Evidence for coexpression of two or more classic neurotransmitters in neurons has increased, but less is known about cotransmission. Ventral tegmental area (VTA) neurons corelease dopamine (DA), the excitatory transmitter glutamate, and the inhibitory transmitter GABA onto target cells in the striatum. Olfactory bulb (OB) short axon cells (SACs) form interglomerular connections and coexpress markers for DA and GABA. Using an optogenetic approach, we provide evidence that mouse OB SACs release both GABA and DA onto external tufted cells (ETCs) in other glomeruli. Optical activation of channelrhodopsin specifically expressed in DAergic SACs produced a GABA_A receptor-mediated monosynaptic inhibitory response, followed by DA–D₁-like receptor-mediated excitatory response in ETCs. The GABA_A receptor-mediated hyperpolarization activates I_h current in ETCs; synaptically released DA increases I_h, which enhances postinhibitory rebound spiking. Thus, the opposing actions of synaptically released GABA and DA are functionally integrated by I_h to generate an inhibition-to-excitation “switch” in ETCs. Consistent with the established role of I_h in ETC burst firing, we show that endogenous DA release increases ETC spontaneous bursting frequency. ETCs transmit sensory signals to mitral/tufted output neurons and drive intraglomerular inhibition to shape glomerulus output to downstream olfactory networks. GABA and DA cotransmission from SACs to ETCs may play a key role in regulating output coding across the glomerular array.

External tufted (ET) cells are juxtaglomerular neurons that spontaneously generate bursts of action potentials, which persist when fast synaptic transmission is blocked. The intrinsic mechanism of this autonomous bursting is unknown. We identified a set of voltage-dependent conductances that cooperatively regulate spontaneous bursting: hyperpolarization-activated inward current (I_h), persistent Na⁺current (I_NaP), low-voltage-activated calcium current (I_L/T) mediated by T- and/or L-type Ca²⁺channels, and large-conductance Ca²⁺-dependent K⁺current (I_BK).I_his important in setting membrane potential and depolarizes the cell toward the threshold ofI_NaPandI_T/L, which are essential to generate the depolarizing envelope that is crowned by a burst of action potentials. Action potentials depolarize the membrane and induce Ca²⁺influx via high-voltage-activated Ca²⁺channels (I_HVA). The combined depolarization and increased intracellular Ca²⁺activatesI_BK, which terminates the burst by hyperpolarizing the membrane. Hyperpolarization activatesI_hand the cycle is regenerated. A novel finding is the role of L-type Ca²⁺channels in autonomous ET cells bursting. A second novel feature is the role of BK channels, which regulate burst duration.I_LandI_BKmay go hand-in-hand, the slow inactivation ofI_LrequiringI_BK-dependent hyperpolarization to deactivate inward conductances and terminate the burst. ET cells receive monosynaptic olfactory nerve input and drive the major inhibitory interneurons of the glomerular circuit. Modulation of the conductances identified here can regulate burst frequency, duration, and spikes per burst in ET cells and thus significantly shape the impact of glomerular circuits on mitral and tufted cells, the output channels of the olfactory bulb.

Sensory processing shapes our perception. In mammals, odor information is encoded by combinatorial activity patterns of olfactory bulb (OB) glomeruli. Glomeruli are richly interconnected by short axon cells (SACs), which form the interglomerular circuit (IGC). It is unclear how the IGC impacts OB output to downstream neural circuits. We combinedin vitroandin vivoelectrophysiology with optogenetics in mice and found the following: (1) the IGC potently and monosynaptically inhibits the OB output neurons mitral/tufted cells (MTCs) by GABA release from SACs: (2) gap junction-mediated electrical coupling is strong for the SAC→MTC synapse, but negligible for the SAC→ETC synapse; (3) brief IGC-mediated inhibition is temporally prolonged by the intrinsic properties of MTCs; and (4) sniff frequency IGC activationin vivogenerates persistent MTC inhibition. These findings suggest that the temporal sequence of glomerular activation by sensory input determines which stimulus features are transmitted to downstream olfactory networks and those filtered by lateral inhibition.SIGNIFICANCE STATEMENTOdor identity is encoded by combinatorial patterns of activated glomeruli, the initial signal transformation site of the olfactory system. Lateral circuit processing among activated glomeruli modulates olfactory signal transformation before transmission to higher brain centers. Using a combination ofin vitroandin vivooptogenetics, this work demonstrates that interglomerular circuitry produces potent inhibition of olfactory bulb output neurons via direct chemical and electrical synapses as well as by indirect pathways. The direct inhibitory synaptic input engages mitral cell intrinsic membrane properties to generate inhibition that outlasts the initial synaptic action.

Odor signals are transmitted to the olfactory bulb by olfactory nerve (ON) synapses onto mitral/tufted cells (MCs) and external tufted cells (ETCs). ETCs, in turn, provide feedforward excitatory input to MCs. MC and ETCs are also regulated by inhibition: intraglomerular and interglomerular inhibitory circuits act at MC and ETC apical dendrites; granule cells (GCs) inhibit MC lateral dendrites via the MC→GC→MC circuit. We investigated the contribution of intraglomerular inhibition to MC and ETCs responses to ON input. ON input evokes initial excitation followed by early, strongly summating inhibitory postsynaptic currents (IPSCs) in MCs; this is followed by prolonged, intermittent IPSCs. The N-methyl-d-aspartate receptor antagonist dl-amino-5-phosphovaleric acid, known to suppress GABA release by GCs, reduced late IPSCs but had no effect on early IPSCs. In contrast, selective intraglomerular block of GABA_A receptors eliminated all early IPSCs and caused a 5-fold increase in ON-evoked MC spiking and a 10-fold increase in response duration. ETCs also receive intraglomerular inhibition; blockade of inhibition doubled ETC spike responses. By reducing ETC excitatory drive and directly inhibiting MCs, intraglomerular inhibition is a key factor shaping the strength and temporal structure of MC responses to sensory input. Sensory input generates an intraglomerular excitation-inhibition sequence that limits MC spike output to a brief temporal window. Glomerular circuits may dynamically regulate this input-output window to optimize MC encoding across sniff-sampled inputs.

Serotonergic neurons in the raphe nuclei constitute one of the most prominent neuromodulatory systems in the brain. Projections from the dorsal and median raphe nuclei provide dense serotonergic innervation of the glomeruli of olfactory bulb. Odor information is initially processed by glomeruli, thus serotonergic modulation of glomerular circuits impacts all subsequent odor coding in the olfactory system. The present study discloses that serotonin (5-HT) produces excitatory modulation of external tufted (ET) cells, a pivotal neuron in the operation of glomerular circuits. The modulation is due to a transient receptor potential (TRP) channel-mediated inward current induced by activation of 5-HT_2Areceptors. This current produces membrane depolarization and increased bursting frequency in ET cells. Interestingly, the magnitude of the inward current and increased bursting inversely correlate with ET cell spontaneous (intrinsic) bursting frequency: slower bursting ET cells are more strongly modulated than faster bursting cells. Serotonin thus differentially impacts ET cells such that the mean bursting frequency of the population is increased. This centrifugal modulation could impact odor processing by: 1) increasing ET cell excitatory drive on inhibitory neurons to increase presynaptic inhibition of olfactory sensory inputs and postsynaptic inhibition of mitral/tufted cells; and/or 2) coordinating ET cell bursting with exploratory sniffing frequencies (5–8 Hz) to facilitate odor coding.