Differential Serotonergic Modulation of Synaptic Inputs to the Olfactory Cortex

Serotonin (5-hydroxytriptamine, 5-HT) is an important monoaminergic neuromodulator involved in a variety of physiological and pathological functions. It has been implicated in the regulation of sensory functions at various stages of multiple modalities, but its mechanisms and functions in the olfactory system have remained elusive. Combining electrophysiology, optogenetics and pharmacology, here we show that afferent (feed-forward) pathway-evoked synaptic responses are boosted, whereas feedback responses are suppressed by presynaptic 5-HT1B receptors in the anterior piriform cortex (aPC) in vitro. Blocking 5-HT1B receptors also reduces the suppressive effects of serotonergic photostimulation of baseline firing in vivo. We suggest that by regulating the relative weights of synaptic inputs to aPC, 5-HT finely tunes sensory inputs in the olfactory cortex.


Introduction
The ability to adapt to an ever-changing environment is key to survival. Sensory systems constantly extract and process signals from both the external world and internal sources to form representations of the environment. This is ultimately used to fine-tune a dynamic behavioral repertoire according to current or anticipated ecological contexts, which is a process that occurs on various timescales and is influenced by previous experience. Various sensory functions are affected by neuromodulators, such as the monoamines. These crucial molecules are present at the peripheral, intermediate, and central stages of every sensory modality. In addition to the widespread monoaminergic innervation of sensory pathways, monoaminergic neurons receive sensory inputs from various modalities [1], resulting in reciprocal interaction between sensory and neuromodulatory systems.
Located in the brainstem raphé nuclei (RN), serotonergic neurons project to various forebrain areas and release serotonin (5-hydroxytryptamine, 5-HT) throughout the entire neuroaxis. 5-HT is implicated in a variety of physiological functions, including the regulation of sensory and motor responses [2][3][4][5], brain states [6][7][8], learning and reward processing [9][10][11], fear responses [12] and social interactions [13,14]. Dysfunctions of the serotonergic system are implicated in several neurological and psychiatric disorders, including depression [15] and epilepsy [16,17]. RN 5-HT fibers densely innervate the primary olfactory cortex (aPC, anterior piriform cortex) with both the dorsal raphe nucleus (DRN) and median raphe nucleus (MRN) neurons contributing to this projection [18]. The most prominent effect of exogenously applied 5-HT in the aPC is a 5HT 1A receptor-mediated hyperpolarization of principal neurons [19] and a 5HT 2 and 5HT 3 receptor-mediated depolarization of local interneurons [20][21][22][23], which coincides with an increase in inhibitory postsynaptic potentials (IPSPs) in principal neurons [24]. Interestingly, the local photostimulation of 5-HT axons has little effect on their membrane potential [25], but can reduce the excitability of principal neurons in the aPC [25,26] and lead to a 5HT 2A receptor-mediated membrane potential depolarization and a subsequent increase in action potential output in interneurons, including perisomatic inhibitory fast-spiking GABAergic neurons [25]. Importantly, the specific stimulation of DRN 5-HT neurons results in a prominent suppression of spontaneous but not odor-evoked activity of most aPC neurons in vivo [2], but the mechanisms involved in this differential action have remained elusive.
Here, we address this question by combining electrophysiology, optogenetics and pharmacology and show that 5-HT can increase responses evoked by feed-forward inputs originating from the OB but suppress feedback inputs originating from various cortical sources. The suppression of feedback synaptic inputs was due to the modulation of glutamate release via presynaptic 5-HT 1B receptors. By this input-specific effect, 5-HT contributes to the fine tuning of sensory computations in general and to olfactory ones in particular.

Discussion
By performing a combination of in vivo and in vitro electrophysiology, optogenetics and pharmacology, we show that (i) 5-HT can differentially affect synaptic inputs to the aPC by suppressing intracortical and increasing afferent inputs, (ii) the suppression of feedback inputs is most likely due to a 5-HT 1B -dependent decrease in glutamate release, and (iii) the suppression of baseline aPC neuronal activity by the specific stimulation of DRN 5-HT neurons can be blocked by the systemic application of 5-HT 1B receptorantagonists.
These results provide a synaptic mechanism for our previous observations that 5-HT can suppress spontaneous but not odor-evoked activity [2] and complement our observations concerning the direct effect of 5-HT on single aPC principal neurons and interneurons [25]. Thus, 5-HT can directly suppress the activity of aPC principal neurons, increase the activity of aPC GABAergic interneurons and increase feed-forward synaptic inputs originating from the olfactory bulb while suppressing feedback inputs originating from various cortical sources including the aPC.
Similar to acetylcholine and noradrenaline [30,31], 5-HT can suppress feedback synaptic responses originating from cortical sources but not afferent (feed-forward) inputs originating from the OB. This differential effect on various inputs is in line with a synapsespecific effect of 5-HT, which is a general feature of this neuromodulator in various brain regions such as the olfactory tubercle [32], the hippocampus [33], and the nucleus accumbens [34].
Testing the effect of 5-HT on the spontaneous and odor-evoked activity of the aPC has led to contrasting results. The selective stimulation of DRN 5-HT neurons resulted in the divisive suppression of spontaneous, but not odor-evoked, spiking activity of anesthetized mice [2]. However, odor-evoked activity was decreased, and spontaneous activity remained unaltered during similar manipulation of 5-HT neurons when monitoring the population Ca 2+ dynamics of aPC principal neurons using fiber photometry in awake mice [26]. Thus, the inhibition of sensory responses seems to be an important general feature of 5-HT across multiple sensory modalities, whereas its effects on spontaneous activity may vary as a function of brain region and state of vigilance.
This study has shed light on the identity of 5-HT receptors involved in the modulation of feedback inputs to the aPC. Both endogenous and exogenous application of 5-HT decreased FB stimulation induced fEPSPs and EPSCs in single neurons. In addition to the reduction in EPSCs in single aPC neurons, bath-applied 5-HT or the local photostimulation of ChR2-expressing DRN axons in the aPC led to an increase in the paired pulse ratio, suggesting a presynaptic site of action. Presynaptic 5-HT 1B receptors are key players in decreasing the release of glutamate in various brain areas [35][36][37][38][39][40][41]. The mechanisms of 5-HT mediated modulation of feed-forward inputs will need to be revealed by future studies.
The differential effect of 5-HT on feed-forward and feedback inputs can have important functional implications. As the afferent inputs to the aPC originate in the OB, these results argue for a locus-specific effect, where 5-HT can differentially modulate neurons and/or synapses located at various levels of the sensory pathway. 5-HT can thus regulate the relative weights of synaptic inputs to aPC influenced by the multiple synaptic input sources of the DRN [1]. One of the major sources of inhibitory and excitatory inputs to DRN neurons is the lateral hypothalamus [8,42] that can broadcast information related to energy balance and arousal to the olfactory system.
In conclusion, two important dichotomy-based motifs seem to empower the neuromodulatory function of 5-HT, its antagonistic effects on excitatory and inhibitory neurons [25], and its pathway-specific synaptic effects: generally suppressing intracortical synapses and sparing feed-forward inputs from the sensory periphery.

Materials and Methods
All experimental procedures were performed in accordance with the European Union Directive (86/609/EEC) and approved by the local ethical committees. Data are presented as mean ± SEM, unless stated otherwise.
For synaptic stimulation, two concentric bipolar stimulating electrodes (FHC, Germany) were positioned in the lateral olfactory tract (LOT) and layer 2 for afferent and associational fiber stimulation, respectively. A recording pipette filled with ACSF (resistance: 4 MΩ) was then positioned above layer 2. Stimulation consisted of brief (0.1 ms) current pulses (10-100 µA). Afferent and associational stimulation was separated by 0.5 s. After obtaining a baseline of field excitatory postsynaptic potentials (fEPSPs), EPSCs or EPSPs serotonin was applied to the recording chamber. ChR2-expressing axons in the aPC were photostimulated through the microscope objective using the epifluorescent illumination via an LED light source (Thorlabs, Germany). Light intensity was set to 0.5 mW. Photostimulation consisted of a 3 s train of 10 ms pulses at 10 Hz. Control and photostimulation trials were intermingled.
For the selective stimulation of DRN 5-HT neurons in vivo, we used similar protocols to the ones in Lottem et al. (2016). Briefly, SERT-cre mice previously (4-8 weeks) injected with 0.5-1 µL of AAV2/1-Flex-ChR2-YFP (AV-1-20298P, University of Pennsylvania, 10 13 GC/mL) in the DRN were anesthetized with urethane (1.2 g/kg) and mounted in a stereotaxic frame. aPC neurons were recorded with a glass electrode (impedance: 8-20 MOhm) filled with saline and connected to a DC amplifier (Axoclamp 2B, Axon Instruments, San Jose, CA, USA). Electrophysiological data were acquired using a Power 1401 and Spike2 software (Cambridge Electronic Design, Cambridge, UK) and stored on a personal computer for offline analysis. Spike sorting was performed using Spike2 software (Cambridge Electronic Design, UK). An optical fiber (200 µm diameter; numerical aperture 0.38) was inserted above the DRN for photostimulation (a 5 s train of 10 ms pulses at 30 Hz, 5 mW). The 5-HT 2B receptor antagonist GR127935 (3 mg/kg, dissolved in saline) was administered intraperitoneally. Statistical significance was assessed by Student's t-test, and a p value below 0.05 was considered significant.