Proc. Nati. Acad. Sci. USA
Vol. 75, No. 10, pp. 5221-5225, October 1978 Neurobiology
f-Endorphin induces nonconvulsive limbic seizures (electroencephalogram/opiates)
STEVEN J. HENRIKSEN*, FLOYD E. BLOOM*, FRANK MCCOY*, NICHOLAS LINGt, AND ROGER GUILLEMINt *Arthur Vining Davis Center for Behavioral Neurobiology and the tLaboratory of Neuroendocrinology, The Salk Institute for Biological Studies, La Jolla, California 92037
Contributed by Floyd E. Bloom, July 24,1978
ABSTRACT The endogenous opioid peptide, P-endorphin, induces nonconvulsive limbic epileptiform activity when administered intraventricularly to rats. Epileptiform activity is elicited by P-endorphin in doses that are devoid of analgesic or behavioral signs. Equimolar intraventricular doses of morphine or of the enkephalin analog [DAIa2,Met5Jenkephalin-NH2 fail to elicit this limbic epileptiform activity. These observations, together with the recent immunohistochemical localization of fl-endorphin to midline limbic structures, suggest that a-endorphin may have an important role in the regulation of limbic excitability. Two classes of opioid peptides have been recently identified in the mammalian pituitary gland and widespread areas of the brain (1-4). Both classes of peptides share common NH2-terminal amino acid sequences with the COOH-terminal portion of a previously identified pituitary hormone fl-lipotropin (5, 6). One class of these neuropeptides, the enkephalin pentapeptides, have amino acid sequences similar to f3-lipotropin(61-65) (1, 4). The second major class, the a-, f3-, and y-endorphins, are structurally identical to f3-lipotropin-(61-76), g-lipotropin-(61-91), and fl-lipotropin-(61-77), respectively (2, 3, 6). Recent evidence suggests that the enkephalins ([Met5]enkephalin and [Leu5]enkephalin) and f3-endorphin are the most potent of the endogenous opioid peptides, while aendorphin appears to be a peptide fragment which originates from the proteolytic breakdown of ,B-endorphin (7). Systemic administration of morphine or other opiate agonists induces a dose-related synchronization of the cortical electroencephalogram (EEG) ranging from intermittent high-voltage spindle bursts (8, 9) to epileptiform discharges and frank convulsions in experimental animals and human beings (10-12). Recently, epileptiform activity has been produced in rats following intracerebral (i.c.) or intraventricular (i.c.v.) administration of [Leu5]enkephalin and [Met5]enkephalin (12, 13). While attempting to use cortical and subcortical EEG changes as a quantifiable index to the actions of endogenous endorphins and synthetic congeners, we observed (14, 15) a predominance of limbic seizures at doses well below those required to produce overt behavioral changes. The present studies were undertaken to investigate the possible role f3-endorphin-mediated processes may have in opiate-induced EEG abnormalities, the possible brain loci underlying limbic epileptogenesis, and the pharmacologic specificity of the EEG changes induced by ,B-endorphin. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
METHODS Sprague-Dawley rats, 250-350 g (n = 85), maint~ained on a 12-hr light-dark cycle, were used in these investigations. Animals were anesthetized with chloral hydrate (400 mg/kg), placed in a Kopf stereotaxic apparatus, and implanted with EEG recording electrodes and intraventricular cannulae (Plastic Products, Inc.) as follows: Bipolar stainless steel or nichrome electrodes were constructed of individual wires measuring 62.5-250 ,um in diameter. Recording sites were dorsal hippocampus, medial septum, central amygdala, dorsal medial thalamus, and preoptic nucleus of the hypothalamus. Stainless steel screw electrodes were placed over the sensory motor and occipital cortex, and a bipolar transcortical electrode pair was positioned over the frontal cortex. Animals were allowed to recover from implantation surgery for 7-14 days before experimental manipulation. EEG recordings were made on Beckman model R511 or Grass model 7B polygraphs. Concomitant magnetic tape records (Honeywell model 7600 FM recorder) were made during selected experiments for off-line EEG frequency analysis. g-Endorphin and other peptides were prepared by solid phase synthesis and purified by methods previously described (16); they were dissolved in sterile Ringer's solution (pH 5.57.0). Sterile Ringer's solution alone was used as a control injection solution. Intracerebroventricular (i.c.v.) injection of freshly prepared solutions was delivered by hand to each animal through a chronic cannula system (Plastic Products) fitted to a 50-Al syringe. Solutions were injected at the rate of 10 gl/min, the total volume never exceeding 15 gl. Baseline EEG and magnetic tape records were made for periods of from 30 min to 6 hr to assess spontaneous EEG alterations. In most cases, control i.c.v. injections (Ringer's solution) were made prior to peptide injections. Peptide solutions were administered at least 30 min after control injections, and EEG and magnetic tape records were continued for at least 30 min. Before, during, and after all injections animals were observed, and spontaneous behavior as well as responses to nociceptive stimulation (tail pinch, corneal reflex) were noted. Rats were given only a single i.c.v. injection series (control and peptide) before they were killed. Histological verification of cannulae and electrode placement was made for each animal. The fQllowing peptides and pharmacological agents were studied: ,B-endorphin, (porcine and human; 0.3-15 nM) i.c.v. and intravenously; [DAla2,Met5]enkephalin-NH2 (3, 10, and 20 nM) i.c.v.; naloxone-HCl (2-10 mg/kg) intraperitoneally (i.p.) and i.c.v.; morphine sulfate (15-75 nM) i.p. and i.c.v.; amphetamine sulfate (2-4 mg/kg) i.p.; apomorphine-HCl (2 mg/kg) i.p.; haloperidol (4 mg/kg) i.p.; and diazepam (5 mg/kg) i.p. Abbreviations: EEG, electroencephalogram; i.c.v., intracerebroventricular; i.p., intraperitoneally. 5221
5222
Neurobiology: Henriksen et al.
Proc. Nati. Acad. Sci. USA 75 (1978)
RESULTS Characterization of electroencephalographic responses to jP-endorphin
f-Endorphin, [DAla2,Met5]enkephalin-NH2, and morphine sulfate all induced epileptiform activity and behavioral abnormalities in rats after i.c.v. administration. However, ,Bendorphin proved to be the most potent epileptogenic agent when compared to these other agents on a molar basis (Table 1). At very low doses (0.3-0.5 nM, i.c.v.) f3-endorphin provoked cortical spindle bursts elicited by a general slowing of EEG frequencies, and complex frequency shifts in subcortical structures. Spectral analysis of EEG frequencies indicated a shift of hippocampal 0 rhythm to higher frequencies and a dramatic increase in low frequency bursts (0-3 Hz) particularly evident in the cortical EEG. The cortical hypersynchronous spindle bursts elicited by administration of j3-endorphin occurred in awake and spontaneously active rats, thus indicating an electrographic dissociation between the EEG and behavior. Rats administered ,B-endorphin in dosages of 1 nM and above developed cortical and subcortical ictal and postictal epileptiform discharges with little or no disturbance in ongoing behavior. These early nonconvulsive seizural episodes developed in the absence of measurable analgesia, rigidity, or loss of righting reflex. However, the latter effects were common at dosages of ,-endorphin greater than 5 nmol. Intravenous doses of ,B-endorphin as high as 20 mg/kg failed to elicit epileptiform activity. At epileptogenic doses of ,B-endorphin, three major classes of electrogenic effects were observed which were dose, time, and site dependent: (i) One-5 min after lateral ventricular administration of 3-5
nmol of O-endorphin, paroxysmal waves (Fig. 1) characteristically emerged from a background of ongoing EEG activity. The initial ictal episode was characterized by a prolonged sequence of high voltage (300,uV-5 mV) paroxysmal waves, three to six per sec, typically of 30-40 sec duration. These sharp waves, seen in cortical as well as subcortical placements, were particularly prominent in the dorsal hippocampus and amygdala. In addition, the absence of the cortical component potentials when recording with local bipolar transcortical electrode arrays and the temporally delayed cortical wave relative
to the subcortical component suggested a subcortical generating locus for those epileptiform events.
(ii) The second phase of ,B-endorphin-induced effects occurred immediately after the initial ictal episode. Epileptiform potentials decreased in frequency in most subcortical recording sites resulting in a predominantly low voltage, highly desynchronized trace. In the hippocampus the EEG developed an isoelectric trace for up to 40 sec, suggesting a temporary local hyperpolarization or total depolarization (Fig. 1D). During this period, other subcortical sites, particularly the amygdala, characteristically discharged single epileptiform waves in the absence of dorsal hippocampal activity (Fig. 1D). Thirty-40 sec after this isoelectric period the hippocampal leads typically began to exhibit epileptiform activity starting with sequentially increasing low amplitude waves building up into large (400 ,uV-5 mV) potentials and/or multispike complexes which occurred at the rate of 30-60 per min (Fig. 1 A and E). This pattern of epileptiform discharge continued for several minutes and was characteristic of the postictal periods. During postictal episodes, cortical and subcortical leads demonstrated generally synchronous epileptiform activity with potentials of stable amplitude reflected in all leads. While cortical potentials tended to follow hippocampal discharge patterns temporally, other subcortical leads, particularly the amygdala, often discharged more complex multispike events in addition to the hippocampal/cortical potentials. Depending on dosage of f3-endorphin and/or the particular ventricular site of administration, multiple secondary ictal episodes occurred. These episodes were usually of shorter duration (10-15 sec) and less complex. As many as 15-20 ictal episodes could be observed in a period of 2 hr in rats after a 10 nM ventricular injection. f3-Endorphin injection into the fourth ventricle elicited identical epileptiform discharges but with a delay in onset (5-15 min). (iii) A third EEG component induced by f3-endorphin occurred between 15 and 30 min after i.c.v. injection when high-voltage synchronous slow waves developed with a concurrent decrease in higher frequency components in the EEG. This was particularly apparent in various cortical traces (Fig. 2). Following the increase in low-frequency high-voltage waves, ictal episodes decreased in frequency or disappeared, and only continuous postictal spiking remained. The duration of this
Table 1. Dose-effect EEG and behavioral profiles after administration of j3-endorphin and other agents Behavioral response Corneal EEG response Dose CTX-HS Ictal P-Ictal M-Ictal reflex WDS Substance Analgesia
g-Endorphin(3) [,B-Lipotropin- (61-91) ] (6) (65) (2) (1)
[DAla2,Met5]Enkephalin amide(2) Morphine sulfate(3)
15 nM
+
+
+
+
+++
-
+++
4-10 nM 3 nM 0.5 nM 20 mg/kg(i.p.)
+ +
+ + -
+ -
+++
+++
+ -
+ +++
-
+ + -
+++
3 nM
+
-
-
-
+
-
+
20 nM
+
+
+
-
++
-
++
26 nM 150 nM
+ +
-
-
-
+
Behavioral seizure
++++++
++
+++
Number of animals used for each test follows agent given. Administration of peptides and morphine was through ventricular cannulae. Naloxone was administered intraperitoneally. EEG responses: ictal, paroxysmal high frequency EEG epileptiform activity occurring 1-5 min after i.c.v. administration of fl-endorphin; M-ictal, reoccurring ictal episodes that follow an initial O-endorphin-induced ictal seizure; P-ictal, individual paroxysmal waves or multiwave complexes occurring intermittently after and/or between ictal episodes; CTX-HS, high voltage slow synchronous EEG activity occurring after administration of ,B-endorphin. Behavioral responses to 3-endorphin and other agents have been arbitrarily quantified by the following scales: (-) no response, (+) detectable response, (++) moderate response, (+++) maximal response. For analgesia, corneal reflex, and the response to pinching the tail strongly between thumb and forefinger were used. WDS (wet-dog-shakes) were measured by the
degree of whole body shaking.
Neurobiology:
Henriksen et al.
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Vol. 75, No. 10, pp. 5221-5225, October 1978 Neurobiology
f-Endorphin induces nonconvulsive limbic seizures (electroencephalogram/opiates)
STEVEN J. HENRIKSEN*, FLOYD E. BLOOM*, FRANK MCCOY*, NICHOLAS LINGt, AND ROGER GUILLEMINt *Arthur Vining Davis Center for Behavioral Neurobiology and the tLaboratory of Neuroendocrinology, The Salk Institute for Biological Studies, La Jolla, California 92037
Contributed by Floyd E. Bloom, July 24,1978
ABSTRACT The endogenous opioid peptide, P-endorphin, induces nonconvulsive limbic epileptiform activity when administered intraventricularly to rats. Epileptiform activity is elicited by P-endorphin in doses that are devoid of analgesic or behavioral signs. Equimolar intraventricular doses of morphine or of the enkephalin analog [DAIa2,Met5Jenkephalin-NH2 fail to elicit this limbic epileptiform activity. These observations, together with the recent immunohistochemical localization of fl-endorphin to midline limbic structures, suggest that a-endorphin may have an important role in the regulation of limbic excitability. Two classes of opioid peptides have been recently identified in the mammalian pituitary gland and widespread areas of the brain (1-4). Both classes of peptides share common NH2-terminal amino acid sequences with the COOH-terminal portion of a previously identified pituitary hormone fl-lipotropin (5, 6). One class of these neuropeptides, the enkephalin pentapeptides, have amino acid sequences similar to f3-lipotropin(61-65) (1, 4). The second major class, the a-, f3-, and y-endorphins, are structurally identical to f3-lipotropin-(61-76), g-lipotropin-(61-91), and fl-lipotropin-(61-77), respectively (2, 3, 6). Recent evidence suggests that the enkephalins ([Met5]enkephalin and [Leu5]enkephalin) and f3-endorphin are the most potent of the endogenous opioid peptides, while aendorphin appears to be a peptide fragment which originates from the proteolytic breakdown of ,B-endorphin (7). Systemic administration of morphine or other opiate agonists induces a dose-related synchronization of the cortical electroencephalogram (EEG) ranging from intermittent high-voltage spindle bursts (8, 9) to epileptiform discharges and frank convulsions in experimental animals and human beings (10-12). Recently, epileptiform activity has been produced in rats following intracerebral (i.c.) or intraventricular (i.c.v.) administration of [Leu5]enkephalin and [Met5]enkephalin (12, 13). While attempting to use cortical and subcortical EEG changes as a quantifiable index to the actions of endogenous endorphins and synthetic congeners, we observed (14, 15) a predominance of limbic seizures at doses well below those required to produce overt behavioral changes. The present studies were undertaken to investigate the possible role f3-endorphin-mediated processes may have in opiate-induced EEG abnormalities, the possible brain loci underlying limbic epileptogenesis, and the pharmacologic specificity of the EEG changes induced by ,B-endorphin. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
METHODS Sprague-Dawley rats, 250-350 g (n = 85), maint~ained on a 12-hr light-dark cycle, were used in these investigations. Animals were anesthetized with chloral hydrate (400 mg/kg), placed in a Kopf stereotaxic apparatus, and implanted with EEG recording electrodes and intraventricular cannulae (Plastic Products, Inc.) as follows: Bipolar stainless steel or nichrome electrodes were constructed of individual wires measuring 62.5-250 ,um in diameter. Recording sites were dorsal hippocampus, medial septum, central amygdala, dorsal medial thalamus, and preoptic nucleus of the hypothalamus. Stainless steel screw electrodes were placed over the sensory motor and occipital cortex, and a bipolar transcortical electrode pair was positioned over the frontal cortex. Animals were allowed to recover from implantation surgery for 7-14 days before experimental manipulation. EEG recordings were made on Beckman model R511 or Grass model 7B polygraphs. Concomitant magnetic tape records (Honeywell model 7600 FM recorder) were made during selected experiments for off-line EEG frequency analysis. g-Endorphin and other peptides were prepared by solid phase synthesis and purified by methods previously described (16); they were dissolved in sterile Ringer's solution (pH 5.57.0). Sterile Ringer's solution alone was used as a control injection solution. Intracerebroventricular (i.c.v.) injection of freshly prepared solutions was delivered by hand to each animal through a chronic cannula system (Plastic Products) fitted to a 50-Al syringe. Solutions were injected at the rate of 10 gl/min, the total volume never exceeding 15 gl. Baseline EEG and magnetic tape records were made for periods of from 30 min to 6 hr to assess spontaneous EEG alterations. In most cases, control i.c.v. injections (Ringer's solution) were made prior to peptide injections. Peptide solutions were administered at least 30 min after control injections, and EEG and magnetic tape records were continued for at least 30 min. Before, during, and after all injections animals were observed, and spontaneous behavior as well as responses to nociceptive stimulation (tail pinch, corneal reflex) were noted. Rats were given only a single i.c.v. injection series (control and peptide) before they were killed. Histological verification of cannulae and electrode placement was made for each animal. The fQllowing peptides and pharmacological agents were studied: ,B-endorphin, (porcine and human; 0.3-15 nM) i.c.v. and intravenously; [DAla2,Met5]enkephalin-NH2 (3, 10, and 20 nM) i.c.v.; naloxone-HCl (2-10 mg/kg) intraperitoneally (i.p.) and i.c.v.; morphine sulfate (15-75 nM) i.p. and i.c.v.; amphetamine sulfate (2-4 mg/kg) i.p.; apomorphine-HCl (2 mg/kg) i.p.; haloperidol (4 mg/kg) i.p.; and diazepam (5 mg/kg) i.p. Abbreviations: EEG, electroencephalogram; i.c.v., intracerebroventricular; i.p., intraperitoneally. 5221
5222
Neurobiology: Henriksen et al.
Proc. Nati. Acad. Sci. USA 75 (1978)
RESULTS Characterization of electroencephalographic responses to jP-endorphin
f-Endorphin, [DAla2,Met5]enkephalin-NH2, and morphine sulfate all induced epileptiform activity and behavioral abnormalities in rats after i.c.v. administration. However, ,Bendorphin proved to be the most potent epileptogenic agent when compared to these other agents on a molar basis (Table 1). At very low doses (0.3-0.5 nM, i.c.v.) f3-endorphin provoked cortical spindle bursts elicited by a general slowing of EEG frequencies, and complex frequency shifts in subcortical structures. Spectral analysis of EEG frequencies indicated a shift of hippocampal 0 rhythm to higher frequencies and a dramatic increase in low frequency bursts (0-3 Hz) particularly evident in the cortical EEG. The cortical hypersynchronous spindle bursts elicited by administration of j3-endorphin occurred in awake and spontaneously active rats, thus indicating an electrographic dissociation between the EEG and behavior. Rats administered ,B-endorphin in dosages of 1 nM and above developed cortical and subcortical ictal and postictal epileptiform discharges with little or no disturbance in ongoing behavior. These early nonconvulsive seizural episodes developed in the absence of measurable analgesia, rigidity, or loss of righting reflex. However, the latter effects were common at dosages of ,-endorphin greater than 5 nmol. Intravenous doses of ,B-endorphin as high as 20 mg/kg failed to elicit epileptiform activity. At epileptogenic doses of ,B-endorphin, three major classes of electrogenic effects were observed which were dose, time, and site dependent: (i) One-5 min after lateral ventricular administration of 3-5
nmol of O-endorphin, paroxysmal waves (Fig. 1) characteristically emerged from a background of ongoing EEG activity. The initial ictal episode was characterized by a prolonged sequence of high voltage (300,uV-5 mV) paroxysmal waves, three to six per sec, typically of 30-40 sec duration. These sharp waves, seen in cortical as well as subcortical placements, were particularly prominent in the dorsal hippocampus and amygdala. In addition, the absence of the cortical component potentials when recording with local bipolar transcortical electrode arrays and the temporally delayed cortical wave relative
to the subcortical component suggested a subcortical generating locus for those epileptiform events.
(ii) The second phase of ,B-endorphin-induced effects occurred immediately after the initial ictal episode. Epileptiform potentials decreased in frequency in most subcortical recording sites resulting in a predominantly low voltage, highly desynchronized trace. In the hippocampus the EEG developed an isoelectric trace for up to 40 sec, suggesting a temporary local hyperpolarization or total depolarization (Fig. 1D). During this period, other subcortical sites, particularly the amygdala, characteristically discharged single epileptiform waves in the absence of dorsal hippocampal activity (Fig. 1D). Thirty-40 sec after this isoelectric period the hippocampal leads typically began to exhibit epileptiform activity starting with sequentially increasing low amplitude waves building up into large (400 ,uV-5 mV) potentials and/or multispike complexes which occurred at the rate of 30-60 per min (Fig. 1 A and E). This pattern of epileptiform discharge continued for several minutes and was characteristic of the postictal periods. During postictal episodes, cortical and subcortical leads demonstrated generally synchronous epileptiform activity with potentials of stable amplitude reflected in all leads. While cortical potentials tended to follow hippocampal discharge patterns temporally, other subcortical leads, particularly the amygdala, often discharged more complex multispike events in addition to the hippocampal/cortical potentials. Depending on dosage of f3-endorphin and/or the particular ventricular site of administration, multiple secondary ictal episodes occurred. These episodes were usually of shorter duration (10-15 sec) and less complex. As many as 15-20 ictal episodes could be observed in a period of 2 hr in rats after a 10 nM ventricular injection. f3-Endorphin injection into the fourth ventricle elicited identical epileptiform discharges but with a delay in onset (5-15 min). (iii) A third EEG component induced by f3-endorphin occurred between 15 and 30 min after i.c.v. injection when high-voltage synchronous slow waves developed with a concurrent decrease in higher frequency components in the EEG. This was particularly apparent in various cortical traces (Fig. 2). Following the increase in low-frequency high-voltage waves, ictal episodes decreased in frequency or disappeared, and only continuous postictal spiking remained. The duration of this
Table 1. Dose-effect EEG and behavioral profiles after administration of j3-endorphin and other agents Behavioral response Corneal EEG response Dose CTX-HS Ictal P-Ictal M-Ictal reflex WDS Substance Analgesia
g-Endorphin(3) [,B-Lipotropin- (61-91) ] (6) (65) (2) (1)
[DAla2,Met5]Enkephalin amide(2) Morphine sulfate(3)
15 nM
+
+
+
+
+++
-
+++
4-10 nM 3 nM 0.5 nM 20 mg/kg(i.p.)
+ +
+ + -
+ -
+++
+++
+ -
+ +++
-
+ + -
+++
3 nM
+
-
-
-
+
-
+
20 nM
+
+
+
-
++
-
++
26 nM 150 nM
+ +
-
-
-
+
Behavioral seizure
++++++
++
+++
Number of animals used for each test follows agent given. Administration of peptides and morphine was through ventricular cannulae. Naloxone was administered intraperitoneally. EEG responses: ictal, paroxysmal high frequency EEG epileptiform activity occurring 1-5 min after i.c.v. administration of fl-endorphin; M-ictal, reoccurring ictal episodes that follow an initial O-endorphin-induced ictal seizure; P-ictal, individual paroxysmal waves or multiwave complexes occurring intermittently after and/or between ictal episodes; CTX-HS, high voltage slow synchronous EEG activity occurring after administration of ,B-endorphin. Behavioral responses to 3-endorphin and other agents have been arbitrarily quantified by the following scales: (-) no response, (+) detectable response, (++) moderate response, (+++) maximal response. For analgesia, corneal reflex, and the response to pinching the tail strongly between thumb and forefinger were used. WDS (wet-dog-shakes) were measured by the
degree of whole body shaking.
Neurobiology:
Henriksen et al.
ICTX
S ad E
Proc. Natl. Acad. Sci. USA 75 (1978)
FROZEN
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