopes are shown in Fig. 2
. The slopes of the total withdrawal score of both 12 and 24 nmol NPY were significantly steeper (P < 0.006) than those of the vehicle groups, indicating an anti-withdrawal effect of NPY at both doses. The withdrawal score following 24 nmol was significantly lower at 5 h post-i.c.v. whereas the 12 nmol dose was not (Fig. 1
).
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A total of three out of 16 rats receiving NPY, as opposed to two out of 17 receiving vehicle, developed seizures during the 5 h observation period. This would appear to rule out an inhibitory effect of NPY on withdrawal seizures. However, this matter cannot be determined in the present study, because withdrawal seizures in the NPY groups occurred before anti-withdrawal effects of NPY were most pronounced. In addition, the number of convulsing rats in the vehicle groups was too small, in the first place, to allow the detection of an anticonvulsant effect.
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DISCUSSION |
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The elucidation of the receptors mediating the anti-withdrawal effects of NPY in the present study awaits the testing of specific NPY receptor ligands. Y1, Y2 and Y5, which are the quantitatively most important NPY receptors in the rat brain, are all potential candidates. Thus, inhibitory effects of NPY against morphine withdrawal are mediated by receptors with a Y5-like receptor profile (Woldbye et al., 1998). Both Y5 and Y2 receptors have been implicated in reduction of neuronal hyperactivity and hyperexcitability associated with seizures (Woldbye et al., 1997
; Vezzani et al., 1999
). The Y1 receptor is another candidate, because it appears to mediate anti-anxiety effects of centrally administered NPY (Heilig et al., 1989
).
Several studies indicate that increased activity at glutamatergic synapses may be centrally involved in the cerebral mechanisms of the ethanol withdrawal syndrome. Thus, in rodents chronically treated with ethanol, glutamatergic N-methyl-d-aspartate (NMDA) receptors are up-regulated in the hippocampus (Trevisan et al., 1994) and glutamate-induced neurotoxicity is increased in cerebellar granule cells (Iorio et al., 1993
). Moreover, increased sensitivity to the neurotoxic and convulsant action of NMDA is present during ethanol withdrawal (Sanna et al., 1993
), and mice bred selectively for their ability to display ethanol withdrawal seizures have increased density of NMDA receptor binding sites (Valverius et al., 1990
). In hippocampal slices, NPY inhibits neuronal activity by presynaptically reducing the release of glutamate (Vezzani et al., 1999
). As the release of glutamate during ethanol withdrawal is increased in several forebrain regions, including the hippocampus (Harms and Woodward, 1995
; Rosetti and Carboni, 1995
; Dahchour and De Witte, 1999
), reduction of glutamate release seems to be one likely mechanism for the anti-withdrawal effect of NPY in the present study.
Because benzodiazepines and barbiturates presently used in the clinic to treat ethanol withdrawal may not sufficiently antagonize the neurotoxic effects of withdrawal (Tsai and Coyle, 1998), drugs with antagonistic effects at glutamate receptors have been suggested as an alternative treatment. In this context, the present study indicates that NPY receptors might deserve attention as a future target for treatment of ethanol withdrawal.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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