![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
NEUROPHARMACOLOGY
Department of Pediatrics, Division of Pediatric Neurology D. Geffen School of Medicine at University of California, Los Angeles, California (A.M., D.S., R.S.); and Department of Neurochemistry, Stockholm University, Stockholm, Sweden (L.L., U.S., Ü.L.).
Received March 22, 2006; accepted May 11, 2006.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
; Mazarati et al., 2004a; Lundström et al., 2005a). Second, chronic overexpression of galanin in the brain mitigated seizure activity (Mazarati et al., 2000; Kokaia et al., 2001; Haberman et al., 2003; Lin et al., 2003). Third, deletion of galanin or galanin receptors through either genetic mutations or antisense technique, resulted in a proconvulsant phenotype (Mazarati et al., 2000, 2004a,b; Jacoby et al., 2002).
Anticonvulsant effects of galanin were shown, by and large, under conditions of acute seizures. However, a desirable property in an antiepileptic drug is the potential ability to prevent epileptogenesis, that is the development of chronic epilepsy (Stables et al., 2003). One of the common forms of drug-resistant epilepsy is temporal lobe epilepsy, in which the primary epileptic focus is located in the hippocampus. Hence, to be effective in temporal lobe epilepsy, an antiepileptic drug should target hippocampal circuitry.
Of the three galanin receptor types known do date, type 1 (GalR1) and type 2 (GalR2) are expressed in the hippocampus (O'Donnell et al., 1999; Mennicken et al.,2004). Activation of both hippocampal GalR1 and GalR2 is anticonvulsant (Mazarati et al., 2004a,b); however, the two types of galanin receptors are coupled to distinct signaling cascades. Similar to other members of the G-protein-coupled receptor family, the signaling mediated by galanin receptors is multifaceted, with apparently only certain mechanisms relevant to their anticonvulsant effects. Thus, coupling of GalR1 to Gi protein opens G-protein-mediated inward rectifier K+ channels (GIRK) or ATP-sensitive K+ channels, which in turn results in presynaptic inhibition of glutamatergic transmission (Mazarati et al., 2000; Counts et al., 2002; Lundström et al., 2005a). The main pathway downstream from GalR2 is through coupling to Gq/11 protein and includes the increase of inositol triphosphate accumulation and the increase of intracellular Ca2+ (Wang et al., 1998, Lundström et al., 2005a); this would presumably stimulate neuronal activity and neurotransmitter release. Such an effect was indeed reported for GalR2-serotonin interaction in the dorsal raphe nucleus (Mazarati et al., 2005). In addition, GalR2 activates mitogen-activated protein kinase through coupling to Go protein (Wang et al., 1998). Furthermore, both GalR1 and GalR2 inhibit cyclic AMP-responsive element-binding protein (CREB), an effect possibly pertaining to the inhibition of long-term potentiation (Badie-Mahdavi et al., 2005).
Modification of epileptogenesis by galanin has been described in a single report, in which galanin-overexpressing mice exhibited a delay in the progression of kindled seizures (Kokaia et al., 2001). However, the study did not address the role of galanin receptor types and downstream signaling pathways that mediated the inhibition of the kindling process.
The present study had two goals. First, we examined whether GalR1 and GalR2 in the hippocampus exerted antiepileptogenic effects. A common approach for evaluating such an effect in antiepileptic drugs is to study their influence on the development of chronic epilepsy resulting from neuronal injury and synaptic reorganization caused by status epilepticus (Löscher, 2002; Morimoto et al., 2004). For our study, however, we used the kindling model of epileptogenesis (Löscher, 2002; Morimoto et al., 2004). Although being different from post-status epilepticus-induced epileptogenesis, kindling represents a useful tool for proving the principle. On the one hand, spontaneous seizures after status epilepticus have random temporal distribution and variable frequency, which requires large number of experimental subjects and prolonged periods of observation to obtain reliable results. On the other hand, kindling offers a controlled situation, in which seizures evolve through the "silent" period to the occurrence and progression of overt limbic seizures in response to repetitive subthreshold electrical stimulation of certain brain areas. More importantly, despite obvious differences in pathophysiological substrates, the two models showed significant overlap in terms of sensitivity to several antiepileptic drugs (Löscher, 2002).
Furthermore, we attempted to outline the mechanisms through which hippocampal GalR1 and GalR2 regulate kindling epileptogenesis. Based on the pathways that might mediate the anticonvulsant effects of galanin, we examined how inhibition of Gi/o protein, GIRK, Gq/11 protein, and intracellular Ca2+ mobilization affected kindling progression and the anticonvulsant effects of GalR1 and GalR2 agonists.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Surgery. Animals were anesthetized with isoflurane and placed in the stereotaxic instrument (model 902; David Kopf Instruments, Tujunga, CA). A bipolar stimulating electrode (PlasticsOne, Roanoke, VA) was implanted into the left ventral hippocampus (4.8 mm posterior and 5.3 mm lateral from bregma and 6.5 mm ventral from the brain surface) (Paxinos and Watson, 1986). A tripolar recording skull electrode (PlasticsOne) was placed on the left side 2 mm anterior from bregma with the ground connected to the screw in the nasal bone. A 5-µl microsyringe model 7105KH (Hamilton, Reno, NV) was placed into the injector of the infusion pump model sp310i (World Precision Instruments, Sarasota, FL), which had been mounted on the arm of the stereotaxic instrument. A hole was drilled on the left, 4.16 mm posterior and 2.5 mm lateral from bregma. The needle of the syringe was lowered 2.5 mm ventral from the brain surface into the CA1 of the hippocampus (Paxinos and Watson, 1986). Five microliters of solution (see below) were infused at a rate of 5 µl/min.
A subcutaneous pocket was made between the rat's shoulders. An ALZET osmotic pump model 2001D (infusion rate 8.3 µl/h, total infusion duration 24 h, and infusion volume 0.2 ml; DURECT Corporation, Cupertino, CA), which had been prefilled with the solution (see below) connected to the infusion cannula of Brain Infusion Kit II (DURECT Corporation) and primed for 2 h in saline at 37°C, was placed into the subcutaneous pocket. The infusion cannula was lowered into the same place in the hippocampus where the solution had been injected through the microsyringe. The electrodes and the cannula were cemented to the skull using Cerebond adhesive (MyNeuroLab.com, St. Louis, MO).
Drug Injections. One of the following substances was injected through the Hamilton microsyringe: a Gi/o protein inhibitor, pertussis toxin (PTX, 0.5 µg in 5 µl, List Biological Laboratories Inc., Campbell, CA) (Bokoch et al., 1983); a blocker of GIRK 1-4 subunits, tertiapin Q (TPQ) (10 nM, 5 µl; Tocris Cookson, Bristol, UK) (Jin and Lu, 1998); a Gq/11 protein inhibitor. ([D-Arg1,D-Trp5,7,9,Leu11]-substance P) (dSP) (10 µM, 5 µl; Bachem California, Torrance, CA) (Mitchell et al., 1995; Sinnett-Smith et al., 2000); an inhibitor of Ca2+ release from endoplasmic reticulum, dantrolene sodium salt (10 µM; Tocris Cookson) (Lauckner et al., 2005); a GalR1/GalR2 antagonist, chimeric peptide M35 [galanin(1-13)-bradykinin(2-9) amide] (10 µM, 5 µl) (Kask et al., 1995). The following compounds were infused through the ALZET osmotic pump: a nonselective GalR1/GalR2 agonist rat galanin(1-29) (5 µM; Bachem California); a preferential GalR1 agonist chimeric peptide M617 (galanin(1-13)-Gln14-bradykinin(2-9)-amide) (5 µM) (synthesized by Lundström et al., 2005b); a preferential GalR2 agonist galanin(2-11) (5 µM; Sigma) (Liu et al., 2001; Lundström et al., 2005a). The peptides were administered alone or in combination with one of the following agents: PTX (0.5 µg); TPQ (5 µM); dSP (5 µM); dantrolene (10 µM); or M35 (10 µM). Selected concentrations for each compound had been optimized in pilot experiments. All substances were dissolved in saline, except dantrolene, which was dissolved in polyethylene glycol 300 (Sigma). Control treatments consisted of the administration of respective vehicles both through the Hamilton microsyringe and from the ALZET osmotic pumps.
Kindling Procedure. We used the rapid kindling protocol, originally described by Lothman et al. (1985). In contrast with conventional kindling, in which electrical stimuli are delivered hours apart and which requires weeks for overt limbic seizures to develop, in rapid kindling the epileptogenesis is compressed to several hours, while still bearing key hallmarks of kindling: appearance and gradual progression of the severity of limbic seizures, and enhanced seizure susceptibility after kindling is complete. Sixteen to 17 h after the implantation of ALZET osmotic pumps (6-7 h before the completion of the infusion), the animals were connected to the DS8000 electrical stimulator via DSI100 stimulus isolators (World Precision Instruments) and to the MP100/EEG100B acquisition system (BIOPAC, Santa Barbara, CA). An electroencephalogram was acquired, AcqKnowledge 3.7 software (BIOPAC) was used. Simultaneously, animals' behavior was recorded by digital video camera. Both an electroencephalogram and behavioral responses were analyzed off-line.
At the beginning of the experiment, afterdischarge threshold and duration were detected by applying trains of electrical stimuli—10-s train duration, 20 Hz, 1-ms pulse duration, square-wave monophasic stimuli, starting with 0.1 mA increments, delivered every 10 min. Ten minutes after the detection of afterdischarge threshold, evident as a high-frequency response of least 2 s duration following the end of the train, animals underwent a rapid kindling procedure. Kindling consisted of 60 trains delivered every 5 min using the parameters described above and the current of 50 µA above the afterdischarge threshold (total procedure duration was 5 h). Behavioral seizures were scored using the following scale: 1, motor arrest and whisker twitching; 2, chewing, head bobbing; 3, forelimb clonus; 4, forelimb clonus and rearing; and 5, rearing and falling. If the animals failed to develop seizures of certain score, 60 (number of stimulations) was assigned. If the animal skipped a certain phase in seizure progression, the number of stimulations required to reach the subsequent phase was also assigned to the skipped phase (e.g., if the animal transitioned from stage 1 to stage 3 seizures without exhibiting stage 2 convulsions, the number of stimulations needed to reach stage 3 was also assigned to stage 2). The number of stimulations required to reach each consecutive seizure score (1 through 5) and the number of full motor seizures (stages 4 and 5) were calculated.
Twenty-four hours after the end of kindling procedure, animals were reconnected to the stimulating/recording system and afterdischarge threshold and afterdischarge duration were detected again. The experimental protocol is summarized in Fig. 1A.
|
Statistical Analysis. Data were analyzed using Prizm 4 software (GraphPad Software Inc., San Diego, CA), and we used a one-way ANOVA followed by the post hoc Dunn's test. P < 0.05 was accepted for statistical significance. Each group included six animals, unless indicated otherwise. As our stimulation/recording system allowed simultaneous processing of four subjects, each set of animals generally included one of three different treatments and one control.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). In the coronal plane the signal spanned across 2 to 3 mm, that is, was present in almost the entire CA1, although the strength of the signal substantially decreased in the portions of CA1 more distant from the injection site. In sections adjacent to the site of injection, an uptake of fluorescein-galanin(1-29) was also observed in the middle part of the upper blade of the dentate gyros (Fig. 1B).
Kindling in Control Animals. In naive rats (n = 10), afterdischarge threshold was 1.1 ± 0.1 mA, and afterdischarge duration was 30.5 ± 2.4 s (Fig. 2A). The kindling procedure led to the occurrence and the progression of behavioral convulsions; it took 5.5 ± 0.5 stimulations to reach stage 1 seizures, 10.7 ± 0.6 stimulations to reach stage 2 seizures, and 15.2 ± 0.7 stimuli to develop the first stage 3 seizure. The first full motor seizure (stage 4) occurred after 23.6 ± 0.5 stimuli, and the first stage 5 seizure was observed after 25.8 ± 0.4 stimulations (Fig. 2B). After the first full motor seizure, animals responded with either stage 4 or 5 convulsions to 17.4 ± 1.8 of stimulations. Twenty-four hours after the last kindling train, all animals showed a decrease of afterdischarge threshold to 0.43 ± 0.1 mA and the increase of afterdischarge duration to 52 ± 2.9 s (p < 0.05 versus the values before kindling) (Fig. 2C). All animals developed behavioral convulsions in response to the threshold stimulation, although none of the rats developed full motor seizures (average seizure score was 2.9 ± 0.2) (Fig. 2D). Rats injected with fluorescein-galanin(1-29) followed the pattern of kindling progression observed in animals treated with rat galanin(1-29), but the data were not included in the statistical analysis.
|
Effects of Galanin Receptor Agonists. Animals treated with galanin(1-29) and M617 exhibited a 60 to 75% increase in afterdischarge threshold, compared with controls (p < 0.05) (Fig. 2A). Galanin(2-11), in contrast, produced a statistically significant decrease in afterdischarge threshold (0.5 ± 0.1 mA), and afterdischarge duration was significantly longer than in the control group (46.0 ± 6.9 s) (Fig. 2A).
During kindling, galanin(1-29) significantly delayed the occurrence of behavioral seizures of all stages compared with controls (Fig. 2B). Furthermore, two of six animals, failed to exhibit stage 5 seizures. Treatment with M617 also produced a significant delay in the development of behavioral seizures; however, the peptide did not block the occurrence of stage 5 convulsions. Galanin(2-11)-treated rats were not different from controls in the development of stage 1 and 2 convulsions, but it took more stimulations, than in controls to develop stage 3 to 5 seizures; in addition, four of six animals failed to exhibit stage 5 convulsions, and the rest developed stage 5 seizure only in response to the last stimulation (Fig. 2B). In the animals that were treated with a combination of M617 and galanin(2-11), kindling followed the pattern similar to that in galanin(1-29)-treated rats (Fig. 2B). The number of full motor seizures was significantly lower in rats that received the injections of galanin(1-29) (0.7 ± 0.2, p < 0.05 versus control), galanin(2-11) (0.6 ± 0.3, p < 0.05 versus control), or a combination of M617 and galanin(2-11) (0.9 ± 0.4, p < 0.05 versus control) with no differences across the three groups. M617-treated animals developed 7.7 ± 1.2 full motor seizures [p < 0.05 versus control, galanin(1-29) and galanin(2-11)]. Testing afterdischarge properties 24 h after kindling revealed that the animals treated with galanin(1-29), with galanin(2-11), or with a M617-galanin(2-11) combination had significantly higher afterdischarge threshold and shorter afterdischarge duration than control animals and those who received M617 (Fig. 2C). Furthermore, the animals of the first three groups either failed to develop behavioral seizures in response to the threshold stimulation or only exhibited stage 1 convulsions [three animals in galanin(1-29)- and two animals in M617 + galanin(2-11)-treated groups].
Effects of M35. Intrahippocampal administration of M35 (n = 5) decreased the afterdischarge threshold (0.56 ± 0.05 mA, p < 0.05 versus control) but did not modify afterdischarge duration. Progression of kindled seizures was not different between M35-treated and control groups (data not shown, n = 5 per group). M35 prevented the increase of afterdischarge threshold induced by M617 but did not affect changes in afterdischarge properties observed after administration of galanin(2-11) (Fig. 3). Coadministration of M35 abolished inhibition of kindling observed after treatment by each of the peptides alone (data not shown).
|
Effects of PTX. Intrahippocampal infusion of PTX alone significantly increased afterdischarge threshold (Fig. 4A). Animals treated with the combination of PTX and galanin(1-29) or with PTX and galanin(2-11) exhibited a significantly longer afterdischarge, compared with both controls and with PTX alone-injected rats. Furthermore, a combination of either of the two peptides with PTX led to a significant reduction of afterdischarge threshold compared with PTX alone. There were no differences between PTX- and PTX + M617-treated animals (Fig. 4A).
|
Twenty-four hours after kindling, PTX and PTX + M617-treated animals still showed lower excitability than controls (Fig. 4C). The animals failed to respond or responded with minimal behavioral seizures to the afterdischarge stimulation. At the same time, kindled animals that had been treated with PTX + galanin(1-29) and PTX + galanin(2-11) were not different from control group in terms of afterdischarge properties. However, afterdischarge threshold was significantly lower (0.9 ± 0.1 and 0.6 ± 0.1 mA, respectively) than in PTX-treated rats (3.2 ± 0.3 mA) (Fig. 4D). Furthermore, in PTX + galanin(1-29)- and PTX + galanin(2-11)-treated animals, the threshold current elicited behavioral seizures (2.4 ± 0.2 and 3.6 ± 0.2, respectively) with severity similar to those is controls and more severe than those in PTX-treated animals (Fig. 4C).
Effects of TPQ. Intrahippocampal infusion of TPQ led to the decrease of afterdischarge threshold (0.15 ± 0.015 mA). Animals treated with a combination of TPQ with galanin(1-29), M617, or galanin(2-11) showed similar decreases in afterdischarge properties (Fig. 5A).
|
Animals that received the TPQ + galanin(1-29) or TPQ + galanin(2-11) combination exhibited kindling rate similar to control group; kindling progression through stages 2 to 5 was significantly slower compared with TPQ-only treated rats (Fig. 5B). The number of stage 4 to 5 seizures in these rats was not different from controls but was significantly lower than in TPQ-treated animals (14.5 ± 1.4 and 18.5 ± 1.1, respectively). Afterdischarge properties and seizure response to the test stimulation 24 h after kindling were similar among all TPQ-treated groups (not shown).
Effects of dSP. Intrahippocampal delivery of dSP altered neither afterdischarge properties nor the rate of kindling epileptogenesis, nor did it affect anticonvulsant profile of M617 (Fig. 6, A and B). However, dSP abolished the galanin(2-11)-induced increase of hippocampal excitability before kindling (Fig. 6A). At the same time, dSP did not affect galanin(2-11)-induced delay of kindling rate and the decrease of the incidence of full motor seizures (0.7 ± 0.3, p < 0.05 versus galanin(2-11 alone). However, adding dSP to the PTX + galanin(2-11) combination inversed the effect of such a treatment from kindling, facilitating (Fig. 4B) to anticonvulsant (Fig. 6B), and abolished the increase of the number of full motor seizures (16.2 ± 0.9 p < 0.05 versus PTX + galanin(2-11). Combined administration of PTX and dSP without galanin receptor ligands affected all examined parameters in the same way as PTX treatment alone (p > 0.05, data not shown).
|
Effects of Dantrolene. Infusion of dantrolene into the hippocampus affected neither afterdischarge threshold nor afterdischarge duration in nonkindled animals (Fig. 6C). Dantrolene did not modify the increase of afterdischarge threshold induced by M617; however, it abolished the increase of hippocampal excitability due to galanin(2-11) (Fig. 6C). Animals that were treated with intrahippocampal dantrolene failed to develop kindling. The maximal severity of seizures was stage 1; such seizures developed in all rats, but only in response to 10 to 19 of 60 stimulations. Animals injected with dantrolene + M617, dantrolene + galanin(2-11), PTX + dantrolene, or PTX + dantrolene + galanin(2-11) showed patterns of kindling progression similar to those of dantrolene only-treated rats (data not shown).
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Effects of PTX, TPQ, dSP, and Dantrolene. Intrahippocampal administration of PTX decreased hippocampal excitability and interfered with the progression of kindling. PTX uncouples Gi/o proteins by catalyzing ADP-ribosylation of 
subunits (Bokoch et al., 1983). ADP-ribosylation has been implicated in kindling epileptogenesis (Suzuki et al., 1997). Furthermore, inhibitory and anticonvulsant effects of galanin, somatostatin, neuropeptide Y, GABA acting at GABAB receptors, glutamate acting at groups II and III metabotropic glutamate receptors, serotonin acting at 5-hydroxytryptamine1A receptors, and adenosine acting at A1 receptors are coupled to Gi-protein (Counts et al., 2002; Wickenden, 2002; Moldrich et al., 2003). Hence, PTX should facilitate, rather than inhibit, seizures. Paradoxically, PTX inhibited kindled seizures not only in our experiments but in earlier studies as well (Watanabe et al., 1991). Inhibition of long-term potentiation (Goh and Pennefather, 1989) in the hippocampus further implicates PTX in inhibiting synaptic activity. Conceivably, signal transduction downstream of PTX affects seizures in different ways, depending on mechanisms predominantly involved in seizure regulation in particular experimental paradigms. PTX inhibition of kindling epileptogenesis is galanin receptor-independent and may recruit other mechanisms relevant to the evolvement of kindled seizures (e.g., cholinergic transmission) (Burchfiel et al., 1979).
Keeping in mind the ubiquity of PTX-mediated intracellular signaling, we narrowed it down to a candidate that might mediate anticonvulsant effects of galanin—GIRK. All of THE neuropeptides and neurotransmitters mentioned above exert their effects through GIRK (Wickenden, 2002). Activation of GIRK inhibits glutamatergic transmission both pre- and postsynaptically through membrane hyperpolarization (Wickenden, 2002). Indeed, injection of TPQ in our experiments yielded a more predictable outcome, as it facilitated kindling epileptogenesis.
Inhibition of Gq/11 by dSP did not affect kindling progression. Gq/11 is coupled to inositol triphosphate production and intracellular Ca2+ release, which should enhance neuronal excitability. Receptors of proconvulsant agents, such as substance P (Liu et al., 1999) and metabotropic glutamate receptors 1 and 5 (Moldrich et al., 2003) are coupled to Gq/11. The absence of the effects of dSP itself is congruent with the previously shown "silent" behavior of this compound: dSP inhibited bombesin-mediated activation of phospholipase C but itself did not affect the enzyme activity (Mitchell et al., 1995). However, direct inhibition of Ca2+ release from endoplasmic reticulum by dantrolene did exert a rather predictable anticonvulsant effect.
Thus, the diversity of signal transduction pathways regulated by G proteins complicates a clear-cut prediction of the net effects of their inhibitors on seizures. However, reducing G-protein coupled signaling cascades to likely candidates of seizure modulation produces more predictable effects. It is noteworthy that such an approach allowed us to correlate regulation of seizures by GalR1 and GalR2 to certain downstream mechanisms.
Role of GalR1. Presumably, activation of GalR1 decreased ambient excitability of the hippocampus and delayed, although did not prevent, kindling epileptogenesis (Table 1). The experiments with PTX and TPQ suggested the involvement of Gi protein and downstream GIRK in the anticonvulsant action of M617 and galanin(1-29), whereas studies with dSP and dantrolene excluded Gq/11 and intracellular Ca2+ as targets for GalR1; such conclusions are in line with previously delineated properties of GalR1 (Wang et al., 1998; Counts et al., 2002; Lundström et al., 2005a). Hence, hippocampal GalR1 exerts disease-modifying but not antiepileptogenic effects in kindling epileptogenesis.
|
Role of GalR2. Galanin(2-11) exhibited strong antikindling effects (Table 1). In fact, the action of galanin(2-11) could be identified as antiepileptogenic, as judged by the complete prevention of both full motor seizures and of a postkindling increase of hippocampal excitability. It should be noted that although galanin(2-11) has equal affinity toward GalR2 and galanin receptor type 3 (Lu et al., 2005), the latter is absent from the hippocampus (Mennicken et al., 2004).
Inhibition of kindling by galanin(2-11) depended on Gi/o, as it was PTX-sensitive, and indeed coupling of GalR2 to Gi/o has been well established (Wang et al., 1998; Lundström et al., 2005a). However, the failure of TPQ to eliminate the anticonvulsant action of galanin(2-11) and galanin(2-19) argued against the involvement of GIRK. Thus, the mechanisms by which galanin(2-11) inhibited kindling require further studies. CREB is one possible candidate: CREB activity was enhanced by kindling in a temporally specific manner (Kashihara et al., 2000), whereas galanin(2-11) inhibited CREB activity (Badie-Mahdavi et al., 2005). At this point it is reasonable to conclude that the anticonvulsant effects of GalR2 are Gi/o protein-coupled but are GIRK-independent.
The result that seemed paradoxical was that treatment with PTX inversed the effects of both galanin(2-11) and galanin(1-29) from anti- to proconvulsant. Furthermore, the effect of the PTX + galanin(2-11) combination was sensitive to both dSP and dantrolene. We speculate that along with a Gi-dependent pathway, which mediates inhibition of kindling, GalR2 in the hippocampus activate Gq/11 and downstream intracellular Ca2+, which ultimately increases neuronal activity and promotes epileptogenesis. When both Gi and Gq/11 are intact, the first pathway dominates, whereas the secondary, excitatory component can be unmasked through Gi inhibition. Such a suggestion was confirmed in the experiments with galanin(1-29), an endogenous neuropeptide that equally acts at GalR1 and GalR2 (GalR1 and GalR2 are equally expressed in the rat hippocampus) (O'Donnell et al., 1999; Mennicken et al., 2004). In the intact hippocampus the effects of galanin(1-29) were congruent with the effects of both GalR1 (decreased ambient excitability) and GalR2 (antiepileptogenic action). Blocking of Gi-dependent cascades changed the effects of galanin(1-29) in such a way that the peptide acted similar to galanin(2-11). Coupling of GalR2 to Gq/11 has been reported (Wang et al., 1998; Lundström et al., 2005a). Furthermore, the activation of Gq/11 inactivates GIRK (Lei et al., 2003), which could further promote seizures. However, the fact that in our experiments both dSP treatment and galanin(2-11) administration did not facilitate kindled seizures proves that the role of Gq/11 in promoting epileptogenesis is secondary to the antiepileptic effects of Gi-coupled pathways.
Blocking of galanin receptors by M35 decreased the afterdischarge threshold and negated the inhibitory effects of M617. Both M617 and M35 are chimeric peptides that contain the C terminus of bradykinin (Kask et al., 1995; Lundström et al., 2005a). The antagonism between the two peptides argues against the involvement of bradykinin receptors in the anticonvulsant effects of M617. Despite the increase of hippocampal excitability by M35, the M35 + galanin(2-11) combination did not show an additive effect, thus suggesting that facilitation of afterdischarge by M35 and galanin(2-11) occurs through different mechanisms. Abolishing the inhibitory effects of M617 and galanin(2-11) on kindling progression by M35 proves the galanin receptor-specific effects of both galanin receptor agonists. M35 itself did not affect the evolution of rapid kindling. Our previous studies indicated that M35 acted as a proconvulsant in pentylenetetrazole seizures and facilitated self-sustaining status epilepticus (Mazarati et al., 1998). The discrepancies mentioned probably reflect differences in the pathophysiology of seizures, depending on experimental model.
Our previous studies suggested that the two galanin receptor subtypes regulated the progression of limbic status epilepticus differently: whereas GalR1 inhibited status epilepticus during the initiation phase, the major anticonvulsant effect of GalR2 was observed during seizure maintenance (Mazarati and Lu, 2005). The present data extend the principle of receptor subtype specificity to the kindling model, when the antiepileptogenic and disease-modifying effects of galanin occur predominantly through GalR2 and GalR1, respectively.
Clearly, our studies did not provide unambiguous conclusions on the mechanisms of seizure modulation by hippocampal galanin receptors. Three major problems can be identified. First, the mechanisms of in vivo epileptogenesis are complex and diverse. Second, currently available pharmacological tools for studying galanin receptors are still not perfect. M617 and galanin(2-11) are the only available agonists of GalR1 and GalR2, respectively, but they do not exhibit absolute preference over galanin receptor subtypes (Lundström et al., 2005a). Third, the signal transduction pathways affected by in vivo administration of G protein inhibitors, which might translate into either anti- or proepileptogenic effects, are diverse. Despite these and possibly other limitations, the results reported here hopefully expand our understanding of how galanin suppresses seizures, bring up the complexity of the effects of the peptides in the hippocampus, and might ultimately be useful for the development of new antiepileptic drugs.
In conclusion, GalR1 and GalR2 in the hippocampus modified kindling epileptogenesis through different downstream signaling cascades, in either a facilitatory or an inhibitory fashion. The net effect of GalR1 and GalR2 activation appears to be antiepileptic; however, hippocampal GalR2 might appear proconvulsant under certain conditions. On this note, our previous studies showed that activation of GalR1 in dorsal raphe nucleus facilitated, rather than inhibited, seizures (Mazarati et al., 2005). The established variety of the effects of galanin receptor subtypes, particularly, the different patterns of anticonvulsant activity (e.g., disease modification versus inhibition of epileptogenesis in the kindling model or regulation of the initiation versus the maintenance of status epilepticus), and, furthermore, the seizure-facilitating effects that occur under certain conditions, should be kept in mind in the development of antiepileptic drugs acting at galanin receptors (Bartfai et al., 2004).
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: GalR1, type 1 galanin receptor(s); GalR2, type 2 galanin receptor(s); GIRK, G-protein-coupled inwardly rectifying K+ channel(s); CREB, cyclic AMP-responsive element-binding protein; PTX, pertussis toxin; TPQ, tertiapin Q; dSP, [D-Arg1,D-Trp5,7,9,Leu11]-substance P; M35, [galanin(1-13)-bradykinin(2-9) amide]; M617, galanin(1-13)-Gln14-bradykinin(2-9)-amide; ANOVA, analysis of variance.
Address correspondence to: Dr. Andréy Mazarati, Department of Pediatrics, Neurology Division, D. Geffen School of Medicine at UCLA, Box 951752, 22-474 MDCC, Los Angeles, CA 90095-1752. E-mail: mazarati{at}ucla.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Badie-Mahdavi H, Lu X, Behrens MM, and Bartfai T (2005) Role of galanin receptor 1 and galanin receptor 2 activation in synaptic plasticity associated with 3',5'-cyclic AMP response element-binding protein phosphorylation in the dentate gyrus: studies with a galanin receptor 2 agonist and galanin receptor 1 knockout mice. Neuroscience 133: 591-604.[CrossRef][Medline]
Bartfai T, Lu X, Badie-Mahdavi H, Barr AM, Mazarati A, Hua X, Yaksh T, Haberhauer G, Trembleau L, Conde Ceide S, et al. (2004) Galmic, a non peptide galanin receptor agonist, affects behaviors in seizure, pain and forced swim tests. Proc Natl Acad Sci USA 101: 10470-10475.
Bokoch GM, Katada T, Northup JK, Hewlett EL, and Gilman AG (1983) Identification of the predominant substrate for ADP-ribosylation by islet activating protein. J Biol Chem 258: 2072-2075.
Burchfiel JL, Duchowny MS, and Duffy FH (1979) Neuronal supersensitivity to acetylcholine induced by kindling in the rat hippocampus. Science (Wash DC) 204: 1096-1098.
Counts SE, McGuire SO, Sortwell CE, Crawley JN, Collier TJ, and Mufson E (2002) Galanin inhibits tyrosine hydroxylase expression in midbrain dopaminergic neurons. J Neurochem 83: 442-451.[CrossRef][Medline]
Goh JW and Pennefather PS (1989) A pertussis toxin-sensitive G protein in hippocampal long-term potentiation. Science (Wash DC) 244: 980-983.
Haberman RP, Samulski RJ, and McCown TJ (2003) Attenuation of seizures and neuronal death by adeno-associated virus vector galanin expression and secretion. Nat Med 9: 1076-1080.[CrossRef][Medline]
Jacoby AS, Hort YJ, Constantinescu G, Shine J, and Iismaa TP (2002) Critical role for GALR1 galanin receptor in galanin regulation of neuroendocrine function and seizure activity. Mol Brain Res 107: 195-200.[Medline]
Jin W and Lu Z (1998) A novel high-affinity inhibitor for inward-rectifier K+ channels. Biochemistry 37: 13291-13299.[CrossRef][Medline]
Kashihara K, Sato K, Akiyama K, Ishihara T, Hayabara T, and Abe K (2000) Temporal profile of CRE DNA-binding activity in the rat hippocampus following a kindling stimulation. Epilepsy Res 40: 171-177.[CrossRef][Medline]
Kask K, Berthold M, Bourne J, Andell S, Langel U, and Bartfai T (1995) Binding and agonist/antagonist actions of M35, galanin(1-13)-bradykinin(2-9)amide chimeric peptide, in Rin m 5F insulinoma cells. Regul Pept 59: 341-348.[CrossRef][Medline]
Kokaia M, Holmberg K, Nanobashvili A, Xu ZQ, Kokaia Z, Lendahl U, Hilke S, Theodorsson E, Kahli U, Bartfai T, et al. (2001) Suppressed kindling epileptogenesis in mice with ectopic overexpression of galanin. Proc Natl Acad Sci USA 98: 14006-14011.
Lauckner JE, Hille B, and Mackie K (2005) The cannabinoid agonist WIN55,212-2 increases intracellular calcium via CB1 receptor coupling to Gq/11 G proteins. Proc Natl Acad Sci USA 102: 19144-19149.
Lei Q, Jones MB, Talley EM, Garrison JC, and Bayliss DA (2003) Molecular mechanisms mediating inhibition of G protein-coupled inwardly-rectifying K+ channels. Mol Cell 15: 1-9.[CrossRef]
Lin EJ, Richichi C, Young D, Baer K, Vezzani A, and During MJ (2003) Recombinant AAV-mediated expression of galanin in rat hippocampus suppresses seizure development. Eur J Neurosci 18: 2087-2092.[CrossRef][Medline]
Liu H, Mazarati AM, Katsumori H, Sankar R, and Wasterlain CG (1999) Substance P is expressed in hippocampal principal neurons during status epilepticus and plays a critical role in the maintenance of status epilepticus. Proc Natl Acad Sci USA 96: 5286-5291.
Liu HX, Brumovsky P, Schmidt R, Brown W, Payza K, Hodzic L, Pou C, Godbout C, and Hökfelt T (2001) Receptor subtype-specific pronociceptive and analgesic actions of galanin in the spinal cord: selective actions via GalR1 and GalR2 receptors. Proc Natl Acad Sci USA 98: 9960-9964.
Löscher W (2002) Animal models of epilepsy for the development of antiepileptogenic and disease-modifying drugs: a comparison of the pharmacology of kindling and post-status epilepticus models of temporal lobe epilepsy. Epilepsy Res 50: 105-123.[CrossRef][Medline]
Lothman EW, Hatlelid JM, Zorumski CF, Conry JA, Moon PF, and Perlin JB (1985) Kindling with rapidly recurring hippocampal seizures. Brain Res 360: 83-91.[CrossRef][Medline]
Lu X, Lundstrom L, and Bartfai T (2005) Galanin(2-11) binds to GalR3 in transfected cell lines: limitations for pharmacological definition of receptor subtypes. Neuropeptides 39: 165-167.[CrossRef][Medline]
Lundström L, Elmquist A, Bartfai T, and Langel U (2005a) Galanin and its receptors in neurological disorders. Neuromol Med 7: 157-180.[CrossRef]
Lundström L, Sollenberg U, Brewer A, Kouya PF, Zheng K, Xu X-J, Sheng X, Robinson JK, Wiesenfeld-Hallin Z, Xu Z-Q, et al. (2005b) A galanin receptor subtype 1 specific agonist. Int J Peptide Res Ther 11: 17-27.[CrossRef]
Mazarati AM, Baldwin RA, Shinmei S, and Sankar R (2005) In vivo interaction between serotonin and galanin type 1 and type 2 receptors in dorsal raphe: implication for limbic seizures. J Neurochem 95: 1495-1503.[CrossRef][Medline]
Mazarati AM, Hohmann JG, Bacon A, Liu H, Sankar R, Steiner RA, Wynick D, and Wasterlain CG (2000) Modulation of hippocampal excitability and seizures by galanin. J Neurosci 20: 6276-6281.
Mazarati AM, Liu H, Soomets U, Sankar R, Shin D, Katsumori H, Langel U, and Wasterlain CG (1998) Galanin modulation of seizures and seizure modulation of hippocampal galanin in animal models of status epilepticus. J Neurosci 18: 10070-10077.
Mazarati AM and Lu X (2005) Regulation of limbic status epilepticus by hippocampal galanin type 1 and type 2 receptors. Neuropeptides 39: 277-280.[CrossRef][Medline]
Mazarati AM, Lu X, Kilk K, Langel U, Wasterlain CG, and Bartfai T (2004a) Galanin type 2 receptors regulate neuronal survival, susceptibility to seizures and seizure-induced neurogenesis in the dentate gyrus. Eur J Neurosci 19: 3235-3244.[CrossRef][Medline]
Mazarati A, Lu X, Shinmei S, Badie-Mahdavi H, and Bartfai T (2004b) Patterns of seizures, hippocampal injury and neurogenesis in three models of status epilepticus in galanin receptor type 1 (galr1) knockout mice. Neuroscience 128: 431-441.[CrossRef][Medline]
Mennicken F, Hoffert C, Pelletier M, Ahmad S, and O'Donnell D (2004) Restricted distribution of galanin receptor 3 (GalR3) mRNA in the adult rat central nervous system. J Chem Neuroanat 24: 257-268.
Mitchell FM, Heasley LE, Qian NX, Zamarripa J, and Johnson GL (1995) Differential modulation of bombesin-stimulated phospholipase C 
and mitogen-activated protein kinase activity by [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P. J Biol Chem 270: 8623-8688.
Moldrich RX, Chapman AG, De Sarro G, and Meldrum BS (2003) Glutamate metabotropic receptors as targets for drug therapy in epilepsy. Eur J Pharmacol 476: 3-16.[CrossRef][Medline]
Morimoto K, Fahnestock M, and Racine RJ (2004) Kindling and status epilepticus models of epilepsy: rewiring the brain. Prog Neurobiol 73: 1-60.[CrossRef][Medline]
O'Donnell D, Ahmad S, Wahlestedt C, and Walker P (1999) Expression of the novel galanin receptor subtype GALR2 in the adult rat CNS: distinct distribution from GALR1. J Comp Neurol 409: 469-481.[CrossRef][Medline]
Paxinos G and Watson C (1986) The Rat Brain in Stereotaxic Coordinates, Academic Press, San Diego, CA.
Sinnett-Smith J, Santiskulvong C, Duque J, and Rozengurt E (2000) [D-Arg1,D-Trp5,7,9,Leu11]substance P inhibits bombesin-induced mitogenic signal transduction mediated by both Gq and G12 in Swiss 3T3cells. Biol Chem 275: 30644-30652.
Stables JP, Bertram E, Dudek FE, Holmes G, Mathern G, Pitkanen A, and White HS (2003) Therapy discovery for pharmacoresistant epilepsy and for disease-modifying therapeutics: summary of the NIH/NINDS/AES models II workshop. Epilepsia 44: 1472-1478.[CrossRef][Medline]
Suzuki K, Iwasa H, Kikuchi S, Sato T, Miyake M, Morinaga N, and Noda M (1997) The contribution of endogenous mono-ADP-ribosylation to kindling-induced epileptogenesis. Brain Res 745: 109-113.[CrossRef][Medline]
Wang S, Hashemi T, Fried S, Clemmons AL, and Hawes BE (1998) Differential intracellular signaling of the GalR1 and GalR2 galanin receptor subtypes. Biochemistry 37: 6711-6717.[CrossRef][Medline]
Watanabe Y, Mori N, and Uemura S (1991) Suppression of kindled seizures following intraamygdaloid injection of pertussis toxin in rats. Neurosci Lett 130: 199-202.[CrossRef][Medline]
Wickenden AD (2002) Potassium channels as anti-epileptic drug targets. Neuropharmacology 43: 1055-1060.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  J. S. Brown Jr. Effects of Bisphenol-A and Other Endocrine Disruptors Compared With Abnormalities of Schizophrenia: An Endocrine-Disruption Theory of Schizophrenia Schizophr Bull, January 1, 2009; 35(1): 256 - 278. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
, p < 0.05 versus M35.
