Neuropeptides ( 1992) 21, 147- 152 0 Longman Group UK Ltd 1992
Endogenous Opioid Peptides in Brain and Pituitary of Rats with Absence Epilepsy W. LASOfi*, B. PRZEWLOCKA”, R. PRZEW4DCKI”
E. L. J. M. VAN LUlJTELAARt,
A. M. L. COENENt
“Neuropeptides Research Department, Institute of Pharmacology, Sciences, Krakdw, Poland; tDepartment of Psychology, University The Netherlands. (Correspondence and reprint requests to WL)
and
Polish Academy of of Nijmegen, Nijmegen,
Abstract -The level of opioid peptides in several brain areas and in the pituitary was estimated in WAG/Rij rats, which are considered to be a genetic animal model for human absence epilepsy. In comparison with three groups of non-epileptic controls, these epileptic rats had an elevated level of the proenkephalin-derived peptide Met-enkephalin-Args-Gly7-Leu* in the mesencephalon and striatum, while the level of the prodynorphin-derived peptide a-neoendorphin was increased in the striatum and hippocampus. In addition various ageand/or strain-related changes in these peptide levels were found in the hippocampus, thalamus, striatum, frontal cortex and neurointermediate lobe of the pituitary. No difference in the hypothalamic P-endorphin level were found between epileptic and non-epileptic rats, though strain- and/or age-related changes in the peptide content were detected in both lobes of the pituitary. The increased level of proenkephalin and prodynorphin opioid peptides in brain structures, essential for the appearance of spike-wave discharges, suggests that these opioid systems, but not proopiomelanocortin one, may play a role in absence epilepsy.
Introduction Some evidence confirms that endogenous opioid systems are involved in seizure phenomena (1, 2) and that opioid peptides have both pro- and anticonvulsant properties (3, 4). Numerous biochemical studies showed regional changes in brain opioid peptide levels following generalized motor seizures (5, 6, 7). However, it remains an open question whether endogenous opioids are also involved in non-convulsive epilepsy. It has been reported that centrally administered enkephalins evoke electroencephalographic and/or behavioral seizures Date received Date accepted
14 August 199 1 20 October 199 1
which resemble human absence epilepsy (8, 9), yet little is known about the involvement of endogenous opioid peptide systems in this type of epilepsy. Recently, it has been proposed that a particular inbred strain of rats, i.e. the WAG/Rij strain, might be a model of absence epilepsy (10, 11). All rats of this strain showed 7- 10 Hz spike-wave discharges in the cortical EEG together with behavioral concomitants. Subsequent pharmacological and electrophysiological studies suggest that these discharges resemble those observed in human absence epilepsy (12). The results from all these studies strongly suggest that rats from the WAG/Rij strain are a suitable genetic model for absence epilepsy in man. In the present study, the level of 147
148 endogenous opioid peptides which represent the proenkephalin, the prodynorphin, and the proopiomelanocortin system were investigated in various brain structures and the pituitary of epileptic WAG/Rij rats and were compared with non-epileptic controls. Methods
Subjects: Male, 6 month-old epileptic WAG/Rij rats (WAG-6) weighing 250-325 g were used. In electrophysiological studies WAG/Rij rats of that age showed a large number of spike-wave discharges (13,14). Three-month-old WAG/Rij rats (WAG-3), which do not exhibit spike-wave discharges, as well as non-epileptic 3- and 6-month-old AC1 (ACI-3 and ACI-6) rats served as controls. 10 rats per each group were used. The animals were decapitated and their brains and pituitaries were quickly removed. Tissue extraction: The hypothalamus, thalamus, hippocampus, striatum, mesencephalon and cortex were carefully dissected. Pituitaries were divided into the anterior and neurointermediate lobes. Tissue parts were weighed and then incubated in 0.1 N HCl (five times the volume of the tissue) at 95°C for 10 min. Following homogenization, the homogenates were centrifuged at 10 000 g for 15 min and the supernatants were adjusted to pH = 7.5. Radioimmunoassays: Aliquots were then assayed for Met-enkephalin-Arg6-Gly’-Leus (MEAGL), aneoendorphin and P-endorphin according to the previously described procedures (15, 16, 17). The antiserum directed against the synthetic a-neoendorphin recognized a- and P-endorphin with high avidity (10% cross-reactivity), but did not crossreact with dynorphin A, Met- or Leu-enkephalin, or P-endorphin (less than 0.01%). The antiserum directed against the human P-endorphin recognized the rat P-endorphin, but did not cross-react with Leuenkephalin, dynorphin or a-neoendorphin. The antiserum directed against MEAGL did not cross-react with dynorphin, a-neoendorphin or P-endorphin. Data analysis: A two-by-two way design was used to test, with an analysis of variance (ANOVA), the two main effects, strain (WAG/Rij and ACI) and age (3 and 6 months), and the strain x age interaction
NEUROPEPTIDES
effect. When a significant interaction between age and strain was established, post-hoc’s according to Duncan (p < 0.05) were used to determine whether the WAG-6 rats differed from three controls. If this interaction was significant and the WAG-6 rats differed from the three controls, the differences in peptide levels were interpreted as being related to the fact that WAG-6 rats were epileptic. Results
The ANOVA for MEAGL levels showed that WAG6 rats had a higher level of the peptide in the mesencephalon (F( 1,36) = 7.77, p < 0.01) and striatum (F( 1,36) = 8.92, p < 0.01). Also a significant interaction was found for the hypothalamus (F( 1,30) = 4.59, p < 0.05), but the post hoc failed to find a significant difference between WAG-6 and the other groups. Additionally, an age effect on the levels in the striatum (F( 1,36) = 4.77, p < 0.05), neurointermediate lobe of the pituitary (F( 1,36) = 5.57, p < 0.05) and frontal cortex (F( 1,34) = 4.40, p < 0.05) was observed. Furthermore, a strain 1 effect for MEAGL levels was found in the striatum (F(1,36) = 36.5, p < O.OOl), thalamus (F(1,34) = 6.92, p < 0.02), and mesencephalon (F( 1,36) = 4.66, p < 0.05) (Table 1). Table 1
Means and standard errors (pmol/g of wet tissue) of the Met-enkephalin-Arg6-Glu’-LeU8 (MEAGL) level in the hippocampus (HIP), mesencephalon (M), striatum (ST), thalamus (TH), frontal cortex (FCX), hypothalamus (HPT) and neurointermediate lobe (NIL) ofthe pituitary in epileptic (WAG-6) and non-epileptic control (ACI-3, ACI-6 and WAG-3) rats AU-3 HIP M ST
TH
FCX HPT NIL
39_+4 118klO 143 i 20 70+12 656 121_+9 524 + 38
ACI-6
47+3 113f5 1262 11 64 & 7 67+3 15Oi 16 454i51
WAG-3
WAG-6
38_+3 114*5 208+29 93*15 60_+6 152_+15 573 f47
39&2 143 f 4’“’ 318 i22’“” 107+15 82 _+7b 117f19” 436 _+37b
*) p< 0.05 (Duncan) WAG-6 group differs from all three control groups: a) p < 05 (ANOVA) strain x age interaction b) p < 0.05 (ANOVA) age effect c) p < 0.05 (ANOVA) strain effect.
The ANOVA for the a-neoendorphin showed that the peptide level in the WAG-6 rats was elevated in the hippocampus (F(1,34)= 18.57, p < 0.001) and
ENDOGENOUS
OPIOID PEPTIDES
IN BRAIN AND PITUITARY
OF RATS WITH ABSENCE
striatum (F( 1,36) = 6.04, p < 0.02). Additionally, the age effect in the hippocampus (F(1,34) = 14.85, p < 0.00 1) and neurointermediate lobe of the pituitary (F( 1,36) = 18.35 p < 0.001) as well as strain effect in the hippocampus(F(1,34) = 7.37,~ < O.Ol)andstriaturn (F( 1,36) = 10.44, p < 0.01) were found (Table 2). Table 2 Means and standard errors of the level of a-neoendorphin (pmol/g of wet tissue) in the hippocampus (HIP), mesencephalon (M), striatum (ST), thalamus (TH), hypothalamus (HPT), neurointermediate (NIL) and pars anterior (AL) lobe of the pituitary in epileptic (WAG-6) and non-epileptic control (ACI-3, ACI-6 and WAG-3) rats
HIP M ST TH HPT NIL AL
AU-3
AU-6
4014 19 i3 48+4 29 f 3 76 f 5 2067 +_90 IlOf 10
3926 25 i 5 41+2 29f4 85 + 5 2393 + 135 104k7
*) p ~0.05 (Duncan) control groups: a) p < 0.05 (ANOVA) b) p < 0.05 (ANOVA) c) p < 0.05 (ANOVA)
WAG-3
32 + 3 28 + 5 53 +_4 32 +4 77 +_4 2039 +_97 104 +4
WAG-6
67 30 80 35 74 2772 105
+ + + + + + +
3”bc 2 12’= 2 4 159b 6
WAG-6 group differs from all three strain x age interaction age effect strain effect.
The ANOVA of /3-endorphin levels showed a significant interaction in the neurointermediate lobe of the pituitary (F( 1,36) = 5.10, p < 0.05), there was however no significant difference between WAG-6 group and that three other groups (in fact the level of the WAG-3 rats was lower than that of the ACI3). Finally, there were significant strain and age effects in the anterior lobe of the pituitary (F(1,36)=6.99, ~~0.02 and F(1,36)= 19.52, p < 0.001, respectively) (Table 3). Table 3 Means and standard errors of the level of P-endorphin in the hypothalamus (HPT) (pmol/g of wet tissue), pars anterior (AL) (nmol/g of wet tissue) and neurointermediate (NIL) lobe of the pituitary (nmol/g wet tissue) in epileptic (WAG-6) and nonepileptic control (ACI-3, ACI-6 and WAG-3) rats A U-3 HP-I-
AL NIL
18k2 22 * 1 373 k 26
AU-6
15 i2 17+ 1 309+ 11
WAG-3
WAG-6
14k2 19fl 303 zk20
17 t4 15 + lk 336 t 26’
a) p
Endogenous Opioid Peptides in Brain and Pituitary of Rats with Absence Epilepsy W. LASOfi*, B. PRZEWLOCKA”, R. PRZEW4DCKI”
E. L. J. M. VAN LUlJTELAARt,
A. M. L. COENENt
“Neuropeptides Research Department, Institute of Pharmacology, Sciences, Krakdw, Poland; tDepartment of Psychology, University The Netherlands. (Correspondence and reprint requests to WL)
and
Polish Academy of of Nijmegen, Nijmegen,
Abstract -The level of opioid peptides in several brain areas and in the pituitary was estimated in WAG/Rij rats, which are considered to be a genetic animal model for human absence epilepsy. In comparison with three groups of non-epileptic controls, these epileptic rats had an elevated level of the proenkephalin-derived peptide Met-enkephalin-Args-Gly7-Leu* in the mesencephalon and striatum, while the level of the prodynorphin-derived peptide a-neoendorphin was increased in the striatum and hippocampus. In addition various ageand/or strain-related changes in these peptide levels were found in the hippocampus, thalamus, striatum, frontal cortex and neurointermediate lobe of the pituitary. No difference in the hypothalamic P-endorphin level were found between epileptic and non-epileptic rats, though strain- and/or age-related changes in the peptide content were detected in both lobes of the pituitary. The increased level of proenkephalin and prodynorphin opioid peptides in brain structures, essential for the appearance of spike-wave discharges, suggests that these opioid systems, but not proopiomelanocortin one, may play a role in absence epilepsy.
Introduction Some evidence confirms that endogenous opioid systems are involved in seizure phenomena (1, 2) and that opioid peptides have both pro- and anticonvulsant properties (3, 4). Numerous biochemical studies showed regional changes in brain opioid peptide levels following generalized motor seizures (5, 6, 7). However, it remains an open question whether endogenous opioids are also involved in non-convulsive epilepsy. It has been reported that centrally administered enkephalins evoke electroencephalographic and/or behavioral seizures Date received Date accepted
14 August 199 1 20 October 199 1
which resemble human absence epilepsy (8, 9), yet little is known about the involvement of endogenous opioid peptide systems in this type of epilepsy. Recently, it has been proposed that a particular inbred strain of rats, i.e. the WAG/Rij strain, might be a model of absence epilepsy (10, 11). All rats of this strain showed 7- 10 Hz spike-wave discharges in the cortical EEG together with behavioral concomitants. Subsequent pharmacological and electrophysiological studies suggest that these discharges resemble those observed in human absence epilepsy (12). The results from all these studies strongly suggest that rats from the WAG/Rij strain are a suitable genetic model for absence epilepsy in man. In the present study, the level of 147
148 endogenous opioid peptides which represent the proenkephalin, the prodynorphin, and the proopiomelanocortin system were investigated in various brain structures and the pituitary of epileptic WAG/Rij rats and were compared with non-epileptic controls. Methods
Subjects: Male, 6 month-old epileptic WAG/Rij rats (WAG-6) weighing 250-325 g were used. In electrophysiological studies WAG/Rij rats of that age showed a large number of spike-wave discharges (13,14). Three-month-old WAG/Rij rats (WAG-3), which do not exhibit spike-wave discharges, as well as non-epileptic 3- and 6-month-old AC1 (ACI-3 and ACI-6) rats served as controls. 10 rats per each group were used. The animals were decapitated and their brains and pituitaries were quickly removed. Tissue extraction: The hypothalamus, thalamus, hippocampus, striatum, mesencephalon and cortex were carefully dissected. Pituitaries were divided into the anterior and neurointermediate lobes. Tissue parts were weighed and then incubated in 0.1 N HCl (five times the volume of the tissue) at 95°C for 10 min. Following homogenization, the homogenates were centrifuged at 10 000 g for 15 min and the supernatants were adjusted to pH = 7.5. Radioimmunoassays: Aliquots were then assayed for Met-enkephalin-Arg6-Gly’-Leus (MEAGL), aneoendorphin and P-endorphin according to the previously described procedures (15, 16, 17). The antiserum directed against the synthetic a-neoendorphin recognized a- and P-endorphin with high avidity (10% cross-reactivity), but did not crossreact with dynorphin A, Met- or Leu-enkephalin, or P-endorphin (less than 0.01%). The antiserum directed against the human P-endorphin recognized the rat P-endorphin, but did not cross-react with Leuenkephalin, dynorphin or a-neoendorphin. The antiserum directed against MEAGL did not cross-react with dynorphin, a-neoendorphin or P-endorphin. Data analysis: A two-by-two way design was used to test, with an analysis of variance (ANOVA), the two main effects, strain (WAG/Rij and ACI) and age (3 and 6 months), and the strain x age interaction
NEUROPEPTIDES
effect. When a significant interaction between age and strain was established, post-hoc’s according to Duncan (p < 0.05) were used to determine whether the WAG-6 rats differed from three controls. If this interaction was significant and the WAG-6 rats differed from the three controls, the differences in peptide levels were interpreted as being related to the fact that WAG-6 rats were epileptic. Results
The ANOVA for MEAGL levels showed that WAG6 rats had a higher level of the peptide in the mesencephalon (F( 1,36) = 7.77, p < 0.01) and striatum (F( 1,36) = 8.92, p < 0.01). Also a significant interaction was found for the hypothalamus (F( 1,30) = 4.59, p < 0.05), but the post hoc failed to find a significant difference between WAG-6 and the other groups. Additionally, an age effect on the levels in the striatum (F( 1,36) = 4.77, p < 0.05), neurointermediate lobe of the pituitary (F( 1,36) = 5.57, p < 0.05) and frontal cortex (F( 1,34) = 4.40, p < 0.05) was observed. Furthermore, a strain 1 effect for MEAGL levels was found in the striatum (F(1,36) = 36.5, p < O.OOl), thalamus (F(1,34) = 6.92, p < 0.02), and mesencephalon (F( 1,36) = 4.66, p < 0.05) (Table 1). Table 1
Means and standard errors (pmol/g of wet tissue) of the Met-enkephalin-Arg6-Glu’-LeU8 (MEAGL) level in the hippocampus (HIP), mesencephalon (M), striatum (ST), thalamus (TH), frontal cortex (FCX), hypothalamus (HPT) and neurointermediate lobe (NIL) ofthe pituitary in epileptic (WAG-6) and non-epileptic control (ACI-3, ACI-6 and WAG-3) rats AU-3 HIP M ST
TH
FCX HPT NIL
39_+4 118klO 143 i 20 70+12 656 121_+9 524 + 38
ACI-6
47+3 113f5 1262 11 64 & 7 67+3 15Oi 16 454i51
WAG-3
WAG-6
38_+3 114*5 208+29 93*15 60_+6 152_+15 573 f47
39&2 143 f 4’“’ 318 i22’“” 107+15 82 _+7b 117f19” 436 _+37b
*) p< 0.05 (Duncan) WAG-6 group differs from all three control groups: a) p < 05 (ANOVA) strain x age interaction b) p < 0.05 (ANOVA) age effect c) p < 0.05 (ANOVA) strain effect.
The ANOVA for the a-neoendorphin showed that the peptide level in the WAG-6 rats was elevated in the hippocampus (F(1,34)= 18.57, p < 0.001) and
ENDOGENOUS
OPIOID PEPTIDES
IN BRAIN AND PITUITARY
OF RATS WITH ABSENCE
striatum (F( 1,36) = 6.04, p < 0.02). Additionally, the age effect in the hippocampus (F(1,34) = 14.85, p < 0.00 1) and neurointermediate lobe of the pituitary (F( 1,36) = 18.35 p < 0.001) as well as strain effect in the hippocampus(F(1,34) = 7.37,~ < O.Ol)andstriaturn (F( 1,36) = 10.44, p < 0.01) were found (Table 2). Table 2 Means and standard errors of the level of a-neoendorphin (pmol/g of wet tissue) in the hippocampus (HIP), mesencephalon (M), striatum (ST), thalamus (TH), hypothalamus (HPT), neurointermediate (NIL) and pars anterior (AL) lobe of the pituitary in epileptic (WAG-6) and non-epileptic control (ACI-3, ACI-6 and WAG-3) rats
HIP M ST TH HPT NIL AL
AU-3
AU-6
4014 19 i3 48+4 29 f 3 76 f 5 2067 +_90 IlOf 10
3926 25 i 5 41+2 29f4 85 + 5 2393 + 135 104k7
*) p ~0.05 (Duncan) control groups: a) p < 0.05 (ANOVA) b) p < 0.05 (ANOVA) c) p < 0.05 (ANOVA)
WAG-3
32 + 3 28 + 5 53 +_4 32 +4 77 +_4 2039 +_97 104 +4
WAG-6
67 30 80 35 74 2772 105
+ + + + + + +
3”bc 2 12’= 2 4 159b 6
WAG-6 group differs from all three strain x age interaction age effect strain effect.
The ANOVA of /3-endorphin levels showed a significant interaction in the neurointermediate lobe of the pituitary (F( 1,36) = 5.10, p < 0.05), there was however no significant difference between WAG-6 group and that three other groups (in fact the level of the WAG-3 rats was lower than that of the ACI3). Finally, there were significant strain and age effects in the anterior lobe of the pituitary (F(1,36)=6.99, ~~0.02 and F(1,36)= 19.52, p < 0.001, respectively) (Table 3). Table 3 Means and standard errors of the level of P-endorphin in the hypothalamus (HPT) (pmol/g of wet tissue), pars anterior (AL) (nmol/g of wet tissue) and neurointermediate (NIL) lobe of the pituitary (nmol/g wet tissue) in epileptic (WAG-6) and nonepileptic control (ACI-3, ACI-6 and WAG-3) rats A U-3 HP-I-
AL NIL
18k2 22 * 1 373 k 26
AU-6
15 i2 17+ 1 309+ 11
WAG-3
WAG-6
14k2 19fl 303 zk20
17 t4 15 + lk 336 t 26’
a) p
