The oxytocin receptor (OXTR) is concentrated in specific brain regions, exemplified by hippocampal subregion CA2, that support social information processing. Oxytocinergic modulation of CA2 directly affects social behavior, yet how oxytocin regulates activity in CA2 remains incompletely understood. We found that OXTR stimulation acts via closure was persistent in CA2 pyramidal cells, whose prolonged burst firing required functional coupling of the OXTR to both Gaq and Gai proteins. Other neuromodulators acted via distinct patterns of G-protein signaling to induce CA2 pyramidal neuron burst firing, underscoring its likely importance. CA2 burst firing impacted hippocampal subregion CA1 where stratum oriens-resident CA1 interneurons were targeted more strongly than CA1 pyramidal cells. Oxytocinergic modulation of interneurons, via CA2 pyramidal cell input and directly, triggered a long-lasting enhancement of CA3-CA1 transmission. Thus, transient activation of oxytocinergic inputs may initiate long-lasting recording of social information. 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89

Understanding how neuromodulators shape neural activity and relate to neuropsychiatric disease are cornerstones of modern neuroscience. Oxytocin, a peptide hormone with well-defined roles in parturition and lactation, has been identified as a modulator of prosocial behavior across the animal kingdom (Donaldson & Young, 2008; Garrison et al., 2012; Gimpl & Fahrenholz, 2001). Increased levels of oxytocin are associated with trust, generosity and facial recognition in humans (Bartz, Zaki, Bolger, & Ochsner, 2011; Kosfeld, Heinrichs, Zak, Fischbacher, & Fehr, 2005; Skuse et al., 2014; Zak, Stanton, & Ahmadi, 2007) , while variations in its receptor and plasma levels have been observed in patients with autism spectrum disorder (ASD)(LoParo & Waldman, 2014; Modahl et al., 1998; Wu et al., 2005). Oxytocin treatment of ASD and other disorders with atypical social behavior has shown variable but encouraging preclinical promise in patients and animal models (Feifel et al., 2010; Penagarikano et al., 2015; Young & Barrett, 2015), prompting further investigation into how oxytocin signals in the brain. A more refined mechanistic understanding of central oxytocin signaling might facilitate the development of more precise and effective therapies.

Expression of the oxytocin receptor (OXTR) is concentrated in specific regions of the brain (Insel & Shapiro, 1992), highlighting pote ntial hubs of social information processing. Coupled with use of transgenic mouse lines (Hidema et al., 2016; Nakajima, Gorlich, & Heintz, 2014; Yoshida et al., 2009), the de velopment of the first specific OXTR antibody (Mitre et al., 2016) has enabled experiments to define what cell-types respond to oxytocin and model how neural circuits underlying social behavior are modulated (Menon et al., 2018; Oettl et al., 2016; Tirko et al., 2018; Xiao, Priest, 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112

Nasenbeny, Lu, & Kozorovitskiy, 2017) . The hippocampal sub-region CA2, which is distinguished from neighboring areas CA1 and CA3 by its anatomy, physiology and gene expression profile (Dudek, Alexander, & Farris, 2016) , is enriched with OXTRs (Lee, Caldwell, Macbeth, Tolu, & Young, 2008; Mitre et al., 2016; Tirko et al., 2018). There, pyramidal cells express the OXTR (Tirko et al., 2018) and their activity is required for the encoding, consolidation and recall of a short-term social memory in mice (Hitti & Siegelbaum, 2014; Meira et al., 2018). CA2 neurons in the dorsal hippocampus (dCA2) primarily send their axonal projections within the hippocampal formation (Cui, Gerfen, & Young, 2013; Hitti & Siegelbaum, 2014), including ventral CA1 (vCA1), which itself has been implicated in social memory (Okuyama, Kitamura, Roy, Itohara, & Tonegawa, 2016). Multiple lines of investigation suggest that projections from dCA2 to vCA1 are critical for social recognition (Meira et al., 2018; Raam, McAvoy, Besnard, Veenema, & Sahay, 2017) , but how CA2 activity modulates CA1 is incompletely understood.

Activation of OXTRs in CA2 is known to increase neuronal excitability and cause local pyramidal neurons to enter into a burst firing mode (Tirko et al., 2018), as well as potentiate synaptic transmission onto CA2 neurons (Pagani et al., 2015). To bridge our understanding of oxytocinergic modulation at the cellular level with observations made in behavioral experiments, we have studied the circuit consequences of oxytocin9s actions in CA2, focusing on which CA2 cell-types are modulated, how the cellular response evolves over time and how CA2 activity propagates to other regions. Our experiments reveal a series of unexpected sequelae that occur in CA2 pyramidal cells upon OXTR activation to produce a persistent change in firing mode that occurs over a behaviorally relevant timescale. This persistent modulation is specific to CA2 pyramidal cells, which, in turn, strongly target stratum oriens-resident interneurons in CA1. Release of endogenous oxytocin into the hippocampus elicits sustained plasticity in CA1 pyramidal cells, a potential circuit mechanism to translate transient oxytocin release into persistent modifications in hippocampal activity as may occur during a social encounter.

Stimulation of hippocampal OXTRs excites both pyramidal cells and parvalbuminexpressing (PV+) interneurons via closure of the <M-current= or IM, which is mediated by potassium-fluxing KCNQ channels (Tirko et al., 2018). However, responses in the two cell-types show remarkably different time-courses (Fig. 1a, b). In these experiments, the specific OXTR agonist Thr4-Gly7-Oxytocin (TGOT, 400 nM) was applied to acute hippocampal slices from adult mice of either sex while whole-cell recordings were made from CA2 cells. TGOT was applied to each slice only once, to avoid receptor internalization as a confounding factor (Busnelli et al., 2012; Gimpl & Fahrenholz, 2001; M. P. Smith et al., 2006) . CA2 pyramidal neurons were identified on the basis of their 131 132 133 134 135 136 137 within 2 min (average time to depolarization: 0.74 ± 0.22 min (PYR) vs. 0.54 ± 0.15 min (PV); Fig. 1c), but only pyramidal cells mounted a response that lasted tens of minutes (as shown in Figs. 1b, d, e, f). As a population, CA2 pyramidal cells exhibited a 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 significantly longer lasting burst response to TGOT stimulation than PV+ interneurons (median burst duration: 18.6 min (PYR) vs. 7.6 min (PV), Fig. 1d). In contrast, PV+ cells consistently showed a large and transient increase in firing (average change in peak firing rate: 20.6 ± 3.5 Hz, n = 12) that roughly matched the duration of TGOT application. Of 12 PV+ cells recorded, all 12 depolarized in response to TGOT application (average peak depolarization: 14.3 ± 1.3 mV, n = 12), suggesting an absence of sub-type specificity in TGOT sensitivity. To define which subclasses of PV+ interneurons were included in our data set, we generated 2D morphological reconstructions after each recording and classified neurons on the basis on their axonal arborizations (Fig. 1g). The response to TGOT was indistinguishable between the two observed PV+ subtypes: the mean depolarization was 10.6±0.9 mV in bistratified neurons (5/9 reconstructed cells) and 9.8±1.6 mV in periso matic targeting neurons (4/9 reconstructed cells)(p = 0.64, unpaired t-test).

In the prefrontal cortex, a specific class of somatostatin-expressing (SST+) interneurons express the OXTR and are implicated in social-sexual behavior (Nakajima et al., 2014). To test for the recruitment of hippocampal SST+ interneurons by OXTR stimulation, we recorded from fluorescently labeled SST-expressing CA2 interneurons in a transgenic mouse line (SST-Cre x Ai9). The TGOT responses were ge nerally small in this group (2.5 ± 0.8 mV, n=9), though 3 of 9 SST+ cells did display bur st firing (Fig. 1 3 Supp. 1). The magnitude of TGOT-induced depolarization was variable even within a defined SST+ subclass: anatomically confirmed OLM (oriens-lacunosum moleculare) interneurons (Fig. 1 3 Supp. 1). 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183

In contrast to the PV+ interneuron response, CA2 pyramidal cell responses were highly variable and often long outlasted the stimulus (Fig. 1). We next considered what mechanisms might underlie the cell type-specific persistence of this response to OXTR stimulation. Because TGOT responses in CA2 pyramidal cells are long-lasting even in the presence of excitatory synaptic blockers (Tirko et al., 2018), we focused on intracellular, not synaptic, signaling mechanisms. We first asked whether or not bursting activity was perpetuated via a <latch= mechanism, whereby once the cell started spiking, it entered a self-perpetuating bursting state. To test this, we forced CA2 pyramidal cells to burst repetitively by injecting ramps of depolarizing current (reaching ~300 pA over 6 s; Fig. 2a). This stimulus caused CA2 PYRs to fire ~35 action potentials, well within the range of what is observed upon OXTR stimulation. Despite multiple forced burst events, the CA2 PYR membrane potential (Vm) remained unchanged without active current injection (pre vs post: -64 ± 1.5 mV vs. -65.2 ± 1.4 mV, p = 0.44, paired t-test; Fig. 2a). Self-perpetuating burst firing was never observed.

In a complementary series of experiments, we asked whether the persistent response was dependent on a continually depolarized membrane potential. After inducing burst firing and depolarization by TGOT application, we held the cell at a hyperpolarized membrane potential (hyperpolarizing the cell by ~8 mV, to near baseline potential, for 200 s; Fig. 2b). To our surprise, this sustained hyperpolarization was unable to trigger a return to the cell9s baseline state (pre vs post: -58.3 ± 1.2 mV vs. -59 ± 1.3 mV, p = 0.32, paired t-test). After cessation of the hyperpolarizing current, cells went right back to a depolarized membrane potential and burst firing (Fig. 2b). Thus, depolarizing the cell was not enough to induce continual burst firing (Fig. 2a) and hyperpolarizing the cell during TGOT-induced bursting was not sufficient to stop previously triggered activity. 187 188 persistent activity. In response to OXTR stimulation, CA2 PYR cell input resistance 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 increased and remained elevated for the duration of the voltage response (Fig. 2c, Fig. 2 3 Supp. 1). Previously, we pharmacologically demonstrated that OXTR stimulation increased input resistance due to closure of the M-channel (Tirko et al., 2018). This observation led us to suspect that sustained channel inhibition might be responsible for the long-lasting depolarization and burst firing, even though swifter recovery from inhibition has been found in other neurons, like sympathetic ganglion cells (Suh & Hille, 2002). To test explicitly for sustained IM inhibition, we applied the M-current opener, retigabine (100 µM), a few minutes after TGOT removal to determine if this could reverse TGOT-induced excitation (Fig. 2d). Input resistance (Rin), an indirect assay of M-current conductance, was sampled every 10 s with a small hyperpolarizing current step (magnified trace, Fig. 2c, d). Application of retigabine promptly reversed the depolarization, repetitive firing and change in Rin induced by TGOT (mean change in Vm: -5.68±0.7 mV, n = 5; Fig. 2d, e). Simply waiting 10 minutes after TGOT application did not result in a comparable repolarization (mean change in Vm: 0.31±0.9 mV, n = 11, p = 0.74; Fig. 2c). The ability of retigabine to reverse TGOT9s long-lasting effects is consistent with OXTR stimulation causing a sustained inhibition of IM.

M-current inhibition is most often driven by depletion of phosphatidylinositol-4,5biphosphate (PIP2) from the plasma membrane by phospholipase C (PLC, Fig. 2f) as KCNQ channels require PIP2 to open (Suh & Hille, 2002; Zhang et al., 2003). We have previously reported that pre-treatment with U73122, commonly employed as a PLC inhibitor, prevents TGOT-mediated excitation (Tirko et al., 2018). We next tested whether U73122 treatment was also able to reverse OXTR-driven depolarization. Unlike 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 retigabine, U73122 was unable to return CA2 PYRs to their resting Vm or input resistance (mean change in Vm following U73122 treatment: 0.31±0.9 mV, n = 11; p = 0.37). These data are consistent with sustained PLC activation being dispensable for sustained M-current inhibition.

Classically, M-current inhibition is induced following activation of Gaq, which directly interacts with PLC to degrade membrane PIP2. The OXTR is capable of coupling to both Gq and Gi a proteins (Gravati et al., 2010; Hoare et al., 1999; Rimoldi et al., 2003; Strakova & Soloff, 1997; Zhou, Lutz, Steffens, Korth, & Wieland, 2007) , raising the possibility that non-canonical G-protein signaling might account for the prolonged inhibition of the M-channel. To explore this idea, we asked 1) whether other neuromodulatory receptors, which signal though Gaq alone, were sufficient to produce sustained bursting in CA2 cells and 2) what G-proteins are required for TGOT-induced burst firing in CA2 pyramidal cells.

The AVP1bR subtype of vasopressin receptor is expressed in CA2 pyramids and is thought to be Gaq-coupled (Pagani et al., 2015), prompting us to compare the voltage response to oxytocin stimulation to that of vasopressin. Application of arginine vasopressin (AVP, 1 µM), depolarized CA2 PYRs and elicited bursts of action potentials that were similar in mean frequency (TGOT v AVP: 24.3±5.6 vs. 12.2±2.8 Hz; p = 0.20, unpaired t-test) and duration (TGOT v AVP: 14.4±1.6 vs. 18.3±2.4 min; p = 0.34, unpaired t-test) to those elicited by TGOT (Fig. 3 3 Supp. 1). The similarity between AVP and TGOT responses suggested that signaling through Gaq proteins alone, via the 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257

AVP1bR, was capable of producing a long-lasting depolarization. While AVP is capable of signaling through both the OXTR and the AVP1bR (Song & Albers, 2018) , application of AVP is capable of producing sustained depolarization in OXTR KO animals (Fig. 3 3 Supp. 2).

Both TGOT and AVP elicit bursting firing in CA2 pyramidal cells, but only TGOT caused highly variable responses. We hypothesized that some of this variability might be due to the activation of multiple G-proteins downstream of the OXTR. To first test whether the OXTR signaled via Gaq, whose activation directly stimulates PLC, we measured the TGOT response in slices pre-treated with the specific Gaq -family inhibitor FR900359 (FR), (Schrage et al., 2015) . FR pre-treatment (1 µM, 1 h) blocked the TGOT response (control v. drug-treated: 5.6±0.8 mV (TGOT) v. 0.9 ±0.5 mV (TGOT+FR); p = 0.001; unpaired t-test), along with that of a positive control, the depolarization induced by the cholinergic agonist carbachol (control v. drug-treated: CCh, 9.6 ±2.6 mV (CCh) vs. 0.9 ±0.8 mV (TGOT+FR); p = 0.009, unpaired t-test; Fig. 3a-c). Carbachol is known to induce burst firing in CA2 pyramidal cells by signaling through M1 and M3 muscarinic receptors that are classically Gaq-coupled (Robert et al., 2020) . FR responsiveness implies the involvement of a Gaq protein in both the TGOT and CCh response, as would be expected from the literature.

In an independent series of experiments, we pre-treated mice with the specific Gai inhibitor pertussis toxin (PTx; intraventricular injection, 24-72 hours before preparing slices). The long pre-treatment with PTx did not alter the electrical properties of CA2

PYRs or the frequency of synaptic input (Fig. 3 - Supp. 3). PTx pre-treatment did, however, blunt the depolarization usually caused by TGOT (control v. drug-treated: 5.6±0.8 mV (TGOT) v. 1.7±0.5 mV (TGOT+PTx) p = 0.02; unpaired t-test), but not that caused by CCh (control v. drug-treated: 9.6 ±2.6 (CCh) v. 8.4±0.8 (CCh+PTx) mV; p = induced increase in input resistance (Fig. 3 - Supp. 4). This PTx sensitivity was specific to pyramidal cells; CA2 PV+ interneurons treated with PTx still responded to TGOT (mean depolarization: 8.8 ± 1.8 mV, n = 3). Furthermore, the PTx sensitivity did not extend to CCh responsiveness (Fig. 3e), indicating that involvement of Gai is not a general prerequisite for persistent bursting.

Sensitivity to both PTx- and FR-treatment suggest that OXTR-induced burst firing in CA2 PYRs requires the activity of Gq and Gi a proteins. While PTx-sensitive proteins are capable of signaling to certain PLC isoforms via bg-proteins (Camps et al., 1992; Katz, Wu, & Simon, 1992) , their activation is not typically kno wn to cause M-current inhibition. It is equally surprising to find a GPCR whose neuronal signaling requires activation of both Gaq and Gai, although there is precedent for such joint dependence in immune cells (Shi et al., 2007) .

Independent of receptor type or G-protein(s) involved, burst firing of CA2 PYRs appears a widespread phenomenon across modulatory systems. To begin to understand the functional significance of CA2 burst firing, we considered how it might propagate to downstream regions. While CA2 PYRs project to numerous areas of the brain, most axonal fibers converge on the neighboring hippocampal sub-region CA1 (Cui et al., 2013; Hitti & Siegelbaum, 2014). To visualize CA2 PYR projections there, we delivered a Cre-dependent virus encoding ChETA-YFP into the CA2 regions of Amigo2-Cre mice and quantified YFP signal density (Fig. 4a). Consistent with reports from other groups (Dudek et al., 2016; Hitti & Siegelbaum, 2014; Kohara et al., 2014; Tamamaki, Abe, & Nojyo, 1988), we observed that axonal projections were densely concentrated in the interneuron-filled stratum oriens (SO) layer of CA1, with substantial projections to the stratum radiatum (SR) as well. Projections to SO showed significantly more labeling than the stratum pyramidale (SP; 24±2.8 (SO) vs. 8.2±1.2 (SP): p = 3.6e-5; paired ttest). Basal CA1 PYR cell dendrites and local interneurons are positioned to be targeted by CA2 axons in the SO layer and direct synaptic connections between CA2 PYRs and CA1 PYRs have been reported previously (Chevaleyre & Siegelbaum, 2010; Hitti & Siegelbaum, 2014; Kohara et al., 2014). To evaluate the effect of hippocampal oxytocin on spontaneous activity in CA1, we recorded from pyramidal cells in current clamp during TGOT bath application, expecting to see synaptically-propagated EPSPs driven by CA2 PYR burst firing (as in example 1, Fig. 4c). To our surprise, however, we observed no change in EPSP frequency across the set of recordings (average change in EPSP frequency: 1±1.3 Hz; p = 0.44, one-sample t-test; Fig. 4c,d). This absence of TGOT-stimulated CA2 PYR drive onto excitatory CA1 cells was independent of distance from CA2 and CA1, and whether or not the CA1 PYR was in the deep or superficial pyramidal layer (data not shown). To understand why we did not observe significant synaptic excitation onto CA1 PYRs during TGOT presentation, we revisited CA2 PYR cell anatomy. As CA2 PYR axons most strongly innervate regions rich in interneurons (Fig. 4a, b), we sought to determine the relative strength of CA2 PYR cell synapses onto 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 excitatory and inhibitory cells in the CA1 SO. In these experiments, we made serial recordings from neighboring CA1 PYR and SO interneuron <pairs=, while optogenetically stimulating CA2 PYR cell fibers and keeping the intensity of light stimulation the same. Consistently, SO-resident interneurons received significantly stronger CA2 input than nearby PYRs (average EPSP amplitude, SO v. PYR: 9.8 ±1.9 vs. 2.2 ±0.4 mV; p = 0.002, paired t-test, n = 10, Fig. 4e, f). Most interneurons fired action potentials in response to a single pulse of blue light, whereas CA1 PYRs were never brought to spike threshold with the same stimulus. As dCA2 projections to vCA1 have been shown to be critical for CA29s role in social recognition (Meira et al., 2018; Raam et al., 2017) , we also tested if this phenomenon would hold in ventral hippocampus, where, indeed, we observed a similar trend (average EPSP amplitude, SO v. PYR: 3.1±1.5 vs. 0.6±0.4 mV; p = 0.17, n = 3, paired t-test). We also considered the possibility that the subset of CA2 PYRs that express the OXTR (OXTR+) might show a different pattern of SO innervation than the cell population as a whole. To test this explicitly, we injected floxed ChETA-YFP into the CA2 sub-region of OXTR-ires-Cre animals and again recorded in CA1 <pairs= while optogenetically stimulating CA2 PYR cell fibers. As CA2 interneurons also express the OXTR and are likely to express virally delivered channelrhodopsin in experiments in the OXTR-ires-Cre line, we clamped the CA1 PYR cell voltage to -70 mV to isolate excitatory currents. Similar to what we observed when stimulating CA2 broadly, there was a trend for CA1 SO interneurons to receive stronger CA2 input than nearby PYRs (average EPSC magnitude, SO v. PYR: 207.1±77.8 vs. 74.9 ±24.2 pA, p = 0.11, n = 5, paired t-test). 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352

Whenever we stimulated CA2 PYRs and recorded in CA1 SO interneurons, we observed a bimodal distribution in the interneuron population response. The majority of interneurons received a strong CA2 PYR cell input, while a minority displayed EPSPs on par with CA1 pyramids. To test if this bimodality was caused by differential targeting of interneuron subtypes by CA2 PYRs, we characterized SO interneurons on the basis of electrophysiological (input resistance and sag ratio as a proxy for Ih current) properties and axonal projection anatomy (Fig. 4 - Supp. 1). In this analysis, we identified multiple interneuron subclasses in our data set. In general, strongly targeted interneurons, (EPSP >5 mV) were characterized by significantly lower input resistance (Rin, strongly v. weakly targeted: 77.7±14.7 v. 165±29.3 M'; p = 0.009, unpaired t-test) and less Ih (Ratio of sag current to steady state, evoked by a hyperpolarizing pulse, strongly v. weakly targeted: 0.13±0.05 v. 0.33±0.08, p = 0.04, unpaired t-test) than those that were weakly targeted (Fig. 4 3 Supp. 1). The physiological properties of strongly targeted interneurons are consistent with features of fast-spiking PV+ interneurons, which may have perisomatic or bistratified axonal projections. Posthoc reconstructions of SO interneurons revealed that the interneurons receiving strong CA2 input classified as perisomatic, but not bistratified (Fig. 4 3 Supp. 1). At least one additional class of interneurons, characterized by strong adaptation and large AHP, was also strongly innervated by CA2 PYRs.

We next asked how strong targeting of CA1 interneurons by CA2 PYRs might locally regulate evoked activity in CA1. First, we considered how acute stimulation of CA2 PYRs influences spike transmission between CA3 and CA1 PYRs, evoked via stimulation of Schaffer Collaterals (SC). To do so, we optogenetically mimicked CA2 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 burst firing by delivering light pulses at 20 Hz for 1 s to ChETA-bearing CA2 fibers in CA1 while simultaneously stimulating the SC (Fig. 5 3 Supp. 1). Baseline spike probability in response to SC stimulation was established over 20 trials, before interleaving every other stimulus with delivery of the blue light. This burst-like stimulation of CA2 PYR fibers had no effect on CA3-CA1 spike transmission or EPSP amplitude (Fig. 5 3 Supp 1), prompting us to ask if optogenetic release of oxytocin, which produces much more persistent burst activity in CA2 (Tirko et al., 2018), can influence CA3-CA1 transmission. Accordingly, we next subjected oxytocinergic fibers, which virally expressed ChETA-YFP and course through the hippocampus, to optogenetic stimulation with blue light pulses (30 Hz for 60 s). After obtaining a 5-minute baseline recording of SC-evoked EPSPs, we stimulated oxytocinergic fibers and observed a sustained, 2-fold increase in SC-evoked EPSP amplitude (post/pre: 2.04±0.4; p = 0.03, one-sample t-test; Fig. 5a,b). This potentiation was not seen when slices were pre-treated with the OXTR antagonist OTA (post/pre: 1.15±0.2; p = 0.392, one-sample t-test; Fig. 5a, b) or the GABA-A receptor blocker bicuculine (post/pre: 1.4±0.4; p = 0.393, one-sample t-test). As an indication of cell health and recording stability, we continuously monitored input resistance, which remained stable throughout the recording period (Fig. 5 3 Supp. 2). The enhancement of evoked CA3-CA1 transmission was not simply due to direct synaptic modulation by oxytocin, insofar as TGOT did not affect the amplitude or dynamics of SC-evoked synaptic currents in CA1 pyramidal cells (Fig. 5 3 Supp. 3). Consistent with a role for interneurons in this phenomenon, we observed a trend for the compound IPSP to enlarge upon oxytocinergic stimulation (Change in net IPSP: 1.07±0.5 mV; p = 0.07, one-sample ttest) that was not observed following OTA pre-treatment (0.59 ±0.5 mV; p = 0.36, onesample t-test; Fig. 5c). Also consistent with activation of interneurons, the SC-evoked EPSP narrowed following optogenetic stimulation (change in EPSP width: -0.99 ±0.4 mV; p = 0.04, one-sample t-test), while OTA pre-treated slices actually showed a broadening of the PSP waveform (0.77±0.19 mV; p = 0.03, one-sample t-test; Fig. 5d). pyramidal cells. Application of oxytocin (TGOT, 400 nM) and vasopressin (AVP, 1 µM) receptor agonists elicits depolarization and burst firing in CA2 PYRs (a, b). Average burst firing rate (c) and duration (d) are comparable between AVP (n = 7 cells / 4 mice) and TGOT (n = 18 cells / 12 mice). Related to Figure 3. pyramidal cells independent of the OXTR. Example recording from a CA2 PYR upon vasopressin (AVP) application in an OXTR KO animal (a). Accompanying input resistance measurements (b). Related to Figure 3. 626 627 628 629 630 631 632 633 pertussis toxin (PTx) or FR900359 (FR) prevents

Input resistance measurements from CA2 PYRs upon TGOT application in control conditions (a), or given pertussis toxin (PTx) pre-treatment (b), or FR900359 (FR) pre-treatment (c). Related to figure

Figure 5 - Supplemental 2. Cellular input resistance is stable during optogenetic recordings. (a) Schaffer collateral (SC) 3 evoked EPSP amplitude in an example CA1 PYR throughout the recording session. Input resistance, which is sampled throughout the recording, is shown below.

The magenta triangle indicates the time of blue light stimulation. (b) Quantification of input resistance before and after optogenetic stimulation. Measurements of EPSP amplitude and input resistance in an example CA1 PYR pre-treated with the OXTR antagonist OTA (1 µM). (d) Group data from OTA-treated cells. P-values are the result of paired ttests. Related to figure 5.

Figure 5 - Supplemental 3. TGOT does not affect directly affect CA3-CA1 transmission. (a) Schaffer collateral evoked EPSC amplitude and paired-pulse ratio (b), before, during and 5 minutes after TGOT treatment. Inset shows data from an example cell. (c) Evoked feed-forward IPSCs and paired-pulse ratio (d) recorded in CA1 PYRs upon SC stimulation. (e) Spontaneous excitatory currents recorded in CA1 pyramidal cells (top) and PV+ interneurons (bottom) under control conditions and with TGOT present (f). Quantification of EPSC frequency across treatment conditions (g, h). P-values are the rMeestuhltoodfsa paired t-test comparing pre and post conditions. PYR data from 4 cells / 2 mice. PV data from 3 cells / 2 mice. Related to Figure 5.

Experimental model All procedures involving animals were approved by the Institutional Animal Care and Use Committee at the New York University Langone Medical Center (NYULMC), and in accordance with guidelines from the National Institutes of Health. Animals were housed in fully equipped facilities in the Science Building, which is operated by NYULMC9s Division of Comparative Medicine. Male and female mice, post-natal days 50 - 90, were used in all experiments. No physiological differences were observed between sexes and data was pooled. Non-transgenic littermates were used as controls in experiments involving transgenic mouse lines. Homozygous Oxytocin-ires-Cre (Jackson Labs; Stock No. 024234), hemizygote Amigo2-Cre mice (Jackson Labs; Stock No. 030215) and homozygous OXTR-ires-Cre (provided by Dr. Katsuhiko Nishimori (Tohoku University)) were used for optogenetic studies. Targeted interneuron recordings were made from the offspring of crosses between either PV-Cre (Jackson Labs; Stock No. 008069) or SSTires-Cre mice (Jackson Labs; Stock No. 013044) with Ai9 mice (Jackson Labs; Stock No. 007909).

We are grateful to Dr. Steven Siegelbaum for generously providing the Amigo2-cre mice. We thank Vincent Robert for expert technical advice and Guoling Tian for excellent technical assistance.We thank Tsien lab members and Drs. Jayeeta Basu, Brian Kobilka, Bertil Hille and Mark Shapiro for helpful discussions.

The authors declare no competing interests. 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877

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