Patch-seq: Past, Present, and Future.

Single-cell transcriptomic approaches are revolutionizing neuroscience. Integrating this wealth of data with morphology and physiology, for the comprehensive study of neuronal biology, requires multiplexing gene expression data with complementary techniques. To meet this need, multiple groups in parallel have developed "Patch-seq," a modification of whole-cell patch-clamp protocols that enables mRNA sequencing of cell contents after electrophysiological recordings from individual neurons and morphologic reconstruction of the same cells. In this review, we first outline the critical technical developments that enabled robust Patch-seq experimental efforts and analytical solutions to interpret the rich multimodal data generated. We then review recent applications of Patch-seq that address novel and long-standing questions in neuroscience. These include the following: (1) targeted study of specific neuronal populations based on their anatomic location, functional properties, lineage, or a combination of these factors; (2) the compilation and integration of multimodal cell type atlases; and (3) the investigation of the molecular basis of morphologic and functional diversity. Finally, we highlight potential opportunities for further technical development and lines of research that may benefit from implementing the Patch-seq technique. As a multimodal approach at the intersection of molecular neurobiology and physiology, Patch-seq is uniquely positioned to directly link gene expression to brain function.

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.

Distinct Evolutionary Trajectories of Neuronal and Hair Cell Nicotinic Acetylcholine Receptors.

The expansion and pruning of ion channel families has played a crucial role in the evolution of nervous systems. Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels with distinct roles in synaptic transmission at the neuromuscular junction, the central and peripheral nervous system, and the inner ear. Remarkably, the complement of nAChR subunits has been highly conserved along vertebrate phylogeny. To ask whether the different subtypes of receptors underwent different evolutionary trajectories, we performed a comprehensive analysis of vertebrate nAChRs coding sequences, mouse single-cell expression patterns, and comparative functional properties of receptors from three representative tetrapod species. We found significant differences between hair cell and neuronal receptors that were most likely shaped by the differences in coexpression patterns and coassembly rules of component subunits. Thus, neuronal nAChRs showed high degree of coding sequence conservation, coupled to greater coexpression variance and conservation of functional properties across tetrapod clades. In contrast, hair cell α9α10 nAChRs exhibited greater sequence divergence, narrow coexpression pattern, and great variability of functional properties across species. These results point to differential substrates for random change within the family of gene paralogs that relate to the segregated roles of nAChRs in synaptic transmission.

α2* Nicotinic acetylcholine receptors influence hippocampus-dependent learning and memory in adolescent mice.

The absence of α2* nicotinic acetylcholine receptors (nAChRs) in oriens lacunosum moleculare (OLM) GABAergic interneurons ablate the facilitation of nicotine-induced hippocampal CA1 long-term potentiation and impair memory. The current study delineated whether genetic mutations of α2* nAChRs (Chrna2L9'S/L9'S and Chrna2KO) influence hippocampus-dependent learning and memory and CA1 synaptic plasticity. We substituted a serine for a leucine (L9'S) in the α2 subunit (encoded by the Chrna2 gene) to make a hypersensitive nAChR. Using a dorsal hippocampus-dependent task of preexposure-dependent contextual fear conditioning, adolescent hypersensitive Chrna2L9'S/L9'S male mice exhibited impaired learning and memory. The deficit was rescued by low-dose nicotine exposure. Electrophysiological studies demonstrated that hypersensitive α2 nAChRs potentiate acetylcholine-induced ion channel flux in oocytes and acute nicotine-induced facilitation of dorsal/intermediate CA1 hippocampal long-term potentiation in Chrna2L9'S/L9'S mice. Adolescent male mice null for the α2 nAChR subunit exhibited a baseline deficit in learning that was not reversed by an acute dose of nicotine. These effects were not influenced by locomotor, sensory or anxiety-related measures. Our results demonstrated that α2* nAChRs influenced hippocampus-dependent learning and memory, as well as nicotine-facilitated CA1 hippocampal synaptic plasticity.

Tracking the molecular evolution of calcium permeability in a nicotinic acetylcholine receptor.

Nicotinic acetylcholine receptors are a family of ligand-gated nonselective cationic channels that participate in fundamental physiological processes at both the central and the peripheral nervous system. The extent of calcium entry through ligand-gated ion channels defines their distinct functions. The α9α10 nicotinic cholinergic receptor, expressed in cochlear hair cells, is a peculiar member of the family as it shows differences in the extent of calcium permeability across species. In particular, mammalian α9α10 receptors are among the ligand-gated ion channels which exhibit the highest calcium selectivity. This acquired differential property provides the unique opportunity of studying how protein function was shaped along evolutionary history, by tracking its evolutionary record and experimentally defining the amino acid changes involved. We have applied a molecular evolution approach of ancestral sequence reconstruction, together with molecular dynamics simulations and an evolutionary-based mutagenesis strategy, in order to trace the molecular events that yielded a high calcium permeable nicotinic α9α10 mammalian receptor. Only three specific amino acid substitutions in the α9 subunit were directly involved. These are located at the extracellular vestibule and at the exit of the channel pore and not at the transmembrane region 2 of the protein as previously thought. Moreover, we show that these three critical substitutions only increase calcium permeability in the context of the mammalian but not the avian receptor, stressing the relevance of overall protein structure on defining functional properties. These results highlight the importance of tracking evolutionarily acquired changes in protein sequence underlying fundamental functional properties of ligand-gated ion channels.

Phylogenetic differences in calcium permeability of the auditory hair cell cholinergic nicotinic receptor.

The α9 and α10 cholinergic nicotinic receptor subunits assemble to form the receptor that mediates efferent inhibition of hair cell function within the auditory sensory organ, a mechanism thought to modulate the dynamic range of hearing. In contrast to all nicotinic receptors, which serve excitatory neurotransmission, the activation of α9α10 produces hyperpolarization of hair cells. An evolutionary analysis has shown that the α10 subunit exhibits signatures of positive selection only along the mammalian lineage, strongly suggesting the acquisition of a unique function. To establish whether mammalian α9α10 receptors have acquired distinct functional properties as a consequence of this evolutionary pressure, we compared the properties of rat and chicken recombinant and native α9α10 receptors. Our main finding in the present work is that, in contrast to the high (pCa(2+)/pMonovalents ∼10) Ca(2+) permeability reported for rat α9α10 receptors, recombinant and native chicken α9α10 receptors have a much lower permeability (∼2) to this cation, comparable to that of neuronal α4ß2 receptors. Moreover, we show that, in contrast to α10, α7 as well as α4 and ß2 nicotinic subunits are under purifying selection in vertebrates, consistent with the conserved Ca(2+) permeability reported across species. These results have important consequences for the activation of signaling cascades that lead to hyperpolarization of hair cells after α9α10 gating at the cholinergic-hair cell synapse. In addition, they suggest that high Ca(2+) permeability of the α9α10 cholinergic nicotinic receptor might have evolved together with other features that have given the mammalian ear an expanded high-frequency sensitivity.

Comparative biology of the amniote vestibular utricle.

The sensory epithelia of the auditory and vestibular systems of vertebrates have shared developmental and evolutionary histories. However, while the auditory epithelia show great variation across vertebrates, the vestibular sensory epithelia appear seemingly more conserved. An exploration of the current knowledge of the comparative biology of the amniote utricle, a vestibular sensory epithelium that senses linear acceleration, shows interesting instances of variability between birds and mammals. The distribution of sensory hair cell types, the position of the line of hair bundle polarity reversal and the properties of supporting cells show marked differences, likely impacting vestibular function and hair cell regeneration potential.

The evolutionary tuning of hearing.

After the transition to life on land, tympanic middle ears emerged separately in different groups of tetrapods, facilitating the efficient detection of airborne sounds and paving the way for high frequency sensitivity. The processes that brought about high-frequency hearing in mammals are tightly linked to the accumulation of coding sequence changes in inner ear genes; many of which were selected during evolution. These include proteins involved in hair bundle morphology, mechanotransduction and high endolymphatic potential, somatic electromotility for sound amplification, ribbon synapses for high-fidelity transmission of sound stimuli, and efferent synapses for the modulation of sound amplification. Here, we review the molecular evolutionary processes behind auditory functional innovation. Overall, the evidence to date supports the hypothesis that changes in inner ear proteins were central to the fine tuning of mammalian hearing.

The Hair Cell α9α10 Nicotinic Acetylcholine Receptor: Odd Cousin in an Old Family.

Nicotinic acetylcholine receptors (nAChRs) are a subfamily of pentameric ligand-gated ion channels with members identified in most eumetazoan clades. In vertebrates, they are divided into three subgroups, according to their main tissue of expression: neuronal, muscle and hair cell nAChRs. Each receptor subtype is composed of different subunits, encoded by paralogous genes. The latest to be identified are the α9 and α10 subunits, expressed in the mechanosensory hair cells of the inner ear and the lateral line, where they mediate efferent modulation. α9α10 nAChRs are the most divergent amongst all nicotinic receptors, showing marked differences in their degree of sequence conservation, their expression pattern, their subunit co-assembly rules and, most importantly, their functional properties. Here, we review recent advances in the understanding of the structure and evolution of nAChRs. We discuss the functional consequences of sequence divergence and conservation, with special emphasis on the hair cell α9α10 receptor, a seemingly distant cousin of neuronal and muscle nicotinic receptors. Finally, we highlight potential links between the evolution of the octavolateral system and the extreme divergence of vertebrate α9α10 receptors.

Loss of Choline Agonism in the Inner Ear Hair Cell Nicotinic Acetylcholine Receptor Linked to the α10 Subunit.

The α9α10 nicotinic acetylcholine receptor (nAChR) plays a fundamental role in inner ear physiology. It mediates synaptic transmission between efferent olivocochlear fibers that descend from the brainstem and hair cells of the auditory sensory epithelium. The α9 and α10 subunits have undergone a distinct evolutionary history within the family of nAChRs. Predominantly in mammalian vertebrates, the α9α10 receptor has accumulated changes at the protein level that may ultimately relate to the evolutionary history of the mammalian hearing organ. In the present work, we investigated the responses of α9α10 nAChRs to choline, the metabolite of acetylcholine degradation at the synaptic cleft. Whereas choline is a full agonist of chicken α9α10 receptors it is a partial agonist of the rat receptor. Making use of the expression of α9α10 heterologous receptors, encompassing wild-type, heteromeric, homomeric, mutant, chimeric, and hybrid receptors, and in silico molecular docking, we establish that the mammalian (rat) α10 nAChR subunit underscores the reduced efficacy of choline. Moreover, we show that whereas the complementary face of the α10 subunit does not play an important role in the activation of the receptor by ACh, it is strictly required for choline responses. Thus, we propose that the evolutionary changes acquired in the mammalian α9α10 nAChR resulted in the loss of choline acting as a full agonist at the efferent synapse, without affecting the triggering of ACh responses. This may have accompanied the fine-tuning of hair cell post-synaptic responses to the high-frequency activity of efferent medial olivocochlear fibers that modulate the cochlear amplifier.

GJB2 and GJB6 genes: molecular study and identification of novel GJB2 mutations in the hearing-impaired Argentinean population.

Mutations in the GJB2 gene are responsible for more than half of all cases of recessive non-syndromic deafness. This article presents a mutation analysis of the GJB2, GJB6, OTOF and MTRNR1 genes in 252 patients with sensorineural non-syndromic hearing loss. Thirty-one different mutations were identified in GJB2 and GJB6 in 86 of the 252 (34%) patients. We describe for the first time two new mutations in GJB2: the missense mutation c.29 T>C (p.Leu10Pro) in the N terminal domain and c.326 G>T (p.Gly109Val) in the intracytoplasmic domain of connexin 26. This work shows the high prevalence of GJB2 mutations in the Argentinean population, with frequencies that are comparable to those of the Mediterranean area. Most important, it adds two novel GJB2 mutations to be taken into consideration in the genetic diagnosis of non-syndromic sensorineural hearing loss.

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.

Properties of mutated murine alpha4beta2 nicotinic receptors linked to partial epilepsy.

We characterized, by electrophysiological methods, two biophysical properties of murine recombinant alpha4beta2 nicotinic acetylcholine receptors (nAChR) bearing a mutation (alpha4:+L264alpha4:beta2 or alpha4:S252Falpha4:beta2) linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Sensitivity to acetylcholine (ACh) was increased by the S252F substitution expressed in heterozygosis (alpha4:S252Falpha4:beta2) but was markedly reduced when this mutation was expressed in homozygosis (S252Falpha4:beta2). ACh sensitivity was not altered by the +L264 insertion. Moreover, receptor desensitization was significantly increased by both mutations expressed in heterozygosis. These results are in general agreement to those of rat and human recombinant receptors bearing the same mutations, thus contributing to validate the use of knock-in mice harboring ADNFLE mutations as models to study this pathology.

Conserved and divergent development of brainstem vestibular and auditory nuclei.

Vestibular function was established early in vertebrates and has remained, for the most part, unchanged. In contrast, each group of tetrapods underwent independent evolutionary processes to solve the problem of hearing on land, resulting in a remarkable mixture of conserved, divergent and convergent features that define extant auditory systems. The vestibuloacoustic nuclei of the hindbrain develop from a highly conserved ground plan and provide an ideal framework on which to address the participation of developmental processes to the evolution of neuronal circuits. We employed an electroporation strategy to unravel the contribution of two dorsoventral and four axial lineages to the development of the chick hindbrain vestibular and auditory nuclei. We compare the chick developmental map with recently established genetic fate-maps of the developing mouse hindbrain. Overall, we find considerable conservation of developmental origin for the vestibular nuclei. In contrast, a comparative analysis of the developmental origin of hindbrain auditory structures echoes the complex evolutionary history of the auditory system. In particular, we find that the developmental origin of the chick auditory interaural time difference circuit supports its emergence from an ancient vestibular network, unrelated to the analogous mammalian counterpart.

The emergence of mesencephalic trigeminal neurons.

BACKGROUND: The cells of the mesencephalic trigeminal nucleus (MTN) are the proprioceptive sensory neurons that innervate the jaw closing muscles. These cells differentiate close to the two key signalling centres that influence the dorsal midbrain, the isthmus, which mediates its effects via FGF and WNT signalling and the roof plate, which is a major source of BMP signalling as well as WNT signalling. METHODS: In this study, we have set out to analyse the importance of FGF, WNT and BMP signalling for the development of the MTN. We have employed pharmacological inhibitors of these pathways in explant cultures as well as utilising the electroporation of inhibitory constructs in vivo in the chick embryo. RESULTS: We find that interfering with either FGF or WNT signalling has pronounced effects on MTN development whilst abrogation of BMP signalling has no effect. We show that treatment of explants with either FGF or WNT antagonists results in the generation of fewer MTN neurons and affects MTN axon extension and that inhibition of both these pathways has an additive effect. To complement these studies, we have used in vivo electroporation to inhibit BMP, FGF and WNT signalling within dorsal midbrain cells prior to, and during, their differentiation as MTN neurons. Again, we find that inhibition of BMP signalling has no effect on the development of MTN neurons. We additionally find that cells electroporated with inhibitory constructs for either FGF or WNT signalling can differentiate as MTN neurons suggesting that these pathways are not required cell intrinsically for the emergence of these neurons. Indeed, we also show that explants of dorsal mesencephalon lacking both the isthmus and roof plate can generate MTN neurons. However, we did find that inhibiting FGF or WNT signalling had consequences for MTN differentiation. CONCLUSIONS: Our results suggest that the emergence of MTN neurons is an intrinsic property of the dorsal mesencephalon of gnathostomes, and that this population undergoes expansion, and maturation, along with the rest of the dorsal midbrain under the influence of FGF and WNT signalling.

Electrical properties and functional expression of ionic channels in cochlear inner hair cells of mice lacking the alpha10 nicotinic cholinergic receptor subunit.

Cochlear inner hair cells (IHCs) release neurotransmitter onto afferent auditory nerve fibers in response to sound stimulation. During early development, synaptic transmission is triggered by spontaneous Ca2+ spikes which are modulated by an efferent cholinergic innervation to IHCs. This synapse is inhibitory and mediated by the alpha9alpha10 nicotinic cholinergic receptor (nAChR). After the onset of hearing, large-conductance Ca2+-activated K+ channels are acquired and both the spiking activity and the efferent innervation disappear from IHCs. In this work, we studied the developmental changes in the membrane properties of cochlear IHCs from alpha10 nAChR gene (Chrna10) "knockout" mice. Electrophysiological properties of IHCs were studied by whole-cell recordings in acutely excised apical turns of the organ of Corti from developing mice. Neither the spiking activity nor the developmental functional expression of voltage-gated and/or calcium-sensitive K+ channels is altered in the absence of the alpha10 nAChR subunit. The present results show that the alpha10 nAChR subunit is not essential for the correct establishment of the intrinsic electrical properties of IHCs during development.

Performance of speech perception after cochlear implantation in DFNB1 patients.

CONCLUSION: There were no apparent differences in speech performance after cochlear implantation between patients with biallelic GJB2 and/or GJB6 mutations and those with deafness of unknown aetiology. These data have important implications for the selection of prognostic indicators of the outcome of cochlear implantation. OBJECTIVE: To compare performance after cochlear implantation in children with mutations in GJB2 (connexin 26) and/or GJB6 (connexin 30) and children with deafness of unknown aetiology. SUBJECTS AND METHODS: Genetic analysis and speech performance evaluation was determined in 24 patients with (n=11) and without (n=13) biallelic GJB2 and/or GJB6 mutations who underwent cochlear implantation. Speech perception skills were measured 12 and 24 months after surgery. Each patient was classified in accordance with the speech perception category (SPC). RESULTS: Overall, the two groups showed similar significant improvement in speech perception after implantation.

Constitutive expression of the alpha10 nicotinic acetylcholine receptor subunit fails to maintain cholinergic responses in inner hair cells after the onset of hearing.

Efferent inhibition of cochlear hair cells is mediated by alpha9alpha10 nicotinic cholinergic receptors (nAChRs) functionally coupled to calcium-activated, small conductance (SK2) potassium channels. Before the onset of hearing, efferent fibers transiently make functional cholinergic synapses with inner hair cells (IHCs). The retraction of these fibers after the onset of hearing correlates with the cessation of transcription of the Chrna10 (but not the Chrna9) gene in IHCs. To further analyze this developmental change, we generated a transgenic mice whose IHCs constitutively express alpha10 into adulthood by expressing the alpha10 cDNA under the control of the Pou4f3 gene promoter. In situ hybridization showed that the alpha10 mRNA is expressed in IHCs of 8-week-old transgenic mice, but not in wild-type mice. Moreover, this mRNA is translated into a functional protein, since IHCs from P8-P10 alpha10 transgenic mice backcrossed to a Chrna10(-/-) background (whose IHCs have no cholinergic function) displayed normal synaptic and acetylcholine (ACh)-evoked currents in patch-clamp recordings. Thus, the alpha10 transgene restored nAChR function. However, in the alpha10 transgenic mice, no synaptic or ACh-evoked currents were observed in P16-18 IHCs, indicating developmental down-regulation of functional nAChRs after the onset of hearing, as normally observed in wild-type mice. The lack of functional ACh currents correlated with the lack of SK2 currents. These results indicate that multiple features of the efferent postsynaptic complex to IHCs, in addition to the nAChR subunits, are down-regulated in synchrony after the onset of hearing, leading to lack of responses to ACh.