Heinz Steffens

Nanoscopy in a living mouse brain.

Super high-resolution microscopy resolves neuron dynamics in the cerebral cortex of a living mouse. We demonstrated superresolution optical microscopy in a living higher animal. Stimulated emission depletion (STED) fluorescence nanoscopy reveals neurons in the cerebral cortex of a mouse with <70-nanometer resolution. Dendritic spines and their subtle changes can be observed at their relevant scales over extended periods of time.

NO mediates microglial response to acute spinal cord injury under ATP control in vivo

To understand the pathomechanisms of spinal cord injuries will be a prerequisite to develop efficient therapies. By investigating acute lesions of spinal cord white matter in anesthetized mice with fluorescently labeled microglia and axons using in vivo two‐photon laser‐scanning microscopy (2P‐LSM), we identified the messenger nitric oxide (NO) as a modulator of injury‐activated microglia. Local tissue damages evoked by high‐power laser pulses provoked an immediate attraction of microglial processes. Spinal superfusion with NO synthase and guanylate cyclase inhibitors blocked these extensions. Furthermore, local injection of the NO‐donor spermine NONOate (SPNO) or the NO‐dependent second messenger cGMP induced efficient migration of microglial cells toward the injection site. High‐tissue levels of NO, achieved by uniform superfusion with SPNO and mimicking extended tissue damage, resulted in a fast conversion of the microglial shape from ramified to ameboid indicating cellular activation. When the spinal white matter was preconditioned by increased, ambient ATP (known as a microglial chemoattractant) levels, the attraction of microglial processes to local NO release was augmented, whereas it was abolished at low levels of tissue ATP. Because both signaling molecules, NO and ATP, mediate acute microglial reactions, coordinated pharmacological targeting of NO and purinergic pathways will be an effective mean to influence the innate immune processes after spinal cord injury.

Synaptic responses of lumbar α-motoneurones to selective stimulation of cutaneous nociceptors and low threshold mechanoreceptors in the spinal cat

Summary1. The reflex projection from afferents of cutaneous nociceptors and low threshold mechanoreceptors onto intracellularly recorded lumbar α-motoneurones was tested in high spinal unanaesthetized cats. Low threshold mechanoreceptors were activated by light stroking of the hairy skin of the foot, nociceptors by radiant heat and in a few cases, for comparison, by pinching of the skin. In each experiment only one cutaneous nerve (Sur, SPC, Saph or Tib) was left intact. 2. Flexor motoneurones (PBSt) generally showed a depolarization together with an increase of synaptic noise to both types of stimulation, the amplitude during nociceptive stimulation being larger in most cases. 3. In extensor motoneurones (GS, ABSm) the responses to noxious and mechanical skin stimulation were more variable. Only a slight depolarization or no change of the level of the membrane potential, together with an increase of synaptic noise, was observed in most cases. Besides that, minor hyperpolarizations or transitions from light depolarization to hyperpolarization were induced during stimulation. The effects of noxious and mechanoreceptive skin stimulation were not strictly related to the effects of electrical stimulation of flexor reflex afferents. 4. It is assumed that particularly for extensor motoneurones, the excitatory and the inhibitory segmental reflex pathways were activated in parallel during skin stimulation. The simultaneous action of the inhibitory pathway in addition to the excitatory one may serve as a mechanism to neutralize unwanted surplus excitation.

Synaptic effects from chemically activated fine muscle afferents upon α-motoneurones in decerebrate and spinal cats

In spinal and decerebrate cats fine muscle afferents (group III and IV) were selectively activated by intra-arterial injection of bradykinin and KCl into the gastrocnemius-soleus muscle. By this method the synaptic responses induced in lumbar alpha-motoneurones by fine muscle afferents could be examined without interference of effects from large afferents. alpha-Motoneurones receiving EPSPs evoked by electrical stimulation of cutaneous and high threshold muscle afferents (mainly flexor motoneurones) responded to the activation of fine muscle afferents with a depolarization of their membrane and an increase in synaptic noise, while motoneurones in which IPSPs were evoked by electrical stimulation of cutaneous and high threshold muscle afferents (mainly extensor motoneurones), responded with hyperpolarization of their membrane. Cells with mixed excitatory-inhibitory electrically induced response characteristic responded with an increase in synaptic noise without substantial change in the level of their membrane potential to chemical stimulation of fine muscle afferents. The results indicate that one function of group III and IV muscle afferents is to participate in the complex reflex control performed by the flexor reflex system.

Contribution of group III and IV muscle afferents to multisensorial spinal motor control in cats.

The contribution of group III and IV muscle afferents to multisensorial segmental reflex pathways was investigated by testing for spatial facilitation between these afferents and non-nociceptive segmental afferents from skin, muscles and joints on postsynaptic potentials (PSPs) in alpha-motoneurones recorded in anaemically decapitated high spinal cats. Group III and IV muscle afferents were activated by intraarterial injection of potassium chloride (320 mM) or bradykinin triacetate (81 microM). Skin, joint and group I-II muscle afferents were stimulated by graded electrical stimulation of various nerves. Conditioning by stimulation of group III and IV muscle afferents spatially facilitated the transmission in segmental reflex pathways from low- to medium-threshold cutaneous and joint afferents as well as from lb and group II muscle afferents. Both excitatory and inhibitory pathways from these afferents were facilitated. Monosynaptic excitation and disynaptic antagonistic inhibition from group Ia afferents remained unaffected. It is concluded that the spatial facilitation observed between group III and IV muscle afferents and the other afferents indicate a convergence from group III and IV muscle afferents and the other afferents on common interneurones in segmental flexor reflex pathways. Under physiological conditions they thus contribute to the multisensorial feedback of the flexor reflex pathways. Pathophysiologically, the observed convergence may aggravate muscle weakness and atrophy of muscles induced by group III and IV muscle afferents.

In Vivo Imaging Reveals Distinct Inflammatory Activity of CNS Microglia versus PNS Macrophages in a Mouse Model for ALS

Mutations in the enzyme superoxide dismutase-1 (SOD1) cause hereditary variants of the fatal motor neuronal disease Amyotrophic lateral sclerosis (ALS). Pathophysiology of the disease is non-cell-autonomous: neurotoxicity is derived not only from mutant motor neurons but also from mutant neighbouring non-neuronal cells. In vivo imaging by two-photon laser-scanning microscopy was used to compare the role of microglia/macrophage-related neuroinflammation in the CNS and PNS using ALS-linked transgenic SOD1G93A mice. These mice contained labeled projection neurons and labeled microglia/macrophages. In the affected lateral spinal cord (in contrast to non-affected dorsal columns), different phases of microglia-mediated inflammation were observed: highly reactive microglial cells in preclinical stages (in 60-day-old mice the reaction to axonal transection was ∼180% of control) and morphologically transformed microglia that have lost their function of tissue surveillance and injury-directed response in clinical stages (reaction to axonal transection was lower than 50% of control). Furthermore, unlike CNS microglia, macrophages of the PNS lack any substantial morphological reaction while preclinical degeneration of peripheral motor axons and neuromuscular junctions was observed. We present in vivo evidence for a different inflammatory activity of microglia and macrophages: an aberrant neuroinflammatory response of microglia in the CNS and an apparently mainly neurodegenerative process in the PNS.

The effect of DOPA and clonidine on reflex pathways from group II muscle afferents to α-motoneurones in the cat.

SummaryIn high spinal cats the transmission in both disynaptic and polysynaptic reflex pathways from group II muscle afferents to motoneurones was effectively depressed by DOPA and clonidine together with an often less pronounced depression of transmission in other FRA reflex pathways. The inhibitory effect of both drugs on the inhibitory group II and FRA pathways was generally less distinct than that on the excitatory ones. The results are in agreement with former observations which suggested that the excitatory and the inhibitory FRA pathways are independently controlled by descending pathways from the brain stem (Holmqvist and Lundberg 1961).

Facilitatory interaction between cutaneous afferents from low threshold mechanoreceptors and nociceptors in segmental reflex pathways to α-motoneurons

EPSPs evoked by air puff stimulation of the hairy skin of the hindlimb or by low strength stimulation of the lateral sural nerve were facilitated by radiant heat of 50-53 degrees C but not by radiant heat of 42-43 degrees C. The results indicate that afferents from low threshold mechanoreceptors and from nociceptors of the skin converge onto common interneurons in reflex pathways to alpha-motoneurons. This would allow a functionally useful cooperation of these two types of afferents in spinal motor control.

Contribution of TTX-resistant C-fibres and Aδ-fibres to nociceptive flexor-reflex and non-flexor-reflex pathways in cats

The contribution of Adelta-fibres and C-fibres activated by noxious heat stimulation of the central pad of the foot to nociceptive spinal flexor reflex pathways (FRA-type) and to nociceptive excitatory reflex pathways to foot extensors (non-FRA type) was investigated in high spinal cats. A-fibres were completely blocked by tetrodotoxin (TTX), leaving C-fibre conduction intact. Thus, effects persisting after TTX were attributed to nociceptive C-fibres while the contribution of nociceptive Adelta-fibres was defined by the difference between those effects and the control effects before TTX. The initial action of noxious stimulation on both types of reflex action was mediated predominantly by Adelta-fibres, while the later action was mainly mediated by C-fibres. In two (out of seven) experiments Adelta-fibres exerted a significant inhibitory influence on the C-fibre action in FRA pathways, but such an inhibitory interaction between the two fibre groups was absent in the non-FRA reflex pathways. The technique of TTX application at the peripheral nerve proved to be a reliable method for a long-lasting selective investigation of C-fibre effects. The results revealed that both Adelta- and C-fibres contributed to nociceptive FRA and non-FRA reflex pathways.

Nanoscopy of filamentous actin in cortical dendrites of a living mouse.

We demonstrate superresolution fluorescence microscopy (nanoscopy) of protein distributions in a mammalian brain in vivo. Stimulated emission depletion microscopy reveals the morphology of the filamentous actin in dendritic spines down to 40 μm in the molecular layer of the visual cortex of an anesthetized mouse. Consecutive recordings at 43-70 nm resolution reveal dynamical changes in spine morphology.