Neuropeptide Y (NPY), one of the most abundant neuropeptides in the brain (Allen et al., 1983; Reichmann and Holzer, 2016; Smith et al., 2019), has robust neuromodulatory effects in the central nervous system (Colmers et al., 1987, Colmers et al., 1985; van den Pol, 2012). NPY mediates a variety of brain functions, such as circuit excitability (Giesbrecht et al., 2010; Silveira et al., 2023) and oscillations (Pollali and Draguhn, 2024), and is implicated in a variety of diseases including epilepsy (Cattaneo et al., 2020, Cattaneo et al., 2024) and stroke (Jiang et al., 2024). NPY is a stress resilience factor (Reichmann and Holzer, 2016; Sajdyk et al., 2008; Silveira Villarroel et al., 2018) and an important mediator of circuits that regulate mental health (Kupcova et al., 2022; Morales-Medina et al., 2010; Rasmusson and Pineles, 2018; Reichmann and Holzer, 2016). NPY is a major anxiolytic neuropeptide in the brain (Heilig et al., 1989; Kautz et al., 2017; Nahvi and Sabban, 2020; Sajdyk et al., 1999; Wu et al., 2011). In humans, a single nucleotide polymorphism in the promoter region of NPY, rs16147, decreases NPY expression and is correlated with enhanced anxiety traits (Sommer et al., 2010; Zhou et al., 2008). NPY has robust anxiolytic properties (Christiansen et al., 2014; Cohen et al., 2012; Heilig et al., 1989; Karlsson et al., 2005) and reduced NPY is implicated in anxiety disorders, the most common form of mental health disorder (Enman et al., 2015; Kautz et al., 2017; Schmeltzer et al., 2016). NPY is reduced in CSF of patients with post-traumatic stress disorder (PTSD) (Sah et al., 2014) and brain tissue of rodent models of PTSD (Cohen et al., 2012). NPY also has anti-depressive properties (Andriushchenko et al., 2022; Redrobe et al., 2002a, Redrobe et al., 2002b), and lower NPY levels are found in patients with major depressive disorder (Bale and Doshi, 2023; Guilloux et al., 2012; Hashimoto et al., 1996; Tural and Iosifescu, 2020) and in rodent models of depression (Heilig, 2004; Jiménez-Vasquez et al., 2007 Jiménez-Vasquez et al., 2000). Multiple antidepressant treatments have been shown to increase NPY levels in rodent models (Bjørnebekk et al., 2010; Husum and Mathé, 2002; Jiménez-Vasquez et al., 2007). NPY itself has been proposed as a potential therapy (Brothers and Wahlestedt, 2010; Sabban et al., 2016); in line with this idea, clinical trials have tested intranasal NPY in modulating symptoms for PTSD (Sayed et al., 2018) and depression (Mathé et al., 2020). Despite the fact that NPY's effects on anxiety and depression have been known for over 30 years (Heilig et al., 1989; Widerlöv et al., 1988), the mechanisms by which NPY modulates synaptic and circuit function to affect mental health are only partially understood.

Anxiety disorders are considered maladaptive forms of learning, and hippocampal dysfunction is implicated in anxiety disorders and PTSD (Çalışkan and Stork, 2019; Cominski et al., 2014; Goosens, 2011). NPY and its receptors are expressed at high levels in hippocampus (Cohen et al., 2012), particularly in the CA1 region of hippocampus (Pickel et al., 1995) that is important for anxiety and fear learning (Engin and Treit, 2007; Engin et al., 2016; Hirsch et al., 2015; Lovett-Barron et al., 2014). Interestingly, within CA1 there is high expression of NPY dense-core vesicles in s. lacunosum moleculare (SLM), the layer of CA1 containing the temporammonic (TA) pathway, a stress-sensitive input pathway (Kallarackal et al., 2013, Kvarta et al., 2015). NPY has long been known to reduce excitatory synaptic transmission of the other main input to CA1, the Schaffer collateral (SC) pathway (Colmers et al., 1988). This effect is through NPY Y2 receptors (Colmers et al., 1991; Greber et al., 1994; McQuiston and Colmers, 1996). Recently, we showed that NPY also inhibits the TA pathway (Li et al., 2017), although the receptor(s) underlying this effect is not yet known.

Injection of NPY into CA1 has anxiolytic effects (Smiałowska et al., 2007) and alleviates anxiety-like behavior in a rodent model of PTSD (Cohen et al., 2012). Both effects are blocked by Y1 receptor antagonism (Cohen et al., 2012; Smiałowska et al., 2007), indicating an essential role for Y1 receptors in CA1 in reducing anxiety. The location of Y1Rs in CA1 that mediate NPY's anxiolytic effects is unknown, and effects of Y1Rs on synaptic function in CA1 have yet to be demonstrated. Until now NPY's only known effects on CA1 synaptic transmission have been via presynaptic Y2 receptors in the SC pathway (Colmers et al., 1991), which mediate NPY's antiepileptic properties. We previously showed that the NPY dose-response relationship is different between TA and SC synapses (Li et al., 2017), suggesting the receptor type might be different between these two inputs to CA1. Consistent with this, immunostaining for Y2Rs shows no expression in CA1 SLM (Hörmer et al., 2018), the site of the TA pathway. CA1 pyramidal cells express Y1R mRNA (Parker and Herzog, 1999, Parker and Herzog, 1998), raising the possibility that Y1Rs in these cells mediate NPY's effects in the TA pathway. Y1R mRNA is also found in entorhinal cortex layer 3 (Kopp et al., 1999), where TA axons originate. We hypothesize that Y1 receptors mediates NPY's effects at TA synapses.

Interestingly, a previous study showed that transgenic rats that overexpress NPY had a reduced number of Y1 receptor binding sites in hippocampus, but not Y2 receptor binding sites (Thorsell et al., 2000). We previously showed that there is impaired sensitivity to exogenous NPY application at TA-CA1 synapses in slices from mice that overexpress NPY entopically (Corder et al., 2020). Surprisingly, these mice displayed heightened avoidance behavior on the elevated plus maze (Corder et al., 2020), rather than the expected reduction in avoidance behavior seen with NPY injection (Cohen et al., 2012). But, NPY overexpression in these same mice did reduce severity of kainate-induced seizures (Ste Marie et al., 2005), potentially suggesting that there are still functional NPY Y2 receptors. It is not yet known whether NPY overexpression impairs NPY Y1 receptors in the TA pathway, nor whether Y2 receptors in the SC pathway remain functional.

Here we demonstrate that the effects of both bath-applied and endogenously-released NPY on TA-CA1 synapses are through NPY Y1 receptors but not Y2 receptors. In addition, NPY overexpression impairs Y1 receptor function in TA in both heterozygous and homozygous NPY overexpression mice. In contrast, NPY overexpression does not impair the function of NPY Y2 receptors at SC-CA1 synapses. Together, these results show that there is pathway-specific regulation by NPY of the inputs to CA1, which are differentially impacted by NPY overexpression.