Galanin is a neuropeptide, which is critically involved in homeostatic processes like controlling arousal, sleep, and regulation of stress. This extensive range of functions aligns with implications of galanin in diverse pathologies, including anxiety disorders, depression, and epilepsy. Here, we investigated the regulatory function of galanin on whole-brain activity in larval zebrafish using wide-field Ca 2+ imaging. Combining this with genetic perturbations of galanin signaling and pharmacologically increasing neuronal activity, we are able to probe actions of galanin across the entire brain. Our findings demonstrate that under unperturbed conditions and during epileptic seizures, galanin exerts a sedative influence on the brain, primarily through the galanin receptor 1 a (_galr1a_). However, exposure to acute stressors like pentylenetetrazole (PTZ) compromises galanin’s sedative effects, leading to overactivation of the brain and increased seizure occurrence. Interestingly, galanin’s impact on seizures appears to be bidirectional, as it can both decrease seizure severity and increase seizure occurrence, potentially through different galanin receptor subtypes. This nuanced interplay between galanin and various physiological processes underscores its significance in modulating stress-related pathways and suggests its potential implications for neurological disorders such as epilepsy. Taken together, our data sheds light on a multifaceted role of galanin, where galanin regulates whole-brain activity but also shapes acute responses to stress.
Similar to classic neurotransmitters, neuropeptides are chemical messengers that are mainly synthesized and released by neurons. In contrast to conventional neurotransmitters, neuropeptides are amino acid chains between 3 and 36 amino acids in length. While classic neurotransmitters are primarily stored in synaptic vesicles, neuropeptides are predominantly stored in large dense-core vesicles (LDCV) and are mostly released during neuronal bursts or high-frequency firing (Lang et al., 2015; van den Pol, 2012). Neuropeptides play a crucial role in modulating neuronal activity and regulating various aspects of neural network function. They act as neuromodulators, exerting long-lasting effects on neuronal excitability, synaptic transmission, and plasticity (Lang et al., 2015; Purves et al., 2019). By influencing the balance between excitatory and inhibitory inputs to neurons, neuropeptides can shape network dynamics and information processing. This is achieved through their interactions with specific receptors and signaling pathways, allowing for precise modulation and optimization of neural activity in response to changing conditions (van den Pol, 2012). Overall, neuropeptides play a pivotal role in orchestrating the complex activity of neuronal networks and maintaining homeostasis within the central nervous system. Elevated neuronal activity is often accompanied by shifts in neuropeptide transcription rates through a phenomenon known as ‘stimulus-secretion-synthesis coupling’ (Douglas, 1968; MacArthur and Eiden, 1996). This intricate interplay between neuropeptide release and biosynthesis likely serves as a crucial mechanism for replenishing neuropeptide stores, given the absence of a mechanism for neuropeptide uptake post-release and their degradation by extracellular peptidases. Furthermore, neuropeptides are well known to contact neurons via volume transmission, where extrasynaptically secreted molecules activate receptors on neurons synaptically unconnected to the releasing neuron. This leads to signal transmission across considerable distances, extending up to multiple microns, targeting multiple neurons in different regions of the brain (Lang et al., 2015; van den Pol, 2012; Jan and Jan, 1982; Atkinson et al., 2021; Nässel, 2009; Ripoll-Sánchez et al., 2023).
Galanin is such a neuropeptide, which is expressed predominantly in the hypothalamus, the center of homeostatic regulation in the brain. Galanin is highly involved in homeostatic processes like controlling arousal and sleep (Woods et al., 2014; Podlasz et al., 2018; Reichert et al., 2019; Gaus et al., 2002; Kroeger et al., 2018; McGinty and Szymusiak, 2003; Ma et al., 2019), but has been shown to regulate stress (Corradi et al., 2022; Juhasz et al., 2014; Hökfelt et al., 2018; Khoshbouei et al., 2002; Picciotto et al., 2010). Moreover, research has demonstrated that galanin-producing neurons in the hypothalamus play a role in regulating both food intake (Schick et al., 1993; Adams et al., 2008; Qualls-Creekmore et al., 2017; Laque et al., 2015; Leibowitz et al., 1998) and parental behavior (Kohl et al., 2018; Wu et al., 2014) in rodents. This extensive range of functions aligns with implications of galanin in diverse pathologies, including anxiety disorders, depression, and epilepsy (Lang et al., 2015; Juhasz et al., 2014; Hökfelt et al., 2018; Kovac and Walker, 2013; Lerner et al., 2008; McColl et al., 2006; Fetissov et al., 2003; Jacoby et al., 2002; Mazarati et al., 2000; Drexel et al., 2018). We found an upregulation of galanin in a recently described novel model of epilepsy (Hotz et al., 2022) that let us hypothesize that galanin may mediate a neuroprotective, net inhibitory effect on epileptic brains.
Here, we investigated the regulatory function of galanin on whole-brain activity in larval zebrafish (_Danio rerio_). Leveraging the transparency of zebrafish during their larval stages, a characteristic that facilitates live imaging, we utilized basic wide-field Ca 2+ imaging methods. Combining this with genetic perturbation of galanin and pharmacologically increasing neuronal activity, we were able to delve into the actions of galanin across the entire brain.
Our findings demonstrate that, under unperturbed conditions and during epileptic seizures, galanin exerts a net inhibitory influence on the brain, which is likely governed by _galanin receptor 1_ a (_galr1a_). However, when faced with an acute stressor like pentylenetetrazole (PTZ), the typically sedative effects of galanin are substantially compromised. We found that exposure to this acute central nervous system stressor results in a galanin-dependent overactivation of the brain that overrides most of galanins sedating actions and increases the occurrence of epileptic seizures. Taken together, our data sheds light on a multifaceted role of galanin, where galanin regulates whole-brain activity but also shapes acute responses to stress.
In prior work, we introduced a novel epilepsy model characterized by recurrent epileptic seizures and interictal neuronal hypoactivity (Figure 1A-D: Hotz et al., 2022). The unexpected observation of locomotor and neuronal hypoactivity in this model stands in contrast to most existing studies in zebrafish that report hyperactivity in epileptic animals (Baraban et al., 2013; Hortopan et al., 2010; Baraban et al., 2005). Intriguingly, recent investigations have demonstrated that overexpression of galanin induces locomotory hypoactivity in zebrafish models (Woods et al., 2014; Podlasz et al., 2018). To explore the potential involvement of galanin in the hypoactivity observed in _eaat2a_ mutants, we conducted qPCR analysis on 5 dpf (days post fertilization) larval brains (Figure 1C). The results revealed a significant 15.4-fold increase in galanin expression compared to wild-type siblings, suggesting an involvement of galanin in the interictal hypoactivity observed in _eaat2a_ mutants. Furthermore, recent findings have shed light on the role of galanin in pharmacologically induced rebound sleep (Reichert et al., 2019). Additionally, it was demonstrated that increasing short-term neuronal activity results in subsequent inactivity that is dependent on galanin (Reichert et al., 2019). To explore whether the application of GABA A receptor antagonist PTZ would also lead to a temporary decrease in whole-brain activity, we exposed 5 dpf larvae to 20 mM PTZ for 1 h, followed by a 2 hr washout period before undergoing Ca 2+ imaging. Remarkably, while PTZ increases swimming activity and induces seizure-like behavior during acute drug exposure (Reichert et al., 2019; Baraban et al., 2005; Baraban et al., 2007), there was a significant reduction in brain activity during PTZ rebound (Figure 1G-K), which was correlated with an increase in galanin expression by 2.5-fold (Figure 1I) compared with non-PTZ-treated larvae. Notably, large Ca 2+ fluctuations (ΔF/F 0>10%) decreased in frequency (Figure 1J), and decreased in amplitude (Figure 1K), while their duration increased (Figure 1L) compared to non-PTZ-treated larvae. For small Ca 2+ fluctuations (ΔF/F 0>5) the frequency also decreased (Figure 1J), while their amplitude and duration were not affected (Figure 1K and L). Together, our results illustrate that the expression of galanin rises in reaction to seizure-like activity in two separate seizure models and is correlated with an overall decrease in whole-brain activity.
(A) Representative calcium signals (_elavl3:GCaMP5G_) recorded across the brain of 5 dpf control (_eaat2a+/+_) larva (blue, top) and _eaat2a-/-_ mutant without seizure activity (red, bottom). (B) Area …
To investigate if galanin by itself is able to reduce whole-brain activity, we employed a transgenic approach by using _hsp70l:gal_ (Woods et al., 2014) expressing larvae. These transgenic fish express galanin under the control of the heat shock promoter _hsp70l_, enabling the induction of galanin expression by upregulating transcripts by over 300 fold (Podlasz et al., 2018). Attempts to induce galanin overexpression through heat shock were discontinued due to a notable impact on the brain activity of wild-type larvae compared to non-exposed wild-type larvae (Figure 2—figure supplement 1). Yet, considering that _hsp70l:gal_ larvae already display a basal elevation of galanin transcripts by approximately eightfold (Podlasz et al., 2018), comparable with the upregulation seen in _eaat2a-/-_ mutants and PTZ rebound (Figure 1C and I), we chose to use larvae without inducing transcription via heat shock. We performed Ca 2+ imaging and found that indeed elevated expression of galanin in _hsp70l:gal_ larvae leads to a decrease in whole-brain activity (Figure 2A) compared to wild-type siblings. We detected a reduction in frequency of Ca 2+ fluctuations for larger Ca 2+ events and a reduction in their amplitude (Figure 2D and E), mirroring our earlier findings in the PTZ rebound (Figure 1G and H). Notably, _hsp70l:gal_ larvae exhibited no difference in small Ca 2+ fluctuations (Figure 2D–F). Subsequent quantification of galanin levels in the brain through qPCR unveiled a 1.8-fold increase in ga
