Vasopressinergic action in the olfactory bulb: Substrates and impact on social odor processing

Social discrimination is a behavioral readout from rats. In the experimental paradigm, rats recognize a rat that they previously interacted with so that they show a preference towards a novel rat over the known one. Since the olfactory bulb is necessary for social discrimination, it is believed to be olfactory discrimination. The intrinsic vasopressin system in the olfactory bulb in the context of social discrimination has been demonstrated with behavioral pharmacology and the discovery of vasopressin expressing cells in the olfactory bulb using a transgenic rat expressing eGFP under the vasopressin promotor. Although it is suggested that bulbar vasopressin cells are a subpopulation of external/superficial tufted cells and vasopressin application reduces firing rates in mitral cells, detailed electrophysiological and morphological properties as well as synaptic inputs to and synaptic outputs from vasopressin cells were undetermined yet. Chapter 2 demonstrates electrophysiological and morphological properties of vasopressin cells. My data contributes to revealing unique electrophysiological properties in vasopressin cells. Accordingly, vasopressin cells had slower membrane time constants and higher input resistances compared to other glutamatergic bulbar neurons. Further, even though vasopressin cells were initially categorized as a subpopulation of external tufted cells, firing patterns upon somatic current injections showed that vasopressin cells are dissimilar to classical external tufted cells. Biocytin filling during recordings enabled reconstruction neurite projections of vasopressin cells. Biocytin labeling combined with immunohistochemical visualization of neurophysin 2, a peptide co-expressed with vasopressin, demonstrated vasopressin expression in not only the soma but also their apical and lateral dendrites. Moreover, I found two subtypes of innervations depending on the number of axons crossing the mitral cell layer (MCL). Accordingly, type 1 cells with multiple axons crossing the MCL had denser arborization in the superficial layers and shorter branch lengths than type 2 cells that had a single axon crossing the MCL. Most neuron types in the olfactory bulb including both excitatory and inhibitory neurons are excitable following electrical olfactory nerve stimulation in acute brain slices. Intriguingly, vasopressin cells were highly inhibited following olfactory nerve stimulation. Further, vasopressin bath application reduced amplitudes of olfactory nerve-evoked inhibitory postsynaptic potentials (IPSPs). However, vasopressin application did not alter the amplitudes of excitatory components in the existence of the GABAA receptor antagonist indicating less inhibition rather than enhanced excitation. These results indicate that olfactory cues alone, i.e., olfactory nerve inputs, are not able to excite vasopressin cells. Therefore, additional inputs than olfactory cues may be required for vasopressin cell excitation. In chapter 3, inputs that are responsible for activating vasopressin cells were further investigated. First, it was revealed that bulbar vasopressin cells are excitable under the in-vivo condition, although no excitation in vasopressin cells was observed in-vitro. Furthermore, vasopressin cells are more activated during social interaction with a novel juvenile rat than during investigating cotton with water in the experiment using immunohistochemical visualization of eGFP and phosphorylated extracellular signal-regulated kinase (pERK), as a neural activity marker. Second, centrifugal neuromodulators, i.e., serotonin, noradrenaline, and acetylcholine, were examined if they can alter olfactory nerve-evoked inhibition in vasopressin cells in-vitro. Indeed, serotonin and noradrenaline reduced amplitudes of evoked IPSPs. However, acetylcholine showed more prominent effects as inhibition was reversed into excitation, i.e., excitatory postsynaptic potentials or action potentials, in 75 % of examined vasopressin cells. I further showed that the muscarinic cholinergic pathway is mainly responsible for effects on vasopressin cells. Lastly, behavioral pharmacology was performed. Local microinjection of atropine, a muscarinic receptor antagonist, into the olfactory bulb impaired social discrimination and additional microinjection of vasopressin after atropine rescued the impairment. Behavioral results indicate that the impairment by atropine is likely due to the blockade of vasopressin release. In conclusion, I suggest that olfactory inputs and acetylcholine synergistically excite vasopressin cells and presumable vasopressin release from them is involved in social discrimination