Arild Njå

Re-innervation of guinea-pig superior cervical ganglion cells by preganglionic fibres arising from different levels of the spinal cord.

1. The ability of preganglionic axons to re‐establish their normal pattern of synaptic connexions with superior cervical ganglion cells has been studied after section of the cervical sympathetic trunk.

DNA injection into single cells of intact mice.

We describe a technique wherein single muscle fibers of intact mice are made transgenic by intracellular injection of DNA expression vectors for the reporter genes lacZ and green fluorescent protein (GFP). Application of in vivo imaging techniques allowed identification of single cells during the injections, and of the same single cells when the muscle was reexposed days or weeks later. DNA concentration by itself had little effect on fiber survival or expression efficacy, but it seemed crucial to exceed a threshold of about 10(6) injected plasmid molecules in order to obtain expression. On the other hand, experiments with coinjection of two different plasmids suggested that relatively few individual molecules were eventually transcribed in expressing cells. Plasmid DNA remained localized to the injection site, and expression was confined to nuclei in the vicinity. Expression was stable, as reporter was detected 2-61 days after injection.

Targeting functional subtypes of spinal motoneurons and skeletal muscle fibers in vivo by intramuscular injection of adenoviral and adeno-associated viral vectors

We report that functional subtypes of spinal motoneurons and skeletal muscle fibers can be selectively transduced using replication-defective adenoviral (ADV) or adeno-associated (AAV) viral vectors. After intramuscular injection in adult rodents, ADV vectors transduced both fast-twitch and slow-twitch skeletal muscle fibers. Intramuscular injection of ADV vectors also caused transduction of spinal motoneurons and dorsal root ganglion cells. However, only neurons innervating the injected muscle were transduced, as shown by co-injection of a retrograde axonal tracer. In adult male rats it is therefore possible to transduce fast or slow spinal motoneurons and muscle fibers selectively since in these animals, the extensor digitorum longus and soleus muscles contain almost exclusively fast or slow motor units, respectively. In rats, AAV vectors transduced muscle fibers in the predominantly fast extensor digitorum longus but not in the predominantly slow soleus muscle. We did not observe any transduction of spinal motoneurons following intramuscular injection of AAV vectors. These results show that physiologically and clinically important subpopulations of cells in the neuromuscular system can be selectively transduced by viral vectors.

The homeodomain transcription factors Islet 1 and HB9 are expressed in adult alpha and gamma motoneurons identified by selective retrograde tracing

To study gene expression in differentiated adult motoneuron subtypes, we used fluorescent dextrans for both anterograde and retrograde axonal tracing in adult rat and mouse. Application of these dyes to the cut distal and proximal ends of small extramuscular nerve branches revealed both the peripheral ramifications and the cell bodies of subsets of motoneurons. We show that the soleus muscle is innervated by two nerve branches, one of which contains gamma motor and sensory axons but no alpha motor axons. By retrograde tracing of this branch, we selectively labelled gamma motoneurons. In adult rat, the nerves innervating the soleus and extensor digitorum longus muscles contain almost exclusively axons innervating slow (type I) and fast (type 2) muscle fibres, respectively. We selectively labelled slow and fast type motoneurons by retrograde tracing of these nerves. With immunocytochemistry we show that adult motoneurons express several homeodomain genes that are associated with motoneuron differentiation during early embryonic development. Combining selective retrograde labelling with immunocytochemistry we compared the expression patterns in alpha and gamma motoneurons. The homeodomain transcription factors Islet 1 and HB9 were expressed in slow and fast alpha motoneurons and in soleus gamma motoneurons. Motoneurons in each population varied in their intensity of the immunostaining, but no factor or combination of factors was unique to any one population.

Two types of synaptic selectivity and their interrelation during sprouting in the guinea‐pig superior cervical ganglion.

1. The synaptic connexions of the guinea‐pig superior cervical ganglion were examined after collateral sprouting provoked by cutting the preganglionic nerve supply from spinal segments T3‐T7. The selective properties of the connexions made by the remaining segments C8, T1 and T2 were explored both with respect to the segmental origin of the preganglionic axons (Njå & Purves, 1977 a) and with respect to their conduction velocity (Wigston, 1983). 2. After sprouting, the ventral root T1 elicited strong sympathetic effects on both the eye and the ear, while the normal response is largely confined to the region of the eye. This effect on the expressed selectivity was confirmed by intracellular recording in vitro, which showed that virtually all the ganglion cells were now innervated by T1. 3. However, neurones that received dominant innervation from T1 were more frequently innervated by sprouted axons from C8 than were neurones whose dominant innervation derived from T2. This indicates that basic mechanisms promoting segmental selectivity were still functioning. 4. The selective innervation of ganglion cells with respect to the conduction velocity of the preganglionic axons was well maintained after sprouting. 5. These results show that during sprouting from a restricted set of preganglionic axons the synaptic partners are still matched according to both types of selectivity. This suggests that the recognition mechanisms which guide neural development are preserved in adult life, but that the differences in the available sets of preganglionic axons lead to different competitive interactions and different resulting patterns of innervation.

Glutamatergic modulation of synaptic-like vesicle recycling in mechanosensory lanceolate nerve terminals of mammalian hair follicles

•  The lanceolate sensory nerve ending of hair follicles is known to contain small (∼50 nm), clear vesicles similar to those of presynaptic terminals, but of unknown function. •  We show that the sensory terminals spontaneously take up and release the fluorescent styryl dye FM1‐43, and also provide other evidence that the dye flux is primarily by recycling of these synaptic‐like vesicles (SLVs). •  FM1‐43 labelling is Ca2+ dependent, and its release is sensitive to α‐latrotoxin, which is known to deplete synaptic vesicles at neuromuscular junctions. •  Responses of hair follicle afferents are not significantly affected by FM1‐43 at a concentration (10 μm) sufficient to label the endings, so the mechanotransduction channel that has previously been shown to be blocked by FM1‐43 permeation in hair cells of the inner ear and in cultured dorsal root ganglion cells is either not responsible for sensory transduction in the lanceolate ending or is in some way protected from exposure to the dye. •  The sensory terminals are relatively enriched in glutamate, presumably within the vesicles. •  Exogenous glutamate increases FM1‐43 labelling, whereas the labelling is strongly inhibited by PCCG‐13, a specific blocker of a non‐canonical phospholipase D‐linked metabotropic glutamate receptor, but not by canonical ionotropic or metabotropic glutamate receptor blockers. It is also inhibited by FIPI, a novel phospholipase D inhibitor. •  The system of SLVs is closely similar to that we have previously described in the muscle spindle, and where we further demonstrated the regulatory action of glutamate on the sensory response to maintained stretch. •  We conclude that an SLV‐mediated glutamatergic system is present in the mechanosensory endings of the primary afferents of lanceolate endings, and it appears to function in a similar way to the autoregulatory system of the muscle spindle.

A microcapsule technique for long-term conduction block of the sciatic nerve by tetrodotoxin.

Tetrodotoxin (TTX) is a selective blocker of voltage-gated Na+ channels that is used to block action potentials in vitro and in vivo. Maintaining a sufficiently high local concentration of TTX in vivo to block conduction in a peripheral nerve is technically demanding and carries a risk of systemic toxicity. We report that slow diffusion of TTX out of a microcapsule (glass capillary) inserted beneath the epineurium of the sciatic nerve, with a loose cuff around the nerve, combines high blocking efficacy with low systemic toxicity in rats and mice. The local anaesthesia and motor paralysis was stable for at least 4-6 weeks. The conduction block was reversible and did not cause any obvious nerve injury. Low cost and simple surgical implementation make this new system an interesting alternative to existing long-term drug delivery methods.

Preserved granulocyte formation and function, as well as bone marrow innervation, in subjects with complete spinal cord injury.

Patients with a spinal cord injury are at risk of infections and is partly attributed to immobilization. Their lymphocyte‐mediated immunity is impaired and the growth of blood progenitor cells is reduced. An adequate immune response depends on granulocytes being mobilized rapidly and activated properly, at the inflammatory site. Possibly this requires a coordinated interaction between the autonomous nervous system and cells within the haematopoietic bone marrow. Granulocyte function in the spinal cord injured has not been evaluated. Although there is evidence that the bone marrow in rodents is innervated, it is uncertain whether human bone marrow is similarly affected. Microscopy and immunolabelling followed by flow cytometry, showed that blood and bone marrow counts of leucocyte subsets were similar in paraplegic, tetraplegic and control subjects (P > 0·05). Neutrophilic migration and oxygen consumption, as well as eosinophil activation, assayed as release of eosinophilic cationic protein or CD69 expression, were not altered after spinal cord injury (P > 0·05). Cryostat sections of human bone marrow biopsies stained positive with glyoxylic acid, indicating the presence of catecholamine‐containing nerves in both the patients and the controls. We conclude that terminal differentiation and formation of granulocytes, as well as their functional capacity, do not depend appreciably on supraspinal nervous regulation.

The selective innervation of guinea‐pig superior cervical ganglion cells by sprouts from intact preganglionic axons.

If the guinea‐pig superior cervical ganglion is partially denervated in a way which spares some preganglionic axons arising from each of the spinal cord segments which normally innervate this ganglion (C8‐T7), sprouting of the intact preganglionic axons occurs without a loss of selective end‐organ responses to stimulation of individual ventral roots (Maehlen & Njå, 1981). In the present work we examine the selective innervation of individual ganglion cells by ventral roots after sprouting and compare it to that in normal ganglia. After sprouting the pattern of ganglion cell innervation by the intact preganglionic axons shows some of the features typical of normal ganglia. Thus each ganglion cell is strongly innervated by one or two neighbouring spinal cord segments, with the adjacent segments contributing a synaptic influence which diminishes with distance from the dominant one. Acutely after the partial denervation there is a tendency for the rostral segments to innervate ganglion cells more strongly than do caudal segments, compared to the situation in normal ganglia. The same is true following sprouting. The pattern of ganglion cell innervation observed after sprouting can be explained if the affinities of ganglion cells for innervation from particular spinal levels are assumed to be unchanged and the shift in the relative availability of different preganglionic axons, caused by the partial denervation, is taken into account. This view was substantiated by statistical analysis based on a model of selective synapse formation in the guinea‐pig superior cervical ganglion. We conclude that the ability of individual ganglion cells to distinguish preganglionic axons arising from different spinal levels is maintained during sprouting. Moreover, the way in which selective recognition and the availability of different preganglionic axons combine to produce particular patterns of innervation appears to be similar in normal development and after partial denervation in maturity.

Selective synaptic connections: significance of recognition and competition in mature sympathetic ganglia

Abstract Neurones in the mammalian superior cervical ganglion are selectively innervated by preganglionic axons arising from different segments of the spinal cord. These connections can be re-established after preganglionic nerve injury in adult life. However, the resulting pattern of innervation is highly dependent on the experimental conditions. This dependency can be used to make inferences about the different mechanisms which combine to guide the formation of neural connections.