Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed

Binary recombinase systems for high-resolution conditional mutagenesis

Conditional mutagenesis using Cre recombinase expressed from tissue specific promoters facilitates analyses of gene function and cell lineage tracing. Here, we describe two novel dual-promoter-driven conditional mutagenesis systems designed for greater accuracy and optimal efficiency of recombination. Co-Driver employs a recombinase cascade of Dre and Dre-respondent Cre, which processes loxP-flanked alleles only when both recombinases are expressed in a predetermined temporal sequence. This unique property makes Co-Driver ideal for sequential lineage tracing studies aimed at unraveling the relationships between cellular precursors and mature cell types. Co-InCre was designed for highly efficient intersectional conditional transgenesis. It relies on highly active trans-splicing inteins and promoters with simultaneous transcriptional activity to reconstitute Cre recombinase from two inactive precursor fragments. By generating native Cre, Co-InCre attains recombination rates that exceed all other binary SSR systems evaluated in this study. Both Co-Driver and Co-InCre significantly extend the utility of existing Cre-responsive allele

c-Maf-positive spinal cord neurons are critical elements of a dorsal horn circuit for mechanical hypersensitivity in neuropathy

Corticospinal tract (CST) neurons innervate the deep spinal dorsal horn to sustain chronic neuropathic pain. The majority of neurons targeted by the CST are interneurons expressing the transcription factor c-Maf. Here, we used intersectional genetics to decipher the function of these neurons in dorsal horn sensory circuits. We find that excitatory c-Maf (c-Maf$^{EX}$) neurons receive sensory input mainly from myelinated fibers and target deep dorsal horn parabrachial projection neurons and superficial dorsal horn neurons, thereby connecting non-nociceptive input to nociceptive output structures. Silencing c-Maf$^{EX}$ neurons has little effect in healthy mice but alleviates mechanical hypersensitivity in neuropathic mice. c-Maf$^{EX}$ neurons also receive input from inhibitory c-Maf and parvalbumin neurons, and compromising inhibition by these neurons caused mechanical hypersensitivity and spontaneous aversive behaviors reminiscent of c-Maf$^{EX}$ neuron activation. Our study identifies c-Maf$^{EX}$ neurons as normally silent second-order nociceptors that become engaged in pathological pain signaling upon loss of inhibitory control

Inhibitory Kcnip2 neurons of the spinal dorsal horn control behavioral sensitivity to environmental cold

Proper sensing of ambient temperature is of utmost importance for the survival of euthermic animals, including humans. While considerable progress has been made in our understanding of temperature sensors and transduction mechanisms, the higher-order neural circuits processing such information are still only incompletely understood. Using intersectional genetics in combination with circuit tracing and functional neuron manipulation, we identified Kcnip2-expressing inhibitory (Kcnip2GlyT2) interneurons of the mouse spinal dorsal horn as critical elements of a neural circuit that tunes sensitivity to cold. Diphtheria toxin-mediated ablation of these neurons increased cold sensitivity without affecting responses to other somatosensory modalities, while their chemogenetic activation reduced cold and also heat sensitivity. We also show that Kcnip2GlyT2 neurons become activated preferentially upon exposure to cold temperatures and subsequently inhibit spinal nociceptive output neurons that project to the lateral parabrachial nucleus. Our results thus identify a hitherto unknown spinal circuit that tunes cold sensitivity. Keywords: circuit; cold; cold allodynia; cold analgesia; cooling; dre recombinase; interneuron; intersectional gene targeting; kcnip2; pai

Immune or genetic-mediated disruption of CASPR2 causes pain hypersensitivity due to enhanced primary afferent excitability

Human autoantibodies to contactin-associated protein-like 2 (CASPR2) are often associated with neuropathic pain, and CASPR2 mutations have been linked to autism spectrum disorders, in which sensory dysfunction is increasingly recognized. Human CASPR2 autoantibodies, when injected into mice, were peripherally restricted and resulted in mechanical pain-related hypersensitivity in the absence of neural injury. We therefore investigated the mechanism by which CASPR2 modulates nociceptive function. Mice lacking CASPR2 (Cntnap2 ) demonstrated enhanced pain-related hypersensitivity to noxious mechanical stimuli, heat, and algogens. Both primary afferent excitability and subsequent nociceptive transmission within the dorsal horn were increased in Cntnap2 mice. Either immune or genetic-mediated ablation of CASPR2 enhanced the excitability of DRG neurons in a cell-autonomous fashion through regulation of Kv1 channel expression at the soma membrane. This is the first example of passive transfer of an autoimmune peripheral neuropathic pain disorder and demonstrates that CASPR2 has a key role in regulating cell-intrinsic dorsal root ganglion (DRG) neuron excitability

Circuit dissection of the role of somatostatin in itch and pain

Stimuli that elicit itch are detected by sensory neurons that innervate the skin. This information is processed by the spinal cord; however, the way in which this occurs is still poorly understood. Here we investigated the neuronal pathways for itch neurotransmission, particularly the contribution of the neuropeptide somatostatin. We find that in the periphery, somatostatin is exclusively expressed in Nppb+ neurons, and we demonstrate that Nppb+somatostatin+ cells function as pruriceptors. Employing chemogenetics, pharmacology and cell-specific ablation methods, we demonstrate that somatostatin potentiates itch by inhibiting inhibitory dynorphin neurons, which results in disinhibition of GRPR+ neurons. Furthermore, elimination of somatostatin from primary afferents and/or from spinal interneurons demonstrates differential involvement of the peptide released from these sources in itch and pain. Our results define the neural circuit underlying somatostatin-induced itch and characterize a contrasting antinociceptive role for the peptide

Calretinin-expressing islet cells are a source of pre- and post-synaptic inhibition of non-peptidergic nociceptor input to the mouse spinal cord

Unmyelinated non-peptidergic nociceptors (NP afferents) arborise in lamina II of the spinal cord and receive GABAergic axoaxonic synapses, which mediate presynaptic inhibition. However, until now the source of this axoaxonic synaptic input was not known. Here we provide evidence that it originates from a population of inhibitory calretinin-expressing interneurons (iCRs), which correspond to lamina II islet cells. The NP afferents can be assigned to 3 functionally distinct classes (NP1–3). NP1 afferents have been implicated in pathological pain states, while NP2 and NP3 afferents also function as pruritoceptors. Our findings suggest that all 3 of these afferent types innervate iCRs and receive axoaxonic synapses from them, providing feedback inhibition of NP input. The iCRs also form axodendritic synapses, and their targets include cells that are themselves innervated by the NP afferents, thus allowing for feedforward inhibition. The iCRs are therefore ideally placed to control the input from non-peptidergic nociceptors and pruritoceptors to other dorsal horn neurons, and thus represent a potential therapeutic target for the treatment of chronic pain and itch

Postnatal loss of Dlk1 imprinting in stem cells and niche astrocytes regulates neurogenesis.

The gene for the atypical NOTCH ligand delta-like homologue 1 (Dlk1) encodes membrane-bound and secreted isoforms that function in several developmental processes in vitro and in vivo. Dlk1, a member of a cluster of imprinted genes, is expressed from the paternally inherited chromosome. Here we show that mice that are deficient in Dlk1 have defects in postnatal neurogenesis in the subventricular zone: a developmental continuum that results in depletion of mature neurons in the olfactory bulb. We show that DLK1 is secreted by niche astrocytes, whereas its membrane-bound isoform is present in neural stem cells (NSCs) and is required for the inductive effect of secreted DLK1 on self-renewal. Notably, we find that there is a requirement for Dlk1 to be expressed from both maternally and paternally inherited chromosomes. Selective absence of Dlk1 imprinting in both NSCs and niche astrocytes is associated with postnatal acquisition of DNA methylation at the germ-line-derived imprinting control region. The results emphasize molecular relationships between NSCs and the niche astrocyte cells of the microenvironment, identifying a signalling system encoded by a single gene that functions coordinately in both cell types. The modulation of genomic imprinting in a stem-cell environment adds a new level of epigenetic regulation to the establishment and maintenance of the niche, raising wider questions about the adaptability, function and evolution of imprinting in specific developmental contexts

The function of Mash1 in the development of the dorsal spinal cord

Titelblatt, Inhalt, Danksagung 1\. Einleitung 6 2\. Material und Methoden 20 3\. Ergebnisse 43 4\. Diskussion 69 5\. Zusammenfassung 78 6\. Literatur 81 6\. ErklärungDie Neurone des dorsalen Rückenmarks integrieren und verschalten sensorische Informationen. Sie entstehen in zwei Phasen der Neurogenese im sich entwickelnden dorsalen Rückenmark. Im Rahmen dieser Arbeit wurde die Funktion des nur in Vorläuferzellen exprimierten bHLH Transkritionsfaktors Mash1 in der Bildung von dorsalen Rückenmarksneuronen untersucht. Während der ersten Phase der Neurogenese entstehen an invarianten Positionen entlang der dorso- ventralen Achse sechs unterschiedliche neuronale Subtypen (dI1- dI6) aus sechs angrenzenden Vorläuferbereichen. Im Rahmen dieser Arbeit konnte gezeigt werden, daß Mash1 die Spezifizierung von zwei der sechs frühgeborenen neuronalen Subtypen (dI3 und dI5) kontrolliert. Mash1 induziert dabei die Expression von Tlx3, einem postmitotischen Selektorgen. In Mash1 mutanten Embryonen exprimieren die eigentlich Tlx3 positiven Neurone fälschlichweise Transkriptionsfaktoren die für Neurone der angrenzenden Subtypen (dI2 bzw. dI4 und dI6) charakteristisch sind. Der Grossteil der Neurone des dorsalen Horns entsteht aber während der zweiten Phase der Neurogenese. Zu diesem Zeitpunkt hat sich der Mash1+ Bereich auf fast die gesamte dorsale Vorläuferschicht ausgedehnt. Aus der Mash1+ Domäne entstehen zwei Neuronensubtypen, dILA und dILB Neurone, in einem Salz-und-Pfeffer-Muster. dILA Neurone differenzieren zu GABAergen und dILB zu glutamatergen Neuronen (Cheng et al. 2004). Die beschriebenen Mechanismen die zur neuralen Diversifikation im Neuralrohr beitragen, beruhen auf der Ausbildung von distinkten Voläuferdomänen. Diese sind durch die Expression unterschiedlicher Transkriptionsfaktorkombinationen gekennzeichnet. Das führt dazu, daß jede Domäne unterschiedliche neuronale Subtypen bildet (Jessell 2000; Briscoe and Ericson 2001). Ein derartiger Mechanismus liefert jedoch keine ausreichende Erklärung für die Entstehung des Salz-und-Pfeffer-Muster, in dem dILA und dILB Neurone aus einer einzigen Domäne hervorgehen. Ich konnte zeigen, daß dILA Neurone nur aus asymmetrischen terminalen oder nichtterminalen Teilungen entstehen, und daß eine Vorläuferzelle in einer terminalen asymmetrischen Teilung eine dILA und eine dILB Zelle bilden kann. Die Mutation von Mash1 beeinflusst die Bildung und Spezifizierung von dILA, nicht aber dILB Neuronen, obwohl, wie in dieser Arbeit gezeigt werden konnte, Mash1 in Vorläufern von dILA und dILB Zellen exprimiert wird. Damit Mash1 seine Funktion in der Bildung und Spezifizierung ausschließlich von dILA Neuronen ausüben kann, muss Mash1 asymmetrisch aktiv sein. Ich konnte weiterhin zeigen, daß der Verlust der spätgeborenen GABAergen dILA Zellen zu Defekten des spinalen Reflexbogens und zur Verminderung der Reflexplastizität führt.Neurons of the dorsal horn relay and integrate sensory information. They arise in two Phases of neurogenesis in the developing dorsal neural tube. The bHLH transcription factor Mash1 is expressed in the progenitors of dorsal spinal interneurons. In the course of the work presented here the function of Mash1 in the generation of dorsal spinal interneurons was analyzed. During the first phase of neurogenesis six different neuron types (dI1-dI6) are born from distinct progenitor domains along the dorsal-ventral axis. Here it could be shown, that Mash1 controls the specification of two of these six early born neuron types (dI3 and dI5). Mash1 is required for the induction of the selector gene Tlx3. In Mash1-/- mice the dI3 and dI5 neurons acquire characteristics of the adjacently born neuron types. The majority of dorsal interneurons is born during the second phase of neurogenesis. During the second phase Mash1 expression expands dorsally and the Mash1+ progenitor domain gives rise to two types of late born dorsal Neurons (dILA and dILB neurons), which arise in a salt and pepper like fashion. dILA mature into GABAergic neurons whereas dILB neurons give rise to the glutamatergic neurons of the dorsal horn (Cheng et al. 2004). Described mechanisms for neuronal diversification relay on the formation of spatially distinct progenitor domains characterized by the expression of different sets of transcription factors. The different transcription factor sets trigger the specification of different neuron types (Jessell 2000; Briscoe and Ericson 2001). These mechanism is not sufficient to explain the generation of two different neuronal types (e.g. dILA and dILB) from the same progenitor domain. Here I show that dILA neurons are always the product of asymmetric cell divisions, either terminal or non-terminal. dILA and dILB cells can therefore be generated from the same progenitor cell in a terminal division. In Mash1 mutant mice the generation and specification of dILA neurons but not dILB neurons is severely impaired, although Mash1 is expressed in the development of dILA and dILB cells. To account for the observed effects of the Mash1 mutation, Mash1 has to exert its effect in a asymmetric manner effecting only one of two possible fates. dILA neurons mature into the GABAergic neurons of the dorsal horn. I could show, that the loss of GABAergic neurons in Mash1 mutant mice results in defects in spinal reflex circuitry and reduces spinal reflex plasticity

Spinally projecting noradrenergic neurons of the locus coeruleus display resistance to AAV2retro-mediated transduction

Background: The locus coeruleus (LC) is the principal source of noradrenaline (NA) in the central nervous system. Projection neurons in the ventral portion of the LC project to the spinal cord and are considered the main source of spinal NA. To understand the precise physiology of this pathway, it is important to have tools that allow specific genetic access to these descending projections. AAV2retro serotype vectors are a potential tool to transduce these neurons via their axon terminals in the spinal cord, and thereby limit the expression of genetic material to the spinal projections from the LC. Here, we assess the suitability of AAV2retro to target these neurons and investigate strategies to increase their labelling efficiency. Results: We show that the neurons in the LC that project to the spinal dorsal horn are largely resistant to transduction with AAV2retro serotype vectors. Compared to Cholera toxin B (CTb) tracing, AAV2retro.eGFP labelled far fewer neurons within the LC and surrounding regions, particularly within neurons that express tyrosine hydroxylase (TH), the rate-limiting enzyme for NA synthesis. We also show that the sensitivity for transduction of this projection can be increased using AAV2retro.eGFP.cre in ROSA26tdTom reporter mice (23% increase), with a higher proportion of the newly revealed neurons expressing TH compared to those directly labelled with AAV2retro containing an eGFP expression sequence. Conclusion: These tracing studies identify limitations in AAV2retro-mediated retrograde transduction of a subset of projection neurons, specifically those that express NA and project to the spinal cord. This is likely to have implications for the study of NA-containing projections as well as other types of projection neuron in the central nervous system. Keywords: Spinal cord; locus coeruleus; noradrenaline; projection neuron