The α₁-adrenoceptor (α₁AR) and the α₂-adrenoceptor (α₂AR), which belong to the G protein-coupled receptor (GPCR) family, have been involved in brain developmental processes and constitute potential therapeutic targets for different neuropathological disorders such as drug addiction, Parkinson’s and Alzheimer’s diseases, or post-traumatic stress (Ghanemi and Hu, 2015; Perez, 2020). The α₁AR predominantly couples to G_q/11, the stimulation of which leads to the activation of phospholipase C (PLC) and the production of inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). IP₃ activates the release of Ca²⁺ into the cytoplasm, whereas DAG activates protein kinase C (PKC) (Hein and Michel, 2007). The α₂AR couples to the G_i/o protein, the activation of which results in the inhibition of adenylyl cyclase and reduction of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) activity (Aantaa et al., 1995). However, each adrenoceptor can couple to multiple signaling pathways. Thus, the α₁AR can activate G_i proteins (Petitcolin et al., 2001) and the α₂AR can couple to G_s proteins (Eason and Liggett, 1995). Furthermore, adrenergic agonists can display ligand-directed signaling bias (Alexander et al., 2023).

Locus coeruleus (LC) is the main noradrenergic nucleus in the central nervous system (CNS) (Foote et al., 1983). It is involved in the regulation of CNS functions, including sleep–wake cycle, attention, memory, and stress-related responses (Berridge and Waterhouse, 2003; Matt et al., 2024). The activity of LC cells is regulated, among others, by the αAR (Schwarz et al., 2015). Both α₁AR and α₂AR have been localized in LC neurons through quantitative autoradiography (Chamba et al., 1991) and RT-PCR (Osborne et al., 2002). In situ hybridization techniques have revealed that the α_1AAR is the main subtype of the α₁AR in the LC (Day et al., 1997). Moreover, a recent immunohistochemical study has shown that the α₁AR colocalizes with tyrosine hydroxylase in LC dendrites, which indicates that the α₁AR is expressed in NA neurons (Luyo et al., 2023). However, there are conflicting data regarding the functional role of the α₁AR in LC neurons. Some authors have suggested that the α₁AR decreases its activity during development and disappears in the adult male rat LC (Finlayson and Marshall, 1984; Williams and Marshall, 1987). In contrast, indirect evidence has suggested that the α₁AR contributes to the excitability of LC neurons observed in the presence of the α₂AR antagonist in adult male rat brain slices (Ivanov and Aston-Jones, 1995). Furthermore, the activation of the α₁AR reduces outward potassium currents induced by α₂AR activation in LC neurons (Osborne et al., 2002). Finally, microdialysis studies have shown that local administration of α₁AR agonist cirazoline increases noradrenaline (NA) in the LC, whereas administration of an α₁AR antagonist decreases it (Pudovkina et al., 2001; Pudovkina and Westerink, 2005).

Although the function of the α₁AR in different brain areas of the CNS and its involvement in several neurological disorders has been studied (Ghanemi and Hu, 2015; Lemmens et al., 2015; Perez, 2020), the role of this receptor in the adult LC remains controversial. Therefore, the aim of this work was to characterize functionally the α₁AR in LC neurons from adult male rats and to examine possible downstream processes linked to receptor activation through single-unit extracellular recordings in brain slices.

The present work was undertaken to investigate the role of the α₁AR in the regulation of the FR of LC neurons and the signaling pathways involved in its effects. Our results reveal that α₁AR activation with the adrenergic agonists NA and PE in the presence of an α₂AR antagonist or with cirazoline stimulates the FR of LC NA cells in adult male rat brain ex vivo. NA-induced stimulation was reduced by the inhibitor of G_i/o protein PTX and the TRP channel blocker 2-APB. However, none of the inhibitors blocked the stimulatory effect induced by cirazoline.

Perfusion with NA or PE inhibits the FR of LC neurons, whereas in the presence of α₂AR antagonist RS 79948, they stimulate the FR. These results show that the inhibitory effects of these adrenergic agonists are mediated by the α₂AR, as previously described (Williams et al., 1985; Williams et al., 1991). Even though PE is mainly considered a selective α₁AR agonist, some studies performed in vascular tissues have shown that PE can activate α₂AR (McGrath et al., 1999; Görnemann et al., 2009; VanLangen et al., 2013). Moreover, PE partially inhibits serotonin release in rat raphe nuclei through α_2AAR activation (Hopwood and Stamford, 2001) and also evokes small-membrane hyperpolarizations in LC neurons (Williams et al., 1985). Finally, it has been recently reported that PE stimulates the cytoplasmatic release of NA via the NA transporter (Al-Khrasani et al., 2022), which could explain the inhibitory effect of PE observed in our experiments due to the presence of large reserve of α₂AR receptors in LC neurons (Pineda et al., 1997).

In the presence of RS 79948 and α₁AR antagonist WB 4101, both the inhibitory and stimulatory effects of NA and PE were blocked, which indicates that the increase in the FR induced by NA or PE after the α₂AR blockade is mediated by the α₁AR. Our data suggest that the α₁AR-mediated excitatory effect of NA in LC neurons in adult rats may be masked by concurrent α₂AR-mediated inhibition becoming apparent only when α₂ARs are blocked by an antagonist. These results are consistent with those of studies that suggest that the activation of the α₁AR may contribute to increase the FR of LC neurons (Ivanov and Aston-Jones, 1995). In contrast, some previous studies have reported that α₁AR-mediated effects in LC neurons are restricted to early developmental stages. This discrepancy may be explained, at least in part, through methodological differences. Notably, earlier studies used techniques such as intracellular recordings in organotypic cultures, which may not fully reflect the physiological properties of mature LC neurons in acute brain slices. Moreover, the studies did not examine the effects of cirazoline or assess NA responses in the presence of an α₂AR antagonist, both key aspects of our experimental design that may have unmasked α₁AR-mediated excitatory effects in adult tissue.

The putative involvement of the β₁AR in the stimulatory effects induced by NA and PE could be considered as both compounds bind to this receptor subtype (Ki ≈ 100–300 nM and 13 μM, respectively). However, in the case of NA, the contribution of the β₁AR appears unlikely as WB 4101—a selective α₁AR antagonist that does not bind to β₁AR—completely blocked the stimulatory effect of NA in the presence of α₂AR antagonist RS 79948. In contrast, in our slice preparations, WB 4101 significantly, but not completely, inhibited the stimulatory effect of PE under the same conditions. This suggests that β₁AR may contribute to the effects of PE, a possibility that cannot be entirely excluded considering their affinity values for different receptors (Ki ≈ 13 µM for β₁AR, 6 µM for α₁AR, and 0.4 µM for α₂AR) and the concentration applied (Chen et al., 1993; Gil and Donello, 2005). Alternatively, the involvement of other receptors (e.g., 5-HT₇ receptor) could not be ruled out (European Molecular Biology Laboratory - European Bioinformatics Institute, 2025). It is important to note, however, that the same concentration of PE (100 µM) has previously been administrated in LC slice preparations to investigate α₁AR-mediated effects, which aligns with the conditions used in our study (Osborne et al., 2002). Furthermore, the concentrations of all drugs used in this study were selected based on their reported Ki values or data from previous electrophysiological studies in brain slices, taking into account that drug affinities observed in isolated radioligand binding assays can differ significantly—often by 10- to 300-fold depending on the drug hydrosolubility—from those used in functionally active slice preparation. We used antagonists RS 79948 and WB 4101 to block α₂AR and α₁AR, respectively, because they have been shown to be rather selective for each α-adrenergic receptor type in previous in vivo or binding studies (Drew, 1982; Michel et al., 1995; Milligan et al., 1997; Mateo et al., 2000; Proudman et al., 2022). In other words, WB 4101 has lower affinity for D₂ (Ki ≈ 123 nM) and α₂AR (Ki ≈ 28–46 nM for α_2B–α_2AAR) than for α₁AR (Ki ≈ 6–8 nM for α_1B and Ki ≈ 0.5 nM for α_1A). We did not use prazosin or terazosin as antagonists due to their lower selectivity for the α_1AAR (Hancock et al., 1995; Yuan et al., 2009). Furthermore, WB 4101 (0.5 µM) blocks the PE-induced effect through the α_1AAR in slices from other brain regions (Hancock et al., 1995; Yuan et al., 2009), which could also occur in the LC.

The α₁AR agonist cirazoline, which also shows moderate affinity for the 5-HT_1A receptor (Ki ≈ 35 nM) and binds to the α₂AR (Ki ≈ 59 nM), stimulated the FR of LC cells. The stimulatory effects induced by cirazoline (1 µM and 10 µM) were blocked by perfusion with α₁AR antagonist WB 4101, supporting the role of the α₁AR in the regulation of the FR of LC neurons. However, at high concentrations (100 µM), cirazoline-induced stimulation was not blocked by WB 4101, which indicates that WB 4101 behaves as a competitive antagonist. The involvement of the non-α₁AR receptor-mediated mechanism in the effect produced by the highest concentration of cirazoline could also be considered, including the activation of imidazoline receptors, 5-HT_1A receptors, and α_2AAR (Angel et al., 1995; Alexander et al., 2023). Thus, in anesthetized rats pretreated with EEDQ (an irreversible α-adrenoceptor antagonist), imidazoline drugs such as clonidine, cirazoline, and rilmenidine stimulate neuronal activity in LC cells through the activation of I₁-imidazoline receptors (Pineda et al., 1993), but it seems to be an indirect effect mediated by imidazoline receptors located on paragigantocellularis neurons that project to the LC (Ruiz-Ortega and Ugedo, 1997). Some imidazoline drugs can stimulate the FR of LC neurons by a non-I₁/I₂ imidazoline receptor located extracellularly (Ugedo et al., 1998). Although this mechanism has not been described for cirazoline, it remains to be studied how cirazoline stimulates the FR of LC neurons after blockade of the α₁AR in our system. The putative contribution of the 5-HT_1A receptor or α₂AR to the observed stimulatory effect could be ruled out as the activation of the 5-HT_1A receptor or α₂AR would reduce rather than increase the FR of LC cells. To address the study of the signaling pathways involved in the stimulatory effect mediated by α₁AR, we used the agonists NA (100 μM; in the presence of the α₂AR antagonist RS 79948 0.1 µM) and cirazoline (10 µM) because their effects were fully blocked after α₁AR antagonism. Even though α₁AR has been considered to be coupled to the G_q/11/PLC/PKC pathway in some areas of the rat brain (Kobayashi et al., 2008), there is evidence showing that this receptor can couple to other G proteins and multiple signaling pathways (Hein and Michel, 2007; Cotecchia, 2010). α₁AR activation can also induce cAMP accumulation and PKA activation, which could modulate PKC (García-Sáinz et al., 2000). The enhancement of intracellular levels of cAMP or application of its analogs is known to increase the FR of LC neurons (Wang and Aghajanian, 1987). The activation of the α₁AR with PE suppresses currents carried by GIRK channels that are opened by Gi/o protein-coupled receptors such as µ opioid receptor or α₂AR (Osborne et al., 2002). In addition, it has been described that TRP channel antagonists suppress inward currents produced after α₁AR activation with phenylephrine in LC neurons of SHR juvenile rats (Igata et al., 2014). Moreover, quantitative real-time PCR analysis found high levels of mRNA expression of TRPC5 (the most abundant type), TRPM7, and TRPM2 channels in the LC (Cui et al., 2011). The drugs that were used to inhibit each signaling pathway, such as Go 6976, PTX, BaCl₂, 2-APB, H-89, and U73122, had been previously used by other authors in rat brain slices at similar concentrations (Chiu et al., 1995; Chessel et al., 1996; Bailey et al., 2009; Murai et al., 2012; Igata et al., 2014; Jolas et al., 2000).

PKC inhibitor Go 6976, PKA inhibitor H-89, and the blockade of GIRK channels with BaCl₂ failed to change the stimulatory effect of NA in the presence of RS 79948. In contrast, G_i/o protein inhibition with PTX and perfusion with the TRPC5 and TRPM7 channel blocker 2-APB significantly reduced the stimulatory effect of NA in the presence of RS 79948, suggesting that the effect induced by NA through α₁AR occurs via a pathway that involves G_i/o proteins and TRP channels. Although there is no evidence describing the α₁AR/G_i/o protein/TRP channel pathway in neurons, some studies performed in different tissues could support this hypothesis. First, α₁AR couples to G_i/o proteins and mediates pertussis toxin-sensitive effects in some vascular systems (Gurdal et al., 1997; Otani et al., 2001; Petitcolin et al., 2001). Second, G_i/o proteins stimulate TRPC5 and TRPM7 channel activities in several cell types (Beech, 2012; Oronowicz et al., 2021). Moreover, TRPC5 channels can be activated by the G_i/o protein-coupled µ opioid receptor (Miller et al., 2011), which is widely expressed in LC neurons, and they also contribute to the development of opioid tolerance in spinal neurons (Chu et al., 2020). Considering the aforementioned studies, we suggest that α₁AR activation by adrenergic agonist NA stimulates the FR of LC neurons through its interaction with G_i/o proteins and TRPC5/TRPM7 channels, and we propose two hypotheses that could explain this mechanism. On the one hand, α₁AR, G_i/o proteins, and TRP channels could be directly coupled. Then, α₁AR activation could regulate G_i/o proteins and lead to the opening of TRP channels, which would induce an inward cationic current to increase the FR. On the other hand, Gi/o proteins could constitutively inhibit TRP channels and α₁AR activation could relieve this inhibition, which would result in TRP channel opening and neuron depolarization.

It has been described that α₁AR activation by PE in LC neurons reduces GIRK channel conductance induced by α₂AR or µ opioid receptors, which are coupled to G_i/o proteins (Osborne et al., 2002). This interaction does not seem to occur in our system as the role of GIRK channels in α₁AR activation was discarded. However, a mechanism involving TRP channels could also explain the observed stimulatory effect of PE on the FR of LC cells as in the case of NA. In contrast to NA-induced stimulation, G_i/o protein inhibitor PTX, TRP channel blocker 2-APB, PKC inhibitors Go 6976 and chelerythrine, PKA inhibitor H89, and PLC inhibitor U73122 failed to reduce the cirazoline stimulatory effect.

Furthermore, Go 6976 and chelerythrine enhanced the effect of cirazoline. Although no studies so far have directly linked PKC inhibition to the increased cirazoline effect, it is plausible to hypothesize that PKC activity exerts a constitutive inhibitory influence on the signaling pathway responsible for α₁AR-mediated increases in the firing rate of LC neurons. Therefore, inhibition of PKC may relieve this suppression, thereby amplifying the stimulatory effect of cirazoline.

The differences in the results regarding the signaling pathways involved in NA or cirazoline stimulatory effects can be explained because these agonists may display functional selectivity. Functional selectivity or biased signaling refers to the ligand-dependent receptor activation of certain signaling pathways over others (Kolb et al., 2022). This property has been widely characterized in many GPCRs, including α₁AR, which has been described by several authors to couple to different G proteins and activate several signaling pathways (Zhong and Minneman, 1999; Alcántara-Hernández et al., 2017; da Silva Junior et al., 2017). Therefore, our results could be explained due to biased signaling as studies performed in CHO-K1 cells expressing α_1aAR found that cirazoline displays the signaling bias toward cAMP accumulation relative to Ca²⁺ release when compared to reference endogenous agonist NA (Evans et al., 2011). Moreover, NA and cirazoline show different affinities for each α₁AR receptor subtype (NA: α_1d > α_1b > α_1a; cirazoline: α_1a > α_1d > α_1b) (Horie et al., 1995; Proudman and Baker, 2021).

Our study has some potential limitations. First, the magnitude of the basal stimulatory effect of NA in the presence of RS 79948 (i.e., in the absence of inhibitors or blockers) appeared to differ across some experimental groups (e.g., Figures 1 vs. Figure 4). However, no changes in experimental conditions could account for this discrepancy. Notably, in most groups, the effect of NA in the presence of RS 79948 was compared to the control NA response recorded in the same neuron, minimizing the impact of this variability on the overall conclusions. Second, consistent with previous studies, only male rats were used in this investigation. Future experiments should aim to characterize α₁AR-mediated effects in female rats to assess potential sex-dependent differences.

α₁AR is involved in several CNS functions and pathologies such as behavioral activity and depression (Stone et al., 2007), pain modulation (Kingery et al., 2002; Nakatsuka et al., 2022), reward processes (Lin et al., 2007), psychostimulant-induced locomotor hyperactivity (Drouin et al., 2002), or neurodegenerative disorders. Some of these have been shown to be related to the LC. In this line, an increase in the neuronal activity induced by the activation of α₁-AR has also been reported in other brain regions, such as in the prefrontal cortex (Datta et al., 2019), the paraventricular nucleus of the hypothalamus (Chen et al., 2006), or the interneurons of the layer CA1 of the hippocampus (Hillman et al., 2009). In the latter case, this activation leads to a decrease in the activity of pyramidal neurons and to an antiepileptic effect. Previous studies have revealed a role of α₂AR in the regulation of the activity of LC neuron (Aghajanian and VanderMaelen, 1982; Pineda et al., 1997) in the adult rat brain, but the function of α₁AR was thought to be reduced during development. Our results reveal a functional importance of α₁AR in the adult rat LC. The α₁AR can be activated by adrenergic agonists NA and PE (after α₂AR blockade) or by α₁AR agonist cirazoline, stimulating the FR of LC neurons. The stimulatory effect induced by NA would occur through a signaling pathway that involves G_i/o proteins and TRPC5/TRPM7 channels. More studies will be required to describe in detail the mechanisms involved in the α₁AR stimulatory effect and the functional role of this receptor in female rats, but our results suggest that the α₁AR receptor in the adult male rat LC could constitute a target in the treatment of several disorders of the CNS.