Distribution of arginine-vasopressin in the trigeminal, dorsal root ganglia and spinal cord of the rat; depletion by capsaicin.

We have measured arginine vasopressin in the neural lobe, the trigeminal ganglion (TG), dorsal root ganglia (DRG), spinal cord, trigeminal and sciatic nerves of the rat by radioimmunoassay. In control rats, the neural lobe contained 1600 pg/mg, the ganglia 52.5, 21.0, 8.5, 4.28, 3.85 pg/mg in the lumbar, sacral, cervical, thoracic, and trigeminal ganglion, respectively, the spinal cord contained 5.1, 4.3, 4.2 and 2.6 pg/mg in the lumbar, thoracic, sacral and cervical cord, respectively and the trigeminal and sciatic nerves contained 3.8 and 13 pg/mg. Neonatal capsaicin treatment depleted about 38-67% of AVP in the ganglia. Residual AVP amounted to 526.8, 30.55, 20.75, 12.88, 4.95, 2.74, 2.14, 7.94 and 2.53 pg/mg in the neural lobe, lumbar, thoracic, sacral, cervical DRG, lumbar, thoracic spinal cord, the sciatic and trigeminal nerves respectively. Capsaicin destroyed about 40.5% of total cells and 52% of AVP-immunoreactive neurons.

Glutamate-immunoreactivity in the trigeminal and dorsal root ganglia, and intraspinal neurons and fibres in the dorsal horn of the rat.

Putative aspartergic and glutamatergic sensory neurons in the rat were identified by autoradiography and immunocytochemistry respectively. Approximately 3% of large L4 dorsal root ganglion neurons (diameter 18-52 microns) accumulated radiolabelled aspartate, whereas all satellite glia had high affinity for the amino acid. Glutamate-immunofluorescent (Glu-FITC) dorsal root ganglia neurons comprised 38.3% at S1, 35.6% at L2, 33.9% at C5 and 28.8% at T6. Numbers of immunoreactive neurons were higher with the more sensitive peroxidase-anti-peroxidase (Glu-PAP) method; and the cell counts totalled 42% (S1), 41.2% (L4), 35% (C5) and 34.6% (T6). The trigeminal ganglion (TG) contained 24% Glu-FITC and 32.3% Glu-PAP positive cells. The majority of glutamate-immunoreactive sensory neurons were small, ranging from 10-35 microns with median diameters of 17.5 microns (C5), 21 microns (S1), 24.2 microns (TG) and 28.5 microns (L2). It is evident therefore, that a subgroup of class B cells are glutamatergic. Glutamate immunoreactivity in the spinal cord was similar in all segments and was localized in the superficial lamina and substantia gelatinosa of the dorsal horn. Stained interneurons were located among the immunoreactive fibres. The dorsolateral funiculus contained dense plexus of immunoreactive fibres which increased in prominence after intraperitoneal injection of L-gluatamate, but penetration of exogenous glutamate into the grey matter was limited. Instead, the meninges and basal layers of the spinal blood vessels were intensely immunoreactive. The studies describe the subtypes of acidic amino acidergic neurons and relates the immunohistochemistry to a functional subclass.

Cytochemistry of the trigeminal and dorsal root ganglia and spinal cord of the rat.

1. The primary sensory neurones have been classified into large light (LLC), type A, small dark (SDC), type B and type C cells on the basis of size, ultrastuctural and immunocytochemical characteristics. 2. Subclassifications have been described according to the configuration and spatial organization of cytoplasmic organelles. 3. Furthermore, the LLC are immunoreactive with a monoclonal antibody, RT97, directed against a neurofilament protein and the SDC are positive with anti-arginine vasopressin (AVP). 4. The majority of the neurochemical substances including substance P (SP), somatostatin (SOM), fluoride resistant acid phosphatase (FRAP), 5-hydroxytryptamine (5-HT) and glutamate were localized to the small and intermediate diameter neurones measuring 9-40 microns. 5. The cytochemistry of the dorsal horn was similar to the dorsal root ganglia (DRG). 6. There is good evidence that substance P (SP) and somatostatin (SOM) are transmitters for a proportion of nociceptive neurones but the neurotransmitters utilized by the rest of the subtypes are unknown. 7. 5-hydroxytryptamine (5-HT) and glutamate may be putative transmitters of the primary sensory neurones as they are localized in 28-30% of the SDC. 8. The wider distribution and extensive coexistence of the neuropeptides is incompatible with neurotransmitter function, but some may be neuromodulators whereas others such as arginine vasopressin (AVP) are useful markers for identifying type B neurones.

A quantitative analysis of the interrelationships between subpopulations of rat sensory neurons containing arginine vasopressin or oxytocin and those containing substance P, fluoride-resistant acid phosphatase or neurofilament protein.

In rat L5 dorsal root ganglia 50% of neurons contained arginine vasopressin-like immunoreactivity and 38% oxytocin-like immunoreactivity, the oxytocin entirely coexisting with the arginine vasopressin. Staining of alternate mirror-image sections with RT97 (an antibody to neurofilament protein, and a marker for large light neurons) and with arginine vasopressin antiserum showed that the two were entirely complementary, thus establishing arginine vasopressin as a marker for all small dark neurons. Mirror-image staining also showed that neurons containing substance P-like immunoreactivity and those containing fluoride-resistant acid phosphatase activity were each contained within the arginine vasopressin-positive population. Arginine vasopressin-like immunoreactivity was axonally transported in the dorsal root and (in greater quantity) in sciatic nerve. Arginine vasopressin-like immunoreactivity was present also in laminae I and II of the dorsal horn of the spinal cord and this reactivity was absent in animals which had been treated neonatally with capsaicin, suggesting that it was contained in primary afferent terminals. These results are discussed in terms of their implications for the classification of primary afferent neurons and of a possible physiological role for arginine vasopressin in these neurons.

Localization of chromatographically characterized oxytocin and arginine-vasopressin to sensory neurones in the rat.

Following treatment with colchicine 50-60% of all neurones in rat trigeminal and L5 spinal ganglia showed oxytocin (OXT)- and arginine-vasopressin (AVP)-like immunoreactivity. Further, OXT and AVP, together with their associated neurophysins, could be isolated from trigeminal ganglia by reversed-phase high-performance liquid chromatography followed by radioimmunoassay.

Localization of 5-hydroxytryptamine to neurons and endoneurial mast cells in rat sensory ganglia.

5-Hydroxytryptamine (5-HT) localization has been studied in the spinal and trigeminal ganglia of adult rats using immunofluorescence, peroxidase-antiperoxidase immunocytochemistry and [3H] 5-HT uptake radioautography, the latter two at the ultrastructural level. Endoneurial mast cells, identified by alcian blue staining, formed 8 and 14% of all mast cells and neurons in spinal and trigeminal ganglia respectively and had a median diameter of 7.6 microns. Light and electron microscopic immunocytochemistry showed that these mast cells contained 5-HT-like immunoreactivity. Some 75% of them accumulated exogenous [3H]5-HT in vitro. A population of small-diameter neurons, which did not stain with alcian blue, was also labelled with anti-5-HT serum and accumulated [3H]5-HT. The possible roles of 5-HT in sensory ganglia are discussed.

Ultrastructural localization of [3H]dopamine in neurons of leech (Haemopis sanguisuga) abdominal ganglia.

Neurons in the cortex of the segmental ganglia of Haemopis sanguisuga accumulated [3H]DOPA in vitro. Electron microscopic autoradiography revealed deposits of silver grains over clusters of electron dense granules measuring 20-35 nM in diameter. [3H]Dopamine (DA) was localized in the giant glial cells. Dopaminergic terminals were labelled with both DOPA and dopamine and the morphology is similar to other DA synapses. The leech could be a useful model for the study of synaptic events at dopaminergic terminals.

Incorporation of [3H]2-deoxyglucose into glycogen in nervous tissues.

Mice were injected with [3H]2-deoxyglucose and after 1 h high molecular weight glycogen was extracted from brain, liver and muscle tissues. 1-2% of the total radioactivity in each tissue was recovered in the glycogen fraction. Isolated buccal ganglia of the pond snail, Planorbis, and isolated abdominal ganglia of the horse leech Haemopis, were exposed in vitro to [3H]2-deoxyglucose for 1 h. 1-10% of the total radioactivity in these tissues was located in the high molecular weight glycogen fraction. Treatment of the extracted labelled glycogen fractions with amyloglucosidase caused release of the label in a manner consistent with the breakdown of labelled glycogen. Ganglia of snail and leech were exposed to [3H]2-deoxyglucose, fixed in glutaraldehyde and osmium tetroxide solutions, and prepared for autoradiography using aqueous histological processing. Light and electron microscope autoradiography showed that over 90% of the label was positively associated with glycogen particles (alpha- and beta-particles). Certain previously published reports on the incorporation of 2-deoxyglucose into glycogen are discussed in relation to these findings. It is concluded that [3H]2-deoxyglucose is partially incorporated into glycogen in nervous tissue; the labelled 2-deoxyglycogen withstands aqueous histological processing and can be visualized directly by autoradiography.

The structure, distribution, and quantitative relationships of the glia in the abdominal ganglia of the horse leech, Haemopis sanguisuga.

The glial cells in abdominal ganglia of the horse leech Haemopis sanguisuga were studied by electron microscopy and analysed quantitatively to evaluate the suitability of this easily obtainable carnivorous species for physiological studies. Each abdominal ganglion contains eight giant glial cells, 12,000-14,000 small glial cells, and approximately 300 neurons. The giant glial cells constituted 44.6% and the small glial cells 6.4% of the ganglion's volume. The giant glial cells contain glycogen and bundles of filaments that are chiefly located in their periphery, close to the neurons into which they send processes. The small glial cells are frequently surrounded by the giant glial cells but also occur around neuronal perikarya and axon tracts, as well as against the basal lamina and connective tissue layers. The small glial cells contain lysosomes and sometimes form a trophospongium with the neurons. A system of extracellular channels, which is continuous with the basal lamina, indents the giant glial cells and extends around parts of the neurons. The extracellular channels contain a matrix that appears very similar to the basal lamina and to the cytoplasm in the processes of the small glial cells. Some of the extracellular channels contain collagen fibrils. Hemidesmosomes join the matrix-filled extracellular channels to both the neurons and the giant glial cells. Ionic lanthanum has a free access to the neurons and glial cells via the extracellular spaces and matrix-filled channels. Areas of synaptic intermingling rarely contain glial cell processes.

High resolution analysis of [3H]2-deoxyglucose incorporation into neurons and glial cells in invertebrate ganglia: histological processing of nervous tissue for selective marking of glycogen.

The 2-deoxyglucose (2-DG) autoradiographic technique, in conjunction with 3H-labelled 2-DG and glutaraldehyde fixation, was applied to the isolated ganglia of the leech and the snail in order to analyse its potential use for the understanding of energy utilization in these simple nervous systems. Approximately 50% of the label is retained in these tissues after histological processing, and the method can be satisfactorily applied at the subcellular level. In both species most neurons progressively accumulate radioactivity over 15 min-1 h, although to different extents. In the leech the glial cells showed greater uptake than the neurons. Paired homologous neurons in contralateral buccal ganglia of the snail were equally labelled. In all structures studied by electron-microscope autoradiography the label was positively associated with glycogen. Freshly extracted glycogen from ganglia previously exposed to 2-DG was significantly labelled (2-10% of total radioactivity in the ganglia); thus the glutaraldehyde fixation did not appear responsible for artifactual binding of the label. The significance of the glycogen labelling and the potential of the technique for metabolic mapping in these nervous systems are discussed.