Neuronal calcium-binding proteins 1/2 localize to dorsal root ganglia and excitatory spinal neurons and are regulated by nerve injury.

Neuronal calcium (Ca(2+))-binding proteins 1 and 2 (NECAB1/2) are members of the phylogenetically conserved EF-hand Ca(2+)-binding protein superfamily. To date, NECABs have been explored only to a limited extent and, so far, not at all at the spinal level. Here, we describe the distribution, phenotype, and nerve injury-induced regulation of NECAB1/NECAB2 in mouse dorsal root ganglia (DRGs) and spinal cord. In DRGs, NECAB1/2 are expressed in around 70% of mainly small- and medium-sized neurons. Many colocalize with calcitonin gene-related peptide and isolectin B4, and thus represent nociceptors. NECAB1/2 neurons are much more abundant in DRGs than the Ca(2+)-binding proteins (parvalbumin, calbindin, calretinin, and secretagogin) studied to date. In the spinal cord, the NECAB1/2 distribution is mainly complementary. NECAB1 labels interneurons and a plexus of processes in superficial layers of the dorsal horn, commissural neurons in the intermediate area, and motor neurons in the ventral horn. Using CLARITY, a novel, bilaterally connected neuronal system with dendrites that embrace the dorsal columns like palisades is observed. NECAB2 is present in cell bodies and presynaptic boutons across the spinal cord. In the dorsal horn, most NECAB1/2 neurons are glutamatergic. Both NECAB1/2 are transported into dorsal roots and peripheral nerves. Peripheral nerve injury reduces NECAB2, but not NECAB1, expression in DRG neurons. Our study identifies NECAB1/2 as abundant Ca(2+)-binding proteins in pain-related DRG neurons and a variety of spinal systems, providing molecular markers for known and unknown neuron populations of mechanosensory and pain circuits in the spinal cord.

How the 1906 Nobel Prize in Physiology or Medicine was shared between Golgi and Cajal.

In 1906 the Nobel Prize in Physiology or Medicine was shared between Camillo Golgi and Ramón y Cajal in recognition of their work on the structure of the nervous system. Golgi's most impressive contribution was his method, described in 1873. This was applied in studies of the cerebellum, the olfactory bulb, hippocampus and the spinal cord. These studies together with his earlier work were included in his Opera Omnia, published in 1903. His method was highly praised by Cajal. His adherence to the reticular theory was opposed by Cajal, however, who had spelled out the neuron theory already in the late 1800s. Cajal's extraordinary contributions to the structure of the nervous system, based largely on the Golgi method and Ehrlich's methylene blue stain, were published in his Textura del Sistema Nerviosa de Hombre y de los Vertebrados, three volumes published from 1897 to 1904. Documents from the Nobel Archives reveal that Kölliker, Retzius and Fürst were the ones who proposed Golgi and Cajal for a shared prize. Golgi was nominated by Hertwig, as well. Cajal was proposed by Ziehen and Holmgren, and also by Retzius, as an alternative to a shared prize. Holmgren, who was commissioned to write the report to the Nobel Committee, found Cajal far superior to Golgi. Sundberg, asked for another evaluation, was more positive to Golgi's contributions than Holmgren. Gadelius supported Holmgren's views. The final vote gave a majority for a shared prize. The prize ceremony and the lectures were described in detail in Cajal's autobiography.

The 1932 and 1944 Nobel Prizes in physiology or medicine: rewards for ground-breaking studies in neurophysiology.

In 1932 Sherrington and Adrian were awarded the Nobel Prize in Physiology or Medicine "for their discoveries regarding the functions of neurons" and in 1944 Erlanger and Gasser were awarded the same prize "for their discoveries relating to the highly differentiated functions of single nerve fibres." Sherrington made important discoveries on the reflex functions of the spinal cord, formulated the concept of the "synapse," defined the principle of the "final common path," studied "reciprocal innervation" and showed that central inhibition was an active phenomenon. He distinguished three types of receptors: extero-, intero-, and proprioceptive, studied the proprioceptive reflexes in the decerebrate animal and mapped their pathways in the spinal cord. Adrian made fundamental discoveries on the function of single nerve fibers, developed new techniques for the amplification of the weak signals and discovered that increased stimulation resulted in increased frequency of the impulses, the amplitude being unaffected. Erlanger and Gasser introduced the cathode-ray oscillograph and demonstrated the existence of three main groups of nerve fibers, A, B, and C, the conduction velocities of which were in approximately linear relationship with the fiber diameter, the A-fibers being the fastest and thickest and the C-fibers the slowest and having the finest diameter. Together the contributions by the four Laureates paved the way to modern neurophysiology.

Suppressive silver methods--a tool for identifying axotomy-induced neuron degeneration.

Suppressive silver methods evolved from empirical observations about 50 years ago that argyrophilia of normal nerve fibers can be suppressed by a short period of oxidation of tissue sections, whereas degenerating nerve fibers in the same preparations were still clearly visible. Based on this property, suppressive silver impregnation became the main technique for investigating pathways in the central nervous system until the early 1970s. Suppressive silver methods were also found to visualize degenerating nerve cell bodies, in addition to degenerating nerve fibers. This possibility has given these methods an important place among current tools for identifying neuronal degeneration in trauma, disease and toxicity. In this article we demonstrate and review the usefulness of suppressive silver methods in identifying neurons undergoing degeneration as a result of peripheral or central axon injury in immature animals. The documentation is based on previously published data from experiments in which silver impregnation was used to demonstrate degeneration of motoneurons following pure motor axon injury or mixed peripheral nerve injury, as well as on new results on degeneration-induced argyrophilia in the inferior olive following cerebellar lesions. We find that silver precipitates resulting from these injuries are localized either to the entire neuronal cytoplasm, to a few (typically two) intranuclear bodies, or to both sites. The findings are discussed in relation to morphological features of apoptosis, necrosis and retrograde neuronal responses. We suggest that suppressive silver methods allow visualization of different processes of neuronal degeneration, and therefore may be a useful adjunct for identifying axotomy-induced neuronal degeneration.

Course of spinocerebellar axons in the ventral and lateral funiculi of the spinal cord with projections to the posterior cerebellar termination area: an experimental anatomical study in the cat, using a retrograde tracing technique.

The course of retrogradely labeled spinocerebellar fibers in the ventral and lateral funiculi of the spinal cord was studied following injections of wheat germ agglutinin-conjugated horseradish peroxidase into the posterior spinocerebellar termination area in the cat. Fibers labeled from unilateral injections into the paramedian lobule were found on the same side in the dorsal part of the lateral funiculus (DLF), corresponding to the dorsal spinocerebellar tract (DSCT), but contralaterally in the ventral part of the lateral funiculus (VLF) and in the ventral funiculus (VF), corresponding to the ventral spinocerebellar tract (VSCT). Following injections into the posterior vermis, labeled fibers were less numerous. Most of them were found in the DSCT and only very few in the VSCT. Previously identified cells of origin of these spinocerebellar tracts were labeled in these experiments and counted. They correlated well with the extents and the locations of the injections that had been made into the two termination sites. These results represent novel detailed information on the location of axons projecting to the two main posterior spinocerebellar termination sites in the spinal white matter in the cat.

Brainstem projections of different branches of the vestibular nerve: an experimental study by transganglionic transport of horseradish peroxidase in the cat. III. The saccular nerve.

The brainstem projection of the saccular nerve was investigated by transganglionic tracing with horseradish peroxidase in the cat. The labeled fibers were located most caudally and laterally (dorsolaterally) in the vestibular nerve root. This was unique when compared with the locations of the fibers from the nerves of the other vestibular-end organs that were studied by the present authors by the same approach previously. In addition, such a comparison revealed a specific location, from lateral to medial, for the fibers from each of the five divisions of the vestibular nerve. In the present study, labeled fibers from the sacculus, of fine caliber, were found close to the restiform body, both medially and laterally, some even penetrating through this structure. Labeled terminals were present in cell group "y". This was unique, compared with the nerves from the other end organs. Such terminals were also found in the four main vestibular nuclei, except for the medial vestibular nucleus, where no labeled terminals could be detected. No labeled terminals were found in the interstitial nucleus of the vestibular nerve. Together with the findings from our previous studies, this suggests that, in contrast to the ampullar nerves, the nerves from the maculae do not project to this structure. This study confirms, but also extends, findings reported from a previous investigation in the cat, using an experimental degeneration technique.

GABA-, glycine-, and glutamate-immunoreactive bouton profiles in apposition to neurons of the central cervical nucleus in the rat.

The neurons of the central cervical nucleus (CCN) convey information about the position and movements of the head, and receive excitatory input from dorsal neck muscles and the labyrinth. Both of these afferent sources form glutamatergic synaptic contacts with CCN neurons. However, these sensory afferent sources can also inhibit CCN neurons. To further elucidate the synaptic organization, we made an electron microscopic investigation, identifying and evaluating the relative frequency of bouton profiles containing the inhibitory transmitters GABA and glycine in apposition to identified CCN neurons. In addition, labeling for glutamate was performed. The identification of the CCN neurons was made possible by injections of retrograde tracer substances into the cerebellum. These substances were made visible by preembedding immunocytochemistry or postembedding immunogold staining. Such staining was also used to detect the three amino acids that were found in boutons apposed to the identified neurons (cf. Ornung et al., J. Comp. Neurol. 1996;365:413-426; Lindå et al., J. Comp. Neurol. 2000;425:10-23). Due to the relatively poor transport of the tracer substances into dendrites of the CCN neurons, the analysis was restricted to the cell body and included bouton profiles in direct apposition to the soma membrane. Data from 10 CCN neurons revealed that about 50% of the apposing bouton profiles were immunoreactive for GABA, and about 34% for glycine. In four neurons, the degree of colocalization of GABA and glycine was determined to be close to 30%. Thus, the vast majority of glycine-labeled profiles also contained GABA, while a considerable fraction of the profiles were immunoreactive for only GABA. The values for glycine immunoreactive bouton profiles presented here may represent somewhat low estimates, depending on the method used. Data from four neurons showed that about 18% of the profiles were labeled for glutamate. The large fraction of purely GABA immunoreactive profiles, or at least a substantial group of them, is suggestive of their derivation from axons descending from the brainstem.

Glutamate and AMPA receptor immunoreactivity in Ia synapses with motoneurons and neurons of the central cervical nucleus.

Axonal tracing and high resolution immunocytochemistry were used to identify transmitter content and postsynaptic receptors in synapses between Ia primary afferents and motoneurons and in neurons of the central cervical nucleus (CCN), respectively, in the rat. The terminals, as well as the target neurons, were identified by postembedding immunogold detection of transganglionically or retrogradely, respectively, transported cholera toxin B subunit (CTB), and in adjacent sections postembedding immunogold was employed to demonstrate glutamate and AMPA receptors in the same synapses. A total of 390 CTB-labelled Ia boutons in apposition to CTB-labelled motoneurons, CCN neurons or unlabelled dendrites in the surrounding neuropil were traced in section series from two animals. A third animal was used as a control. In the motor nucleus, a majority of the synapses were with medium-sized dendrites, whereas in the CCN the distribution was skewed towards fine-calibre dendrites. In both nuclei, somatic and juxtasomatic synapses were quite infrequent (<10%). All of the CTB-labelled Ia boutons recovered in the sections incubated for glutamate (n=323) were enriched with glutamate immunoreactivity. One hundred and fifty of these disclosed synaptic contact in at least two ultrathin sections. In this sample, 50% (33-59%) appeared immunoreactive to receptor sub-units GluR1-4 in at least two ultrathin sections, whereas 35% were labelled in one section only. Distribution of gold particles relative to presynaptic and postsynaptic membrane profiles (n=23) revealed a close correlation between AMPA immunoreactivity and the postsynaptic membrane of the synapse. Finally, immunogold particles signalling GluR1 were observed much less frequently than particles signalling GluR2/3 or GluR4. Our results provide additional strong evidence that chemical transmission at Ia synapses is mediated by glutamate and identify GluR2/3 and GluR4 as important postsynaptic receptors.

Peripheral axotomy induces only very limited sprouting of coarse myelinated afferents into inner lamina II of rat spinal cord.

Peripheral axotomy-induced sprouting of thick myelinated afferents (A-fibers) from laminae III-IV into laminae I-II of the spinal cord is a well-established hypothesis for the structural basis of neuropathic pain. However, we show here that the cholera toxin B subunit (CTB), a neuronal tracer used to demonstrate the sprouting of A-fibers in several earlier studies, also labels unmyelinated afferents (C-fibers) in lamina II and thin myelinated afferents in lamina I, when applied after peripheral nerve transection. The lamina II afferents also contained vasoactive intestinal polypeptide and galanin, two neuropeptides mainly expressed in small dorsal root ganglion (DRG) neurons and C-fibers. In an attempt to label large DRG neurons and A-fibers selectively, CTB was applied four days before axotomy (pre-injury-labelling), and sprouting was monitored after axotomy. We found that only a small number of A-fibers sprouted into inner lamina II, a region normally innervated by C-fibers, but not into outer lamina II or lamina I. Such sprouts made synaptic contact with dendrites in inner lamina II. Neuropeptide Y (NPY) was found in these sprouts in inner lamina II, an area very rich in Y1 receptor-positive processes. These results suggest that axotomy-induced sprouting from deeper to superficial layers is much less pronounced than previously assumed, in fact it is only marginal. This limited reorganization involves large NPY immunoreactive DRG neurons sprouting into the Y1 receptor-rich inner lamina II. Even if quantitatively small, it cannot be excluded that this represents a functional circuitry involved in neuropathic pain.