Rapid presynaptic maturation in naturally regenerating axons of the adult mouse olfactory nerve.

Successful neuronal regeneration requires the reestablishment of synaptic connectivity. This process requires the reconstitution of presynaptic neurotransmitter release, which we investigate here in a model of entirely natural regeneration. After toxin-induced injury, olfactory sensory neurons in the adult mouse olfactory epithelium can regenerate fully, sending axons via the olfactory nerve to reestablish synaptic contact with postsynaptic partners in the olfactory bulb. Using electrophysiological recordings in acute slices, we find that, after initial recontact, functional connectivity in this system is rapidly established. Reconnecting presynaptic terminals have almost mature functional properties, including high release probability and strong capacity for presynaptic inhibition. Release probability then matures quickly, rendering reestablished terminals functionally indistinguishable from controls just 1 week after initial contact. These data show that successful synaptic regeneration in the adult mammalian brain is almost a "plug-and-play" process, with presynaptic terminals undergoing a rapid phase of functional maturation as they reintegrate into target networks.

Developing and maintaining a nose-to-brain map of odorant identity.

Olfactory sensory neurons (OSNs) in the olfactory epithelium of the nose transduce chemical odorant stimuli into electrical signals. These signals are then sent to the OSNs' target structure in the brain, the main olfactory bulb (OB), which performs the initial stages of sensory processing in olfaction. The projection of OSNs to the OB is highly organized in a chemospatial map, whereby axon terminals from OSNs expressing the same odorant receptor (OR) coalesce into individual spherical structures known as glomeruli. This nose-to-brain map of odorant identity is built from late embryonic development to early postnatal life, through a complex combination of genetically encoded, OR-dependent and activity-dependent mechanisms. It must then be actively maintained throughout adulthood as OSNs experience turnover due to external insult and ongoing neurogenesis. Our review describes and discusses these two distinct and crucial processes in olfaction, focusing on the known mechanisms that first establish and then maintain chemospatial order in the mammalian OSN-to-OB projection.

Brief sensory deprivation triggers plasticity of dopamine-synthesising enzyme expression in genetically labelled olfactory bulb dopaminergic neurons.

In the glomerular layer of the olfactory bulb, local dopaminergic interneurons play a key role in regulating the flow of sensory information from nose to cortex. These dual dopamine- and GABA-releasing cells are capable of marked experience-dependent changes in the expression of neurotransmitter-synthesising enzymes, including tyrosine hydroxylase (TH). However, such plasticity has most commonly been studied in cell populations identified by their expression of the enzyme being studied and after long periods of sensory deprivation. Here, instead, we used brief 1- or 3-day manipulations of olfactory experience in juvenile mice, coupled with a conditional genetic approach that labelled neurons contingent upon their expression of the dopamine transporter (DAT-tdTomato). This enabled us to evaluate the potential for rapid changes in neurotransmitter-synthesising enzyme expression in an independently identified neuronal population. Our labelling strategy showed good specificity for olfactory bulb dopaminergic neurons, while revealing a minority sub-population of non-dopaminergic DAT-tdTomato cells that expressed the calcium-binding protein calretinin. Crucially, the proportions of these neuronal subtypes were not affected by brief alterations in sensory experience. Short-term olfactory manipulations also produced no significant changes in immunofluorescence or whole-bulb mRNA for the GABA-synthesising enzyme GAD67/Gad1. However, in bulbar DAT-tdTomato neurons, brief sensory deprivation was accompanied by a transient, small drop in immunofluorescence for the dopamine-synthesising enzyme dopa decarboxylase (DDC) and a sustained decrease for TH. Deprivation also produced a sustained decrease in whole-bulb Th mRNA. Careful characterisation of an independently identified, genetically labelled neuronal population therefore enabled us to uncover rapid experience-dependent changes in dopamine-synthesising enzyme expression.

Development of the mammalian main olfactory bulb.

The mammalian main olfactory bulb is a crucial processing centre for the sense of smell. The olfactory bulb forms early during development and is functional from birth. However, the olfactory system continues to mature and change throughout life as a target of constitutive adult neurogenesis. Our Review synthesises current knowledge of prenatal, postnatal and adult olfactory bulb development, focusing on the maturation, morphology, functions and interactions of its diverse constituent glutamatergic and GABAergic cell types. We highlight not only the great advances in the understanding of olfactory bulb development made in recent years, but also the gaps in our present knowledge that most urgently require addressing.

Brief Sensory Deprivation Triggers Cell Type-Specific Structural and Functional Plasticity in Olfactory Bulb Neurons.

Can alterations in experience trigger different plastic modifications in neuronal structure and function, and if so, how do they integrate at the cellular level? To address this question, we interrogated circuitry in the mouse olfactory bulb responsible for the earliest steps in odor processing. We induced experience-dependent plasticity in mice of either sex by blocking one nostril for one day, a minimally invasive manipulation that leaves the sensory organ undamaged and is akin to the natural transient blockage suffered during common mild rhinal infections. We found that such brief sensory deprivation produced structural and functional plasticity in one highly specialized bulbar cell type: axon-bearing dopaminergic neurons in the glomerular layer. After 24 h naris occlusion, the axon initial segment (AIS) in bulbar dopaminergic neurons became significantly shorter, a structural modification that was also associated with a decrease in intrinsic excitability. These effects were specific to the AIS-positive dopaminergic subpopulation because no experience-dependent alterations in intrinsic excitability were observed in AIS-negative dopaminergic cells. Moreover, 24 h naris occlusion produced no structural changes at the AIS of bulbar excitatory neurons, mitral/tufted and external tufted cells, nor did it alter their intrinsic excitability. By targeting excitability in one specialized dopaminergic subpopulation, experience-dependent plasticity in early olfactory networks might act to fine-tune sensory processing in the face of continually fluctuating inputs.SIGNIFICANCE STATEMENT Sensory networks need to be plastic so they can adapt to changes in incoming stimuli. To see how cells in mouse olfactory circuits can change in response to sensory challenges, we blocked a nostril for just one day, a naturally relevant manipulation akin to the deprivation that occurs with a mild cold. We found that this brief deprivation induces forms of axonal and intrinsic functional plasticity in one specific olfactory bulb cell subtype: axon-bearing dopaminergic interneurons. In contrast, intrinsic properties of axon-lacking bulbar dopaminergic neurons and neighboring excitatory neurons remained unchanged. Within the same sensory circuits, specific cell types can therefore make distinct plastic changes in response to an ever-changing external landscape.

Protocol for Patch-Seq of Small Interneurons.

Obtaining electrophysiological recordings and gene expression information from the same neuron (Patch-seq) brings forward a unique opportunity to study the transcriptional correlates of functional properties and vice versa. Here, we provide a detailed Patch-seq protocol tailored to the specialized demands of studying small interneurons. Focusing on the technically demanding process of transitioning between patch recordings and cell extraction, our protocol describes and troubleshoots steps for successfully collecting small interneurons, allowing for multi-modal Patch-seq interrogation of this crucial cell type.

Non-neuronal expression of SARS-CoV-2 entry genes in the olfactory system suggests mechanisms underlying COVID-19-associated anosmia.

Altered olfactory function is a common symptom of COVID-19, but its etiology is unknown. A key question is whether SARS-CoV-2 (CoV-2) - the causal agent in COVID-19 - affects olfaction directly, by infecting olfactory sensory neurons or their targets in the olfactory bulb, or indirectly, through perturbation of supporting cells. Here we identify cell types in the olfactory epithelium and olfactory bulb that express SARS-CoV-2 cell entry molecules. Bulk sequencing demonstrated that mouse, non-human primate and human olfactory mucosa expresses two key genes involved in CoV-2 entry, ACE2 and TMPRSS2. However, single cell sequencing revealed that ACE2 is expressed in support cells, stem cells, and perivascular cells, rather than in neurons. Immunostaining confirmed these results and revealed pervasive expression of ACE2 protein in dorsally-located olfactory epithelial sustentacular cells and olfactory bulb pericytes in the mouse. These findings suggest that CoV-2 infection of non-neuronal cell types leads to anosmia and related disturbances in odor perception in COVID-19 patients.

Embryonic and postnatal neurogenesis produce functionally distinct subclasses of dopaminergic neuron.

Most neurogenesis in the mammalian brain is completed embryonically, but in certain areas the production of neurons continues throughout postnatal life. The functional properties of mature postnatally generated neurons often match those of their embryonically produced counterparts. However, we show here that in the olfactory bulb (OB), embryonic and postnatal neurogenesis produce functionally distinct subpopulations of dopaminergic (DA) neurons. We define two subclasses of OB DA neuron by the presence or absence of a key subcellular specialisation: the axon initial segment (AIS). Large AIS-positive axon-bearing DA neurons are exclusively produced during early embryonic stages, leaving small anaxonic AIS-negative cells as the only DA subtype generated via adult neurogenesis. These populations are functionally distinct: large DA cells are more excitable, yet display weaker and - for certain long-latency or inhibitory events - more broadly tuned responses to odorant stimuli. Embryonic and postnatal neurogenesis can therefore generate distinct neuronal subclasses, placing important constraints on the functional roles of adult-born neurons in sensory processing.

Myosin II activity is required for structural plasticity at the axon initial segment.

In neurons, axons possess a molecularly defined and highly organised proximal region - the axon initial segment (AIS) - that is a key regulator of both electrical excitability and cellular polarity. Despite existing as a large, dense structure with specialised cytoskeletal architecture, the AIS is surprisingly plastic, with sustained alterations in neuronal activity bringing about significant alterations to its position, length or molecular composition. However, although the upstream activity-dependent signalling pathways that lead to such plasticity have begun to be elucidated, the downstream mechanisms that produce structural changes at the AIS are completely unknown. Here, we use dissociated cultures of rat hippocampus to show that two forms of AIS plasticity in dentate granule cells - long-term relocation, and more rapid shortening - are completely blocked by treatment with blebbistatin, a potent and selective myosin II ATPase inhibitor. These data establish a link between myosin II and AIS function, and suggest that myosin II's primary role at the structure may be to effect activity-dependent morphological alterations.

Evaluating Tools for Live Imaging of Structural Plasticity at the Axon Initial Segment.

The axon initial segment (AIS) is a specialized neuronal compartment involved in the maintenance of axo-dendritic polarity and in the generation of action potentials. It is also a site of significant structural plasticity-manipulations of neuronal activity in vitro and in vivo can produce changes in AIS position and/or size that are associated with alterations in intrinsic excitability. However, to date all activity-dependent AIS changes have been observed in experiments carried out on fixed samples, offering only a snapshot, population-wide view of this form of plasticity. To extend these findings by following morphological changes at the AIS of individual neurons requires reliable means of labeling the structure in live preparations. Here, we assessed five different immunofluorescence-based and genetically-encoded tools for live-labeling the AIS of dentate granule cells (DGCs) in dissociated hippocampal cultures. We found that an antibody targeting the extracellular domain of neurofascin provided accurate live label of AIS structure at baseline, but could not follow rapid activity-dependent changes in AIS length. Three different fusion constructs of GFP with full-length AIS proteins also proved unsuitable: while neurofascin-186-GFP and NaVß4-GFP did not localize to the AIS in our experimental conditions, overexpressing 270kDa-AnkyrinG-GFP produced abnormally elongated AISs in mature neurons. In contrast, a genetically-encoded construct consisting of a voltage-gated sodium channel intracellular domain fused to yellow fluorescent protein (YFP-NaVII-III) fulfilled all of our criteria for successful live AIS label: this construct specifically localized to the AIS, accurately revealed plastic changes at the structure within hours, and, crucially, did not alter normal cell firing properties. We therefore recommend this probe for future studies of live AIS plasticity in vitro and in vivo.

A role for CaV1 and calcineurin signaling in depolarization-induced changes in neuronal DNA methylation.

Direct manipulations of neuronal activity have been shown to induce changes in DNA methylation (DNAm), although little is known about the cellular signaling pathways involved. Using reduced representation bisulfite sequencing, we identify DNAm changes associated with moderate chronic depolarization in dissociated rat hippocampal cultures. Consistent with previous findings, these changes occurred primarily in the vicinity of loci implicated in neuronal function, being enriched in intergenic regions and underrepresented in CpG-rich promoter regulatory regions. We subsequently used 2 pharmacological interventions (nifedipine and FK-506) to test whether the identified changes depended on 2 interrelated signaling pathways known to mediate multiple forms of neuronal plasticity. Both pharmacological manipulations had notable effects on the extent and magnitude of depolarization-induced DNAm changes indicating that a high proportion of activity-induced changes are likely to be mediated by calcium entry through L-type CaV1 channels and/or downstream signaling via the calcium-dependent phosphatase calcineurin.

Rapid Modulation of Axon Initial Segment Length Influences Repetitive Spike Firing.

Neurons implement a variety of plasticity mechanisms to alter their function over timescales ranging from seconds to days. One powerful means of controlling excitability is to directly modulate the site of spike initiation, the axon initial segment (AIS). However, all plastic structural AIS changes reported thus far have been slow, involving days of neuronal activity perturbation. Here, we show that AIS plasticity can be induced much more rapidly. Just 3 hr of elevated activity significantly shortened the AIS of dentate granule cells in a calcineurin-dependent manner. The functional effects of rapid AIS shortening were offset by dephosphorylation of voltage-gated sodium channels, another calcineurin-dependent mechanism. However, pharmacological separation of these phenomena revealed a significant relationship between AIS length and repetitive firing. The AIS can therefore undergo a rapid form of structural change over timescales that enable interactions with other forms of activity-dependent plasticity in the dynamic control of neuronal excitability.

A distinct subtype of dopaminergic interneuron displays inverted structural plasticity at the axon initial segment.

The axon initial segment (AIS) is a specialized structure near the start of the axon that is a site of neuronal plasticity. Changes in activity levels in vitro and in vivo can produce structural AIS changes in excitatory cells that have been linked to alterations in excitability, but these effects have never been described in inhibitory interneurons. In the mammalian olfactory bulb (OB), dopaminergic interneurons are particularly plastic, undergoing constitutive turnover throughout life and regulating tyrosine hydroxylase expression in an activity-dependent manner. Here we used dissociated cultures of rat and mouse OB to show that a subset of bulbar dopaminergic neurons possess an AIS and that these AIS-positive cells are morphologically and functionally distinct from their AIS-negative counterparts. Under baseline conditions, OB dopaminergic AISs were short and located distally along the axon but, in response to chronic 24 h depolarization, lengthened and relocated proximally toward the soma. These activity-dependent changes were in the opposite direction to both those we saw in non-GABAergic OB neurons and those reported previously for excitatory cell types. Inverted AIS plasticity in OB dopaminergic cells was bidirectional, involved all major components of the structure, was dependent on the activity of L-type CaV1 calcium channels but not on the activity of the calcium-activated phosphatase calcineurin, and was opposed by the actions of cyclin-dependent kinase 5. Such distinct forms of AIS plasticity in inhibitory interneurons and excitatory projection neurons may allow considerable flexibility when neuronal networks must adapt to perturbations in their ongoing activity.

Calcineurin signaling mediates activity-dependent relocation of the axon initial segment.

The axon initial segment (AIS) is a specialized neuronal subcompartment located at the beginning of the axon that is crucially involved in both the generation of action potentials and the regulation of neuronal polarity. We recently showed that prolonged neuronal depolarization produces a distal shift of the entire AIS structure away from the cell body, a change associated with a decrease in neuronal excitability. Here, we used dissociated rat hippocampal cultures, with a major focus on the dentate granule cell (DGC) population, to explore the signaling pathways underlying activity-dependent relocation of the AIS. First, a pharmacological screen of voltage-gated calcium channels (VGCCs) showed that AIS relocation is triggered by activation of L-type Cav1 VGCCs with negligible contribution from any other VGCC subtypes. Additional pharmacological analysis revealed that downstream signaling events are mediated by the calcium-sensitive phosphatase calcineurin; inhibition of calcineurin with either FK506 or cyclosporin A totally abolished both depolarization- and optogenetically-induced activity-dependent AIS relocation. Furthermore, calcineurin activation is sufficient for AIS plasticity, because expression of a constitutively active form of the phosphatase resulted in relocation of the AIS of DGCs without a depolarizing stimulus. Finally, we assessed the role of calcineurin in other forms of depolarization-induced plasticity. Neither membrane resistance changes nor spine density changes were affected by FK506 treatment, suggesting that calcineurin acts via a separate pathway to modulate AIS plasticity. Together, these results emphasize calcineurin as a vital player in the regulation of intrinsic plasticity as governed by the AIS.

Short- and long-term plasticity at the axon initial segment.

The axon initial segment (AIS) is a highly specialized neuronal subregion that is the site of action potential initiation and the boundary between axonal and somatodendritic compartments. In recent years, our understanding of the molecular structure of the AIS, its maturation, and its multiple fundamental roles in neuronal function has seen major advances. We are beginning to appreciate that the AIS is dynamically regulated, both over short timescales via adaptations in ion channel function, and long timescales via activity-dependent structural reorganization. Here, we review results from this emerging field highlighting how structural and functional plasticity relate to the development of the initial segment, and to neuronal disorders linked to AIS dysfunction.