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Keywords:

  • Nociceptin;
  • Opioid receptor like-1;
  • Kindling;
  • Kainate;
  • Knockout

Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Summary:  Purpose: To investigate the role of orphanin FQ/nociceptin (OFQ/N) in epilepsy, we analyzed (a) proOFQ/N (the OFQ/N precursor) and ORL-1 (the OFQ/N receptor) messenger RNA (mRNA) levels in the kainate and in the kindling models of epilepsy in the rat; and (b) seizure expression in proOFQ/N knockout mice.

Methods: Epilepsy models: kainate and kindling. Northern blot analysis, radioactive in situ hybridization.

Results: Increased proOFQ/N mRNA levels were found in the thalamus (reticular nucleus) after kainate administration. In contrast, ORL-1 gene expression decreased dramatically in the amygdala, hippocampus, thalamus, and cortex after kainate administration. OFQ/N knockout mice displayed reduced susceptibility to kainate-induced seizures, in that (a) lethality was reduced, (b) latency to generalized seizure onset was significantly prolonged, and (c) behavioral seizure scores were significantly reduced. Furthermore, kindling progression was delayed in OFQ/N–/– mice.

Conclusions: These data indicate that limbic seizures are associated with increased OFQ/N release in multiple brain areas, causing downregulation of ORL-1 receptors and activation of OFQ/N biosynthesis in selected areas, and support the notion that the OFQ/N-ORL-1 system may play a facilitatory role in ictogenesis and in epileptogenesis.

Orphanin FQ or nociceptin (OFQ/N) (1,2) is a neuropeptide that activates an orphan receptor of the opioid family, ORL-1 (3). OFQ/N has been implicated in a variety of biologic functions, including nociception, locomotor activity, stress, and anxiety (4). Moreover, there is evidence suggesting that OFQ/N may serve a role in the control of limbic seizures: (a) the gene encoding the precursor for OFQ/N (proOFQ/N) and the ORL-1 gene are constitutively expressed in some forebrain areas thought to be implicated in limbic seizures, such as the granule cells of the hippocampus (5,6); (b) kainate-induced seizures dramatically increase proOFQ/N messenger RNA (mRNA) levels in the thalamic reticular nucleus (7); and (c) OFQ/N modulates neuronal excitability and neurotransmitter release in the hippocampus (4,8).

In this study, we investigated whether seizure expression is altered in mice made OFQ/N deficient by targeted disruption of the gene coding for the OFQ/N precursor (9). Furthermore, we tested whether ORL-1 and proOFQ/N gene expressions are altered in the kainate and kindling models of limbic seizures in the rat.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Adult OFQ/N knockout mice (9) and male Sprague–Dawley rats (300–350 g) were used for experiments. Procedures involving animals were carried out in accordance with European Community and national laws and policies.

Kainate was administered i.p. (20 mg/kg in mice, 10 mg/kg in rats), and the behavior was observed for 2 h thereafter. Seizures were classified as follows (10): 1, chewing and drooling; 2, head nodding; 3, unilateral forelimb clonus; 4, bilateral forelimb clonus; 5, bilateral forelimb and/or hindlimb clonus with falling; 6, running or jumping seizure; 7, tonic hindlimb extension; and 8, death. For Northern blot analysis, rats were killed at different times (3–24 h) after kainate administration, and their hippocampi and thalami dissected and frozen. For in situ hybridization, rats were killed 6 h after kainate injection, and their brains frozen in isopentane. The methods used for Northern blot and in situ hybridization were described previously (7).

For kindling, a bipolar electrode was implanted in the right amygdala of mice and rats under ketamine anesthesia. After 7 days of postoperative recovery, afterdischarge (AD) threshold was determined by application of 1-s trains of bipolar pulses (1 ms, 60 Hz) at 1-min intervals, beginning at 40 μA and increasing by 20 μA until an electrographic seizure was observed. Beginning the following day, animals received a single 1-s train of bipolar pulses (1 ms, 60 Hz, 25% above the AD threshold) once daily. The evoked behavioral seizures were classified as described earlier. Kindling criteria were three consecutive class 4 or 5 seizures.

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Northern blot analysis revealed increased proOFQ/N mRNA levels in the thalamus (but not in the hippocampus) 3–24 h after kainate administration, with maximal effect (twofold increase over basal levels) reached at 6 h. No variation in proOFQ/N mRNA levels was observed 1–6 h after a stimulus-evoked kindled seizure. In contrast, ORL-1 gene expression was found to decrease dramatically (–50%) both in the thalamus and hippocampus beginning 6 h after kainate administration (data not shown). In situ hybridization analysis confirmed and extended these findings. Kainate was found to evoke a dramatic (fourfold) increase in proOFQ/N mRNA levels selectively in the thalamic reticular nucleus. A small, nonsignificant increase in proOFQ/N gene expression was found in the dentate gyrus of the hippocampus after kindled seizures. In contrast, ORL-1 mRNA levels decreased in a dramatic (–50%) and widespread (amygdala, hippocampus, thalamus, cortex) manner 6 h after kainate injection (data not shown).

To examine the involvement of the OFQ/N system in seizures and epileptogenesis, 10 homozygous OFQ/N–/– and 10 OFQ/N+/+ littermate mice were injected with 20 mg/kg i.p. kainate, and their behavior and EEG recorded for 2 h thereafter. As shown in Table 1, OFQ/N knockout mice displayed reduced susceptibility to kainate-induced seizures, in that (a) lethality was reduced, (b) latency to generalized seizure onset was significantly delayed, and (c) behavioral seizure score (two distinct scoring systems) was significantly reduced. Kindling development also was retarded in OFQ/N-deficient mice.

Table 1. 
 Seizure index11Cumulative seizure scoreDeath (% total)Latency (min ± s.e.)
OFQ/N +/+ (n = 10)4.6 ± 0.2133 ± 215016 ± 4
OFQ/N −/− (n = 10)3.4 ± 0.3**58 ± 14**1041 ± 5**

DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

ProOFQ/N and ORL-1 gene-expression data suggest that limbic seizures are associated with increased OFQ/N release in multiple brain areas, causing downregulation of ORL-1 receptors and activation of OFQ/N biosynthesis in selected areas (notably in the thalamic reticular nucleus). In vivo microdialysis experiments are ongoing to verify this hypothesis directly. The experiments on kainite and kindling in OFQ/N-deficient mice support the notion that the OFQ/N-ORL-1 system may play a facilitatory role in epileptogenesis and in seizure expression. To test this hypothesis, experiments are ongoing to evaluate kainate seizure expression and kindling development in normal animals after administration of selective ORL-1–receptor antagonists.

Acknowledgment: This study was supported by a grant from the Italian Ministry for the University and Scientific Research (MURST 60%, M.S.).

REFERENCES

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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