Markus Rothermel - Academia.edu (original) (raw)

Papers by Markus Rothermel

Research paper thumbnail of Connections of the superior colliculus to shoulder muscles of the rat: a dual tracing study

Frontiers in Neuroanatomy, 2013

Previous investigations indicate that the superior colliculus (SC) is involved in the initiation ... more Previous investigations indicate that the superior colliculus (SC) is involved in the initiation and execution of forelimb movements. In the present study we investigated the tectofugal, in particular the tecto-reticulo-spinal projections to the shoulder and arm muscles in the rat. We simultaneously retrogradely labeled the premotor neurons in the brainstem by injection of the pseudorabies virus PrV Bartha 614 into the m. rhomboideus minor and m. acromiodeltoideus, and anterogradely visualized the tectofugal projections by intracollicular injection of the tracer FITC dextrane. Our results demonstrate that the connection of the SC to the skeletal muscles of the forelimb is at least trisynaptic. This was confirmed by long survival times after virus injections into the muscles (98-101 h) after which numerous neurons in the deep layers of the SC were labeled. Transsynaptically retrogradely labeled brainstem neurons connected disynaptically to the injected muscles with adjacent tectal terminals were predominantly located in the gigantocellular nuclear complex of the reticular formation. In addition, putative relay neurons were found in the caudal part of the pontine reticular nucleus. Both tectal projections to the nucleus gigantocellularis and the pontine reticular nucleus were bilateral but ipsilaterally biased. We suggest this projection to be involved in more global functions in motivated behavior like general arousal allowing fast voluntary motor activity.

Research paper thumbnail of Chemosensory properties of murine nasal and cutaneous trigeminal neurons identified by viral tracing

BMC Neuroscience, 2006

Background: Somatosensation of the mammalian head is mainly mediated by the trigeminal nerve that... more Background: Somatosensation of the mammalian head is mainly mediated by the trigeminal nerve that provides innervation of diverse tissues like the face skin, the conjunctiva of the eyes, blood vessels and the mucouse membranes of the oral and nasal cavities. Trigeminal perception encompasses thermosensation, touch, and pain. Trigeminal chemosensation from the nasal epithelia mainly evokes stinging, burning, or pungent sensations. In vitro characterization of trigeminal primary sensory neurons derives largely from analysis of complete neuronal populations prepared from sensory ganglia. Thus, functional properties of primary trigeminal afferents depending on the area of innervation remain largely unclear.

Research paper thumbnail of Imaging Activity in Neurons and Glia with a Polr2a-Based and Cre-Dependent GCaMP5G-IRES-tdTomato Reporter Mouse

Research paper thumbnail of Cholinergic Inputs from Basal Forebrain Add an Excitatory Bias to Odor Coding in the Olfactory Bulb

Journal of Neuroscience, 2014

Cholinergic modulation of central circuits is associated with active sensation, attention, and le... more Cholinergic modulation of central circuits is associated with active sensation, attention, and learning, yet the neural circuits and temporal dynamics underlying cholinergic effects on sensory processing remain unclear. Understanding the effects of cholinergic modulation on particular circuits is complicated by the widespread projections of cholinergic neurons to telencephalic structures that themselves are highly interconnected. Here we examined how cholinergic projections from basal forebrain to the olfactory bulb (OB) modulate output from the first stage of sensory processing in the mouse olfactory system. By optogenetically activating their axons directly in the OB, we found that cholinergic projections from basal forebrain regulate OB output by increasing the spike output of presumptive mitral/tufted cells. Cholinergic stimulation increased mitral/tufted cell spiking in the absence of inhalation-driven sensory input and further increased spiking responses to inhalation of odorless air and to odorants. This modulation was rapid and transient, was dependent on local cholinergic signaling in the OB, and differed from modulation by optogenetic activation of cholinergic neurons in basal forebrain, which led to a mixture of mitral/tufted cell excitation and suppression. Finally, bulbar cholinergic enhancement of mitral/tufted cell odorant responses was robust and occurred independent of the strength or even polarity of the odorant-evoked response, indicating that cholinergic modulation adds an excitatory bias to mitral/tufted cells as opposed to increasing response gain or sharpening response spectra. These results are consistent with a role for the basal forebrain cholinergic system in dynamically regulating the sensitivity to or salience of odors during active sensing of the olfactory environment.

Research paper thumbnail of Transgene Expression in Target-Defined Neuron Populations Mediated by Retrograde Infection with Adeno-Associated Viral Vectors

Journal of Neuroscience, 2013

Tools enabling the manipulation of well defined neuronal subpopulations are critical for probing ... more Tools enabling the manipulation of well defined neuronal subpopulations are critical for probing complex neuronal networks. Cre recombinase (Cre) mouse driver lines in combination with the Cre-dependent expression of proteins using viral vectors--in particular, recombinant adeno-associated viral vectors (rAAVs)--have emerged as a widely used platform for achieving transgene expression in specified neural populations. However, the ability of rAAVs to further specify neuronal subsets on the basis of their anatomical connectivity has been reported as limited or inconsistent. Here, we systematically tested a variety of widely used neurotropic rAAVs for their ability to mediate retrograde gene transduction in the mouse brain. We tested pseudotyped rAAVs of several common serotypes (rAAV 2/1, 2/5, and 2/9) as well as constructs both with and without Cre-dependent expression switches. Many of the rAAVs tested--in particular, though not exclusively, Cre-dependent vectors--showed a robust capacity for retrograde infection and transgene expression. Retrograde expression was successful over distances as large as 6 mm and in multiple neuron types, including olfactory projection neurons, neocortical pyramidal cells projecting to distinct targets, and corticofugal and modulatory projection neurons. Retrograde infection using transgenes such as ChR2 allowed for optical control or optically assisted electrophysiological identification of neurons defined genetically as well as by their projection target. These results establish a widely accessible tool for achieving combinatorial specificity and stable, long-term transgene expression to isolate precisely defined neuron populations in the intact animal.

Research paper thumbnail of Nasal Chemosensory-Stimulation Evoked Activity Patterns in the Rat Trigeminal Ganglion Visualized by In Vivo Voltage-Sensitive Dye Imaging

PLoS ONE, 2011

Mammalian nasal chemosensation is predominantly mediated by two independent neuronal pathways, th... more Mammalian nasal chemosensation is predominantly mediated by two independent neuronal pathways, the olfactory and the trigeminal system. Within the early olfactory system, spatiotemporal responses of the olfactory bulb to various odorants have been mapped in great detail. In contrast, far less is known about the representation of volatile chemical stimuli at an early stage in the trigeminal system, the trigeminal ganglion (TG), which contains neurons directly projecting to the nasal cavity. We have established an in vivo preparation that allows high-resolution imaging of neuronal population activity from a large region of the rat TG using voltage-sensitive dyes (VSDs). Application of different chemical stimuli to the nasal cavity elicited distinct, stimulus-category specific, spatiotemporal activation patterns that comprised activated as well as suppressed areas. Thus, our results provide the first direct insights into the spatial representation of nasal chemosensory information within the trigeminal ganglion imaged at high temporal resolution.

Research paper thumbnail of Neuronal pathways of viral invasion in mice after intranasal inoculation of pseudorabies virus PrV-9112C2 expressing bovine herpesvirus 1 glycoprotein B

Journal of Neurovirology, 2006

give evidence for viral entry receptors present in swine but not mice OSNs. Journal of NeuroVirol... more give evidence for viral entry receptors present in swine but not mice OSNs. Journal of NeuroVirology (2006) 12, 60-64.

Research paper thumbnail of Anterograde transsynaptic tracing in the murine somatosensory system using Pseudorabies virus (PrV): A “live-cell”-tracing tool for analysis of identified neurons in vitro

Journal of Neurovirology, 2007

The Pseudorabies virus (PrV) strain Bartha is widely used as a tool for retrograde transneuronal ... more The Pseudorabies virus (PrV) strain Bartha is widely used as a tool for retrograde transneuronal tracing in mammals. Traced neurons can be identified in cell culture allowing the analysis of their physiological features ("live-cell"-tracing). Compared to PrV-Bartha, PrV-Kaplan is known for higher virulence and transsynaptic spread in both retrograde and anterograde direction. Herein we assess the authors assess PrV-Kaplan for transsynaptic anterograde "live-cell"-tracing. Following intranasal application in mice, labelled trigeminal and brainstem neurons could be identified in vitro. Detailed electrophysiological analysis indicated that viral infection did not affect neuronal properties, making PrV-Kaplan eligible for functional analysis of identified neurons within somatosensory systems.

Research paper thumbnail of Advanced tracing tools: functional neuronal expression of virally encoded fluorescent calcium indicator proteins

Journal of Neurovirology, 2009

Pseudorabies virus (PrV) strains such as PrV-Bartha and its marker protein-expressing variants ha... more Pseudorabies virus (PrV) strains such as PrV-Bartha and its marker protein-expressing variants have been used in numerous studies as retrograde transneuronal tracing tools, defining the synaptic organization of mammalian neuronal circuits. However, the possibilities for functional examination of virus-infected neurons are limited to electrophysiological approaches or bulk loading strategies using calcium-sensitive dyes. Herein we report the generation and functional characterization of three PrV-Bartha-derived recombinant virus mutants that express different fluorescent calcium indicator proteins (FCIPs). All three generated virus recombinants are able to infect murine trigeminal neurons and express the corresponding FCIP (GCaMP2, camgaroo-2, or inverse pericam). Functionality of these virally expressed constructs was verified by using confocal Ca-imaging technologies. These FCIP-expressing virus recombinants provide a new tool for the functional analysis of whole circuits of synaptically connected neurons in vitro and in vivo.

Research paper thumbnail of Chemosensory information processing between keratinocytes and trigeminal neurons

Journal of Biological Chemistry, 2014

Trigeminal fibers terminate within the facial mucosa and skin and transmit tactile, proprioceptiv... more Trigeminal fibers terminate within the facial mucosa and skin and transmit tactile, proprioceptive, chemical, and nociceptive sensations. Trigeminal sensations can arise from the direct stimulation of intraepithelial free nerve endings or indirectly through information transmission from adjacent cells at the peripheral innervation area. For mechanical and thermal cues, communication processes between skin cells and somatosensory neurons have already been suggested. High concentrations of most odors typically provoke trigeminal sensations in vivo but surprisingly fail to activate trigeminal neuron monocultures. This fact favors the hypothesis that epithelial cells may participate in chemodetection and subsequently transmit signals to neighboring trigeminal fibers. Keratinocytes, the major cell type of the epidermis, express various receptors that enable reactions to multiple environmental stimuli. Here, using a co-culture approach, we show for the first time that exposure to the odorant chemicals induces a chemical communication between human HaCaT keratinocytes and mouse trigeminal neurons. Moreover, a supernatant analysis of stimulated keratinocytes and subsequent blocking experiments with pyrodoxalphosphate-6-azophenyl-2',4'-disulfonate revealed that ATP serves as the mediating transmitter molecule released from skin cells after odor stimulation. We show that the ATP release resulting from Javanol® stimulation of keratinocytes was mediated by pannexins. Consequently, keratinocytes act as chemosensors linking the environment and the trigeminal system via ATP signaling.

Research paper thumbnail of In vivo monitoring of chemically evoked activity patterns in the rat trigeminal ganglion

Frontiers in Systems Neuroscience, 2013

Albeit lacking a sense of smell, anosmic patients maintain a reduced ability to distinguish diffe... more Albeit lacking a sense of smell, anosmic patients maintain a reduced ability to distinguish different volatile chemicals by relying exclusively on their trigeminal system (TS). To elucidate differences in the neuronal representation of these volatile substances in the TS, we performed voltage-sensitive dye imaging (VSDI) in the rat trigeminal ganglion (TG) in vivo. We demonstrated that stimulus-specific patterns of bioelectrical activity occur within the TG upon nasal administration of ten different volatile chemicals. With regard to spatial differences between the evoked trigeminal response patterns, these substances could be sorted into three groups. Signal intensity and onset latencies were also dependent on the administered stimulus and its concentration. We conclude that particular compounds detected by the TS are represented by (1) a specific spatial response pattern, (2) the signal intensity, and (3) onset latencies within the pattern. Jointly, these trigeminal representations may contribute to the surprisingly high discriminative skills of anosmic patients.

Research paper thumbnail of Connections of the superior colliculus to shoulder muscles of the rat: a dual tracing study

Frontiers in Neuroanatomy, 2013

Previous investigations indicate that the superior colliculus (SC) is involved in the initiation ... more Previous investigations indicate that the superior colliculus (SC) is involved in the initiation and execution of forelimb movements. In the present study we investigated the tectofugal, in particular the tecto-reticulo-spinal projections to the shoulder and arm muscles in the rat. We simultaneously retrogradely labeled the premotor neurons in the brainstem by injection of the pseudorabies virus PrV Bartha 614 into the m. rhomboideus minor and m. acromiodeltoideus, and anterogradely visualized the tectofugal projections by intracollicular injection of the tracer FITC dextrane. Our results demonstrate that the connection of the SC to the skeletal muscles of the forelimb is at least trisynaptic. This was confirmed by long survival times after virus injections into the muscles (98-101 h) after which numerous neurons in the deep layers of the SC were labeled. Transsynaptically retrogradely labeled brainstem neurons connected disynaptically to the injected muscles with adjacent tectal terminals were predominantly located in the gigantocellular nuclear complex of the reticular formation. In addition, putative relay neurons were found in the caudal part of the pontine reticular nucleus. Both tectal projections to the nucleus gigantocellularis and the pontine reticular nucleus were bilateral but ipsilaterally biased. We suggest this projection to be involved in more global functions in motivated behavior like general arousal allowing fast voluntary motor activity.

Research paper thumbnail of Muscarinic receptors modulate dendrodendritic inhibitory synapses to sculpt glomerular output

The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 8, 2015

Cholinergic [acetylcholine (ACh)] axons from the basal forebrain innervate olfactory bulb glomeru... more Cholinergic [acetylcholine (ACh)] axons from the basal forebrain innervate olfactory bulb glomeruli, the initial site of synaptic integration in the olfactory system. Both nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors (mAChRs) are expressed in glomeruli. The activation of nAChRs directly excites both mitral/tufted cells (MTCs) and external tufted cells (ETCs), the two major excitatory neurons that transmit glomerular output. The functional roles of mAChRs in glomerular circuits are unknown. We show that the restricted glomerular application of ACh causes rapid, brief nAChR-mediated excitation of both MTCs and ETCs in the mouse olfactory bulb. This excitation is followed by mAChR-mediated inhibition, which is blocked by GABAA receptor antagonists, indicating the engagement of periglomerular cells (PGCs) and/or short axon cells (SACs), the two major glomerular inhibitory neurons. Indeed, selective activation of glomerular mAChRs, with ionotropic Glu...

Research paper thumbnail of Functional imaging of cortical feedback projections to the olfactory bulb

Frontiers in Neural Circuits, 2014

Processing of sensory information is substantially shaped by centrifugal, or feedback, projection... more Processing of sensory information is substantially shaped by centrifugal, or feedback, projections from higher cortical areas, yet the functional properties of these projections are poorly characterized. Here, we used genetically-encoded calcium sensors (GCaMPs) to functionally image activation of centrifugal projections targeting the olfactory bulb (OB). The OB receives massive centrifugal input from cortical areas but there has been as yet no characterization of their activity in vivo. We focused on projections to the OB from the anterior olfactory nucleus (AON), a major source of cortical feedback to the OB. We expressed GCaMP selectively in AON projection neurons using a mouse line expressing Cre recombinase (Cre) in these neurons and Cre-dependent viral vectors injected into AON, allowing us to image GCaMP fluorescence signals from their axon terminals in the OB. Electrical stimulation of AON evoked large fluorescence signals that could be imaged from the dorsal OB surface in vivo. Surprisingly, odorants also evoked large signals that were transient and coupled to odorant inhalation both in the anesthetized and awake mouse, suggesting that feedback from AON to the OB is rapid and robust across different brain states. The strength of AON feedback signals increased during wakefulness, suggesting a state-dependent modulation of cortical feedback to the OB. Two-photon GCaMP imaging revealed that different odorants activated different subsets of centrifugal AON axons and could elicit both excitation and suppression in different axons, indicating a surprising richness in the representation of odor information by cortical feedback to the OB. Finally, we found that activating neuromodulatory centers such as basal forebrain drove AON inputs to the OB independent of odorant stimulation. Our results point to the AON as a multifunctional cortical area that provides ongoing feedback to the OB and also serves as a descending relay for other neuromodulatory systems.

Research paper thumbnail of Connections of the superior colliculus to shoulder muscles of the rat: a dual tracing study

Frontiers in Neuroanatomy, 2013

Previous investigations indicate that the superior colliculus (SC) is involved in the initiation ... more Previous investigations indicate that the superior colliculus (SC) is involved in the initiation and execution of forelimb movements. In the present study we investigated the tectofugal, in particular the tecto-reticulo-spinal projections to the shoulder and arm muscles in the rat. We simultaneously retrogradely labeled the premotor neurons in the brainstem by injection of the pseudorabies virus PrV Bartha 614 into the m. rhomboideus minor and m. acromiodeltoideus, and anterogradely visualized the tectofugal projections by intracollicular injection of the tracer FITC dextrane. Our results demonstrate that the connection of the SC to the skeletal muscles of the forelimb is at least trisynaptic. This was confirmed by long survival times after virus injections into the muscles (98-101 h) after which numerous neurons in the deep layers of the SC were labeled. Transsynaptically retrogradely labeled brainstem neurons connected disynaptically to the injected muscles with adjacent tectal terminals were predominantly located in the gigantocellular nuclear complex of the reticular formation. In addition, putative relay neurons were found in the caudal part of the pontine reticular nucleus. Both tectal projections to the nucleus gigantocellularis and the pontine reticular nucleus were bilateral but ipsilaterally biased. We suggest this projection to be involved in more global functions in motivated behavior like general arousal allowing fast voluntary motor activity.

Research paper thumbnail of Chemosensory properties of murine nasal and cutaneous trigeminal neurons identified by viral tracing

BMC Neuroscience, 2006

Background: Somatosensation of the mammalian head is mainly mediated by the trigeminal nerve that... more Background: Somatosensation of the mammalian head is mainly mediated by the trigeminal nerve that provides innervation of diverse tissues like the face skin, the conjunctiva of the eyes, blood vessels and the mucouse membranes of the oral and nasal cavities. Trigeminal perception encompasses thermosensation, touch, and pain. Trigeminal chemosensation from the nasal epithelia mainly evokes stinging, burning, or pungent sensations. In vitro characterization of trigeminal primary sensory neurons derives largely from analysis of complete neuronal populations prepared from sensory ganglia. Thus, functional properties of primary trigeminal afferents depending on the area of innervation remain largely unclear.

Research paper thumbnail of Imaging Activity in Neurons and Glia with a Polr2a-Based and Cre-Dependent GCaMP5G-IRES-tdTomato Reporter Mouse

Research paper thumbnail of Cholinergic Inputs from Basal Forebrain Add an Excitatory Bias to Odor Coding in the Olfactory Bulb

Journal of Neuroscience, 2014

Cholinergic modulation of central circuits is associated with active sensation, attention, and le... more Cholinergic modulation of central circuits is associated with active sensation, attention, and learning, yet the neural circuits and temporal dynamics underlying cholinergic effects on sensory processing remain unclear. Understanding the effects of cholinergic modulation on particular circuits is complicated by the widespread projections of cholinergic neurons to telencephalic structures that themselves are highly interconnected. Here we examined how cholinergic projections from basal forebrain to the olfactory bulb (OB) modulate output from the first stage of sensory processing in the mouse olfactory system. By optogenetically activating their axons directly in the OB, we found that cholinergic projections from basal forebrain regulate OB output by increasing the spike output of presumptive mitral/tufted cells. Cholinergic stimulation increased mitral/tufted cell spiking in the absence of inhalation-driven sensory input and further increased spiking responses to inhalation of odorless air and to odorants. This modulation was rapid and transient, was dependent on local cholinergic signaling in the OB, and differed from modulation by optogenetic activation of cholinergic neurons in basal forebrain, which led to a mixture of mitral/tufted cell excitation and suppression. Finally, bulbar cholinergic enhancement of mitral/tufted cell odorant responses was robust and occurred independent of the strength or even polarity of the odorant-evoked response, indicating that cholinergic modulation adds an excitatory bias to mitral/tufted cells as opposed to increasing response gain or sharpening response spectra. These results are consistent with a role for the basal forebrain cholinergic system in dynamically regulating the sensitivity to or salience of odors during active sensing of the olfactory environment.

Research paper thumbnail of Transgene Expression in Target-Defined Neuron Populations Mediated by Retrograde Infection with Adeno-Associated Viral Vectors

Journal of Neuroscience, 2013

Tools enabling the manipulation of well defined neuronal subpopulations are critical for probing ... more Tools enabling the manipulation of well defined neuronal subpopulations are critical for probing complex neuronal networks. Cre recombinase (Cre) mouse driver lines in combination with the Cre-dependent expression of proteins using viral vectors--in particular, recombinant adeno-associated viral vectors (rAAVs)--have emerged as a widely used platform for achieving transgene expression in specified neural populations. However, the ability of rAAVs to further specify neuronal subsets on the basis of their anatomical connectivity has been reported as limited or inconsistent. Here, we systematically tested a variety of widely used neurotropic rAAVs for their ability to mediate retrograde gene transduction in the mouse brain. We tested pseudotyped rAAVs of several common serotypes (rAAV 2/1, 2/5, and 2/9) as well as constructs both with and without Cre-dependent expression switches. Many of the rAAVs tested--in particular, though not exclusively, Cre-dependent vectors--showed a robust capacity for retrograde infection and transgene expression. Retrograde expression was successful over distances as large as 6 mm and in multiple neuron types, including olfactory projection neurons, neocortical pyramidal cells projecting to distinct targets, and corticofugal and modulatory projection neurons. Retrograde infection using transgenes such as ChR2 allowed for optical control or optically assisted electrophysiological identification of neurons defined genetically as well as by their projection target. These results establish a widely accessible tool for achieving combinatorial specificity and stable, long-term transgene expression to isolate precisely defined neuron populations in the intact animal.

Research paper thumbnail of Nasal Chemosensory-Stimulation Evoked Activity Patterns in the Rat Trigeminal Ganglion Visualized by In Vivo Voltage-Sensitive Dye Imaging

PLoS ONE, 2011

Mammalian nasal chemosensation is predominantly mediated by two independent neuronal pathways, th... more Mammalian nasal chemosensation is predominantly mediated by two independent neuronal pathways, the olfactory and the trigeminal system. Within the early olfactory system, spatiotemporal responses of the olfactory bulb to various odorants have been mapped in great detail. In contrast, far less is known about the representation of volatile chemical stimuli at an early stage in the trigeminal system, the trigeminal ganglion (TG), which contains neurons directly projecting to the nasal cavity. We have established an in vivo preparation that allows high-resolution imaging of neuronal population activity from a large region of the rat TG using voltage-sensitive dyes (VSDs). Application of different chemical stimuli to the nasal cavity elicited distinct, stimulus-category specific, spatiotemporal activation patterns that comprised activated as well as suppressed areas. Thus, our results provide the first direct insights into the spatial representation of nasal chemosensory information within the trigeminal ganglion imaged at high temporal resolution.

Research paper thumbnail of Neuronal pathways of viral invasion in mice after intranasal inoculation of pseudorabies virus PrV-9112C2 expressing bovine herpesvirus 1 glycoprotein B

Journal of Neurovirology, 2006

give evidence for viral entry receptors present in swine but not mice OSNs. Journal of NeuroVirol... more give evidence for viral entry receptors present in swine but not mice OSNs. Journal of NeuroVirology (2006) 12, 60-64.

Research paper thumbnail of Anterograde transsynaptic tracing in the murine somatosensory system using Pseudorabies virus (PrV): A “live-cell”-tracing tool for analysis of identified neurons in vitro

Journal of Neurovirology, 2007

The Pseudorabies virus (PrV) strain Bartha is widely used as a tool for retrograde transneuronal ... more The Pseudorabies virus (PrV) strain Bartha is widely used as a tool for retrograde transneuronal tracing in mammals. Traced neurons can be identified in cell culture allowing the analysis of their physiological features ("live-cell"-tracing). Compared to PrV-Bartha, PrV-Kaplan is known for higher virulence and transsynaptic spread in both retrograde and anterograde direction. Herein we assess the authors assess PrV-Kaplan for transsynaptic anterograde "live-cell"-tracing. Following intranasal application in mice, labelled trigeminal and brainstem neurons could be identified in vitro. Detailed electrophysiological analysis indicated that viral infection did not affect neuronal properties, making PrV-Kaplan eligible for functional analysis of identified neurons within somatosensory systems.

Research paper thumbnail of Advanced tracing tools: functional neuronal expression of virally encoded fluorescent calcium indicator proteins

Journal of Neurovirology, 2009

Pseudorabies virus (PrV) strains such as PrV-Bartha and its marker protein-expressing variants ha... more Pseudorabies virus (PrV) strains such as PrV-Bartha and its marker protein-expressing variants have been used in numerous studies as retrograde transneuronal tracing tools, defining the synaptic organization of mammalian neuronal circuits. However, the possibilities for functional examination of virus-infected neurons are limited to electrophysiological approaches or bulk loading strategies using calcium-sensitive dyes. Herein we report the generation and functional characterization of three PrV-Bartha-derived recombinant virus mutants that express different fluorescent calcium indicator proteins (FCIPs). All three generated virus recombinants are able to infect murine trigeminal neurons and express the corresponding FCIP (GCaMP2, camgaroo-2, or inverse pericam). Functionality of these virally expressed constructs was verified by using confocal Ca-imaging technologies. These FCIP-expressing virus recombinants provide a new tool for the functional analysis of whole circuits of synaptically connected neurons in vitro and in vivo.

Research paper thumbnail of Chemosensory information processing between keratinocytes and trigeminal neurons

Journal of Biological Chemistry, 2014

Trigeminal fibers terminate within the facial mucosa and skin and transmit tactile, proprioceptiv... more Trigeminal fibers terminate within the facial mucosa and skin and transmit tactile, proprioceptive, chemical, and nociceptive sensations. Trigeminal sensations can arise from the direct stimulation of intraepithelial free nerve endings or indirectly through information transmission from adjacent cells at the peripheral innervation area. For mechanical and thermal cues, communication processes between skin cells and somatosensory neurons have already been suggested. High concentrations of most odors typically provoke trigeminal sensations in vivo but surprisingly fail to activate trigeminal neuron monocultures. This fact favors the hypothesis that epithelial cells may participate in chemodetection and subsequently transmit signals to neighboring trigeminal fibers. Keratinocytes, the major cell type of the epidermis, express various receptors that enable reactions to multiple environmental stimuli. Here, using a co-culture approach, we show for the first time that exposure to the odorant chemicals induces a chemical communication between human HaCaT keratinocytes and mouse trigeminal neurons. Moreover, a supernatant analysis of stimulated keratinocytes and subsequent blocking experiments with pyrodoxalphosphate-6-azophenyl-2',4'-disulfonate revealed that ATP serves as the mediating transmitter molecule released from skin cells after odor stimulation. We show that the ATP release resulting from Javanol® stimulation of keratinocytes was mediated by pannexins. Consequently, keratinocytes act as chemosensors linking the environment and the trigeminal system via ATP signaling.

Research paper thumbnail of In vivo monitoring of chemically evoked activity patterns in the rat trigeminal ganglion

Frontiers in Systems Neuroscience, 2013

Albeit lacking a sense of smell, anosmic patients maintain a reduced ability to distinguish diffe... more Albeit lacking a sense of smell, anosmic patients maintain a reduced ability to distinguish different volatile chemicals by relying exclusively on their trigeminal system (TS). To elucidate differences in the neuronal representation of these volatile substances in the TS, we performed voltage-sensitive dye imaging (VSDI) in the rat trigeminal ganglion (TG) in vivo. We demonstrated that stimulus-specific patterns of bioelectrical activity occur within the TG upon nasal administration of ten different volatile chemicals. With regard to spatial differences between the evoked trigeminal response patterns, these substances could be sorted into three groups. Signal intensity and onset latencies were also dependent on the administered stimulus and its concentration. We conclude that particular compounds detected by the TS are represented by (1) a specific spatial response pattern, (2) the signal intensity, and (3) onset latencies within the pattern. Jointly, these trigeminal representations may contribute to the surprisingly high discriminative skills of anosmic patients.

Research paper thumbnail of Connections of the superior colliculus to shoulder muscles of the rat: a dual tracing study

Frontiers in Neuroanatomy, 2013

Previous investigations indicate that the superior colliculus (SC) is involved in the initiation ... more Previous investigations indicate that the superior colliculus (SC) is involved in the initiation and execution of forelimb movements. In the present study we investigated the tectofugal, in particular the tecto-reticulo-spinal projections to the shoulder and arm muscles in the rat. We simultaneously retrogradely labeled the premotor neurons in the brainstem by injection of the pseudorabies virus PrV Bartha 614 into the m. rhomboideus minor and m. acromiodeltoideus, and anterogradely visualized the tectofugal projections by intracollicular injection of the tracer FITC dextrane. Our results demonstrate that the connection of the SC to the skeletal muscles of the forelimb is at least trisynaptic. This was confirmed by long survival times after virus injections into the muscles (98-101 h) after which numerous neurons in the deep layers of the SC were labeled. Transsynaptically retrogradely labeled brainstem neurons connected disynaptically to the injected muscles with adjacent tectal terminals were predominantly located in the gigantocellular nuclear complex of the reticular formation. In addition, putative relay neurons were found in the caudal part of the pontine reticular nucleus. Both tectal projections to the nucleus gigantocellularis and the pontine reticular nucleus were bilateral but ipsilaterally biased. We suggest this projection to be involved in more global functions in motivated behavior like general arousal allowing fast voluntary motor activity.

Research paper thumbnail of Muscarinic receptors modulate dendrodendritic inhibitory synapses to sculpt glomerular output

The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 8, 2015

Cholinergic [acetylcholine (ACh)] axons from the basal forebrain innervate olfactory bulb glomeru... more Cholinergic [acetylcholine (ACh)] axons from the basal forebrain innervate olfactory bulb glomeruli, the initial site of synaptic integration in the olfactory system. Both nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors (mAChRs) are expressed in glomeruli. The activation of nAChRs directly excites both mitral/tufted cells (MTCs) and external tufted cells (ETCs), the two major excitatory neurons that transmit glomerular output. The functional roles of mAChRs in glomerular circuits are unknown. We show that the restricted glomerular application of ACh causes rapid, brief nAChR-mediated excitation of both MTCs and ETCs in the mouse olfactory bulb. This excitation is followed by mAChR-mediated inhibition, which is blocked by GABAA receptor antagonists, indicating the engagement of periglomerular cells (PGCs) and/or short axon cells (SACs), the two major glomerular inhibitory neurons. Indeed, selective activation of glomerular mAChRs, with ionotropic Glu...

Research paper thumbnail of Functional imaging of cortical feedback projections to the olfactory bulb

Frontiers in Neural Circuits, 2014

Processing of sensory information is substantially shaped by centrifugal, or feedback, projection... more Processing of sensory information is substantially shaped by centrifugal, or feedback, projections from higher cortical areas, yet the functional properties of these projections are poorly characterized. Here, we used genetically-encoded calcium sensors (GCaMPs) to functionally image activation of centrifugal projections targeting the olfactory bulb (OB). The OB receives massive centrifugal input from cortical areas but there has been as yet no characterization of their activity in vivo. We focused on projections to the OB from the anterior olfactory nucleus (AON), a major source of cortical feedback to the OB. We expressed GCaMP selectively in AON projection neurons using a mouse line expressing Cre recombinase (Cre) in these neurons and Cre-dependent viral vectors injected into AON, allowing us to image GCaMP fluorescence signals from their axon terminals in the OB. Electrical stimulation of AON evoked large fluorescence signals that could be imaged from the dorsal OB surface in vivo. Surprisingly, odorants also evoked large signals that were transient and coupled to odorant inhalation both in the anesthetized and awake mouse, suggesting that feedback from AON to the OB is rapid and robust across different brain states. The strength of AON feedback signals increased during wakefulness, suggesting a state-dependent modulation of cortical feedback to the OB. Two-photon GCaMP imaging revealed that different odorants activated different subsets of centrifugal AON axons and could elicit both excitation and suppression in different axons, indicating a surprising richness in the representation of odor information by cortical feedback to the OB. Finally, we found that activating neuromodulatory centers such as basal forebrain drove AON inputs to the OB independent of odorant stimulation. Our results point to the AON as a multifunctional cortical area that provides ongoing feedback to the OB and also serves as a descending relay for other neuromodulatory systems.