Galanin contributes to monoaminergic dysfunction and to dependent neurobehavioral comorbidities of epilepsy

Abstract

Status epilepticus (SE) in rats, along with chronic epilepsy, leads to the development of behavioral impairments resembling depressive disorder and/or attention deficit/hyperactivity disorder (ADHD), thus reflecting respective comorbidities in epilepsy patients. Suppressed neurotransmitter tone in the raphe nucleus (RN)-prefrontal cortex (PFC) serotonergic pathway and in the locus coeruleus (LC)-PFC noradrenergic pathway underlies depressive- and impulsive-like behavioral deficits respectively. We examined possible mechanisms leading to the monoamine dysfunction in brainstem efferents, namely modulatory effects of the neuropeptide galanin on serotonin (5-HT) and norepinephrine (NE) signaling. SE was induced in young adult male Wistar rats by LiCl and pilocarpine. Epileptic rats were categorized vis-à-vis behavioral deficits as not impaired, "depressed" and "impulsive". Depressive- and impulsive-like behaviors were examined in the forced swimming test (FST). The strength of serotonergic transmission in RN-PFC and of noradrenergic transmission in LC-PFC was analyzed using in vivo fast scan cyclic voltammetry. Galanin receptor type 1 (GalR1)/type 2 (GalR2) antagonist M40, and a preferential GalR2 antagonist M871 were administered over 3days locally into either RN or LC by means of ALZET osmotic minipumps connected to locally implanted infusion cannulas. Intra-RN injection of M40 improved serotonergic tone and depressive-like behavior in epileptic "depressed" rats. Intra-LC injection of M40 improved noradrenergic tone and impulsive-like behavior in epileptic "impulsive" rats. The effects of M40 were only observed in impaired subjects. The treatment did not modify neurotransmission and behavior in naïve and epileptic not impaired rats; in "depressed" rats the effects were limited to serotonergic transmission and immobility, while in "impulsive" rats - to noradrenergic transmission and struggling behavior. Intra-RN administration of M871 exacerbated depressive-like behavior, but had no effects on any other of the examined parameters in any category of animals. These findings suggest that endogenous galanin, acting through GalR1 may be involved in the pathophysiology of epilepsy-associated depression and ADHD via inhibiting RN-PFC serotonergic and LC-PFC noradrenergic transmissions respectively.

Keywords: Attention deficit/hyperactivity disorder; Comorbidity; Depression; Epilepsy; Galanin; Locus coeruleus; Norepinephrine; Prefrontal cortex; Raphe nucleus; Serotonin.

Fig. 1. Study design

Experiment design Explanations in Methods.

Fig. 2. Baseline behavior in the FST

Immobility and struggle are shown for animals of each category during the FST1, performed at the end of the baseline seizure monitoring and before the randomization for GalR ligand injections. Data are presented as Mean±SEM. Sample sizes: naïve: 39; Epilepsy, not impaired: 32; Epilepsy, “depressed” 30; Epilepsy, “impulsive”: 30. ****- p<0.0001 vs, Naïve (One Way ANOVA plus Dunnet’s multiple comparison test).

Fig. 3. Effects of galanin receptor blockers administered in raphe nucleus on behavior

Saline, a GalR1/GalR2 blocker M40, or a GalR2 blocker M871 were infused in RN of naïve rats and epileptic animals classified as not impaired, “depressed” and “impulsive” based on their performance in the FST (see Methods). A. Effects on the immobility. Immobility time was significantly increased in epileptic “depressed” rats (Saline). Blockade of RN GalR1/GalR2 (M40) significantly shortened immobility time in these animals only (p>0.05 vs Naïve). Conversely, preferential blockade of GalR2 further increased the immobility time. Neither of the GalR blockers affected immobility in animals of other categories. B. Effects on the struggle. Struggling time was significantly increased in epileptic “impulsive” rats. Neither M40, nor M871 had any effects on struggling behavior in animals of all groups. Data are presented as Mean±SEM. *-p<0.05 vs. Naïve for the same treatment; †- p<0.05 vs. respective category of Saline-treated rats. Two-Way ANOVA plus Tukey’s multiple comparisons test. Immobility: Interaction F (6, 51) = 6.245, p<0.0001; treatment factor F (2, 51) = 14.09, p < 0.0001; behavior factor F (3, 51) = 42.49, p<0.0001. Struggle: Interaction F (6, 51) = 0.1038, p = 0.9956; treatment factor F (2, 51) = 0.1104, p = 0.8957; behavior factor F (3, 51) = 137.0, p < 0.0001.

Fig. 4. Effects of galanin receptor blockers administered in the raphe nucleus on serotonergic and noradrenergic transmission

The strength of serotonergic tone in RN-PFC and of noradrenergic tone in LC-PFC are shown for the same animals as in Fig. 1. A. Effects on serotonergic transmission. Epileptic “depressed” rats were characterized by the suppressed serotonergic tone, evident as lower amount of 5-HT released from PFC in response to the RN stimulation (Saline). Blockade of RN GalR1/GalR2 (M40) significantly improved serotonergic tone in “depressed” rats, while blockade of GalR2 (M871) had no effects. Neither of the GalR blockers affected responses in animals of other categories. B. Effects on noradrenergic transmission. Noradrenergic tone in LC-PFC was suppressed in epileptic “impulsive” subjects (Saline). Neither of the treatments had any effects on noradrenergic transmission in animals of all groups. Data are presented as Mean±SEM *-p<0.05 vs. Naïve in the same treatment; †- p<0.05 vs. respective category of Saline-treated rats. Two-Way ANOVA plus Tukey’s multiple comparisons test. Serotonergic transmission: Interaction F (6, 51) = 2.371, p = 0.0426; treatment factor F (2, 51) = 5.952, p = 0.0047; behavior factor F (3, 51) = 25.24, p < 0.0001. Noradrenergic transmission: Interaction F (6, 51) = 1.021, p = 0.4226; treatment factor: F (2, 51) = 0.1458, p = 0.8647; behavior factor F (3, 51) = 44.92, p < 0.0001.

Fig. 5. Behavior and serotonergic transmission after a washout following the administration of M40 in raphe nucleus

A. Behavior. Immobility in the FST was examined 3 days (i.e. during drug delivery) and 2 weeks (i.e. after a 10 day-washout) after the start of intra-RN infusion of a GalR1/GalR2 blocker M40 in epileptic not-impaired and “depressed” rats. During M40 administration immobility time was similar between the two groups, and in the range observed in naïve animals (compare with Naïve, Fig. 1). After the washout, immobility time in “depressed” animals was significantly longer and in the range observed in “depressed” saline-treated rats (compared with Fig 1, Epilepsy, “depressed”). Data are presented as Mean±SEM *- p<0.05 “M40, 1-week washout” vs. “M40, 3 days”; †- p<0.05 Epilepsy, “depressed” vs. Epilepsy, not impaired. Two Way ANOVA with multiple comparisons plus Sidak’s multiple comparisons test. Interaction: F (1, 8) = 20.20, p = 0.002; time factor F (1, 8) = 24.59, p = 0.0011; Behavior factor F (1, 8) = 70.32, p = 0.0001. B. Serotonergic transmission. After the second FST (shown on A), serotonergic tone was measured in RN-PFC. In epileptic “depressed” subjects, the strength of responses was lowers than in epileptic not impaired rats. In both groups, data were in the same range as in respective saline-treated rats (compare with Fig. 2A). *-p<0.05 (Mann-Whitney test).

Fig. 6. Effects of galanin receptor blockers administered in the locus coeruleus on behavior

Saline, a GalR1/GalR2 blocker M40, or a GalR2 blocker M871 were infused in LC of naïve rats (n=6 for each treatment) and epileptic animals classified as not impaired, “depressed” and “impulsive” based on their performance in the FST (see Methods). A. Effects on the struggle. Blockade of LC GalR1/GalR2 (M40) significantly shortened the duration of struggling behavior in epileptic “impulsive” rats, although the behavior was still significantly more represented than in naïve animals. Blockade of GalR2 (M871) did not modify the struggling behavior. None of GalR blockers affected the examined behavior in animals of other groups. B. Effects on the immobility. No effects on either of GalR blockers were observed in animals of all groups. Data are presented as Mean±SEM *-p<0.05 vs. Naïve in the same treatment; †- p<0.05 vs. respective category of Saline-treated rats. Two-Way ANOVA plus Tukey’s multiple comparisons test. Struggle: Interaction F (6, 53) = 17.25, p< 0.0001; treatment factor F (2, 54) = 15.3 , p < 0.0001; behavior factor F (3, 53) = 146.7, p < 0.0001. Immobility: Interaction F (6, 51) = 0.1544, p = 0.9873; treatment factor F (2, 51) = 0.1423, p = 0.8677; behavior factor F (3, 51) = 35.51, p < 0.0001.

Fig. 7. Effects of galanin receptor blockers administered in the locus coeruleus on serotonergic and noradrenergic transmission

The strength of noradrenergic tone in LC-PFC and of serotonergic tone in RN-PFC in LC-PFC are shown in the same animals as in Fig. 6. A. Effects on noradrenergic transmission. In epileptic “impulsive” rats, blockade of LC GalR1/GalR2 (M40) significantly strengthened noradrenergic tone, although it remained weaker than in naïve subjects. Blockade of GalR2 (M871) had no effect on the examined parameter. Neither of the GalR blockers modified noradrenergic transmission in animals of other groups. B. Effects on serotonergic transmission. Neither of the treatments had any effects on serotonergic tone in animals of all groups. Data are presented as Mean±SEM. *-p<0.05 vs. Naïve in the same treatment; †- p<0.05 vs. respective category of Saline-treated rats. Two-Way ANOVA plus Tukey’s multiple comparisons test. Noradrenergic transmission: Interaction F (6, 51) = 2.116, p = 0.0674; treatment factor F (2, 51) = 2.073, p = 0.1363; behavior factor F (3, 51) = 38.83, p < 0.0001. Serotonergic transmission: Interaction F (6, 51) = 0.8145, p = 0.5637; treatment F (2, 51) = 1.107, p = 0.3382; behavior factor F (3, 51) = 31.24, p< 0.0001.