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Sympathetic nerve activity often fluctuates with the respiratory cycle, but the central neurons that impose this respiratory modulation have not been conclusively identified. In the present study, we used intracellular recording and dye-filling to identify expiratory neurons in the Bötzinger complex. Our aim was to see if Bötzinger neurons project towards putative cardiovascular neurons in the rostral ventrolateral medulla. In the first series of experiments, histochemistry and immunohistochemistry were used to reveal the labelled Bötzinger neurons and neurons immunoreactive for tyrosine hydroxylase. Two out of four Bötzinger neurons had axon varicosities that were closely apposed to tyrosine hydroxylase-immunoreactive neurons with cell bodies located within 0.6 mm caudal to the facial nucleus (three and five close appositions, respectively). In a second series of studies, rats were injected with cholera toxin B into the intermediolateral cell column of the spinal cord 4-7 days before the electrophysiological recording. Eight of the fourteen labelled Bötzinger neurons had a direct projection towards cholera toxin B-labelled neurons in the rostral ventrolateral medulla. Close appositions were found on both somata and proximal dendrites (5 +/- 2 close appositions/neuron, n = 8). The present study supports the idea that a direct projection from Bötzinger neurons to presympathetic neurons in the rostral medulla plays a role in the respiratory modulation of sympathetic nerve activity.

Axosomatic and axodendritic synapses occur on sympathetic preganglionic neurons, but it is not yet known whether their axons receive synaptic input, which could be particularly effective at regulating sympathetic outflow. Here, we examined retrogradely labelled sympathetic preganglionic axons to see if they received synapses. Cholera toxin B subunit (CTB) or CTB conjugated to horseradish peroxidase (CTB-HRP) was used to label neurons projecting to the rat or rabbit superior cervical ganglion, the rat adrenal medulla, or the rabbit stellate ganglion. At the light microscopic level, small groups of CTB-immunoreactive axons travelled through the ventral horn near its lateral boundary, with occasional axons taking a more medial course. The axons passed through the ventrolateral funiculus to exit at the ventral roots. In parasagittal section, a few axons branched within the ventral horn, sending processes rostrally and caudally for short distances before they turned ventrally to exit the spinal cord. At the ultrastructural level, CTB-immunoreactive rat and rabbit sympathetic preganglionic axons were almost exclusively unmyelinated. In contrast, labelling with CTB-HRP revealed both myelinated and unmyelinated axons in the ventral horn, the ventrolateral white matter, and the ventral roots. CTB-HRP also allowed the detection of the initial segment of a sympathetic preganglionic axon. Synapses, with vesicles clustered presynaptically and membrane specializations postsynaptically, were found on some unmyelinated CTB-immunoreactive axons. Occasional axons received several synapses. Synapses were most common on CTB-containing axons just ventral to the intermediolateral cell column. One synapse was found on an axon within 2 microns of its origin from a proximal dendrite. Rare synapses were found several hundred micrometers ventral to the intermediolateral cell column. One branching axon had synapses just below the branch point on both the main axon and the axonal branch. These findings indicate an extensive synaptic input to the axons of at least some sympathetic preganglionic neurons. These axoaxonic synapses could have a profound effect on sympathetic activity.

The input from serotonin-containing nerve fibres to rabbit sympathetic preganglionic neurons projecting to either the superior cervical ganglion or the adrenal medulla was investigated by combining retrograde tracing with the B subunit of cholera toxin and immunocytochemistry for serotonin. There were pronounced rostrocaudal variations in the density of serotonin fibres in the rabbit intermediolateral cell column from T1 to L4; maximum numbers of fibres were found in T3-6 and L3-4 and minimum numbers in T1 and T10-12. By light microscopy, retrogradely labelled sympathetic preganglionic neurons projecting to the superior cervical ganglion or the adrenal medulla received variable densities of close appositions from serotonin-immunoreactive fibres. Some neurons from each population received many close appositions, whereas others received moderate numbers or few appositions. Appositions occurred on the cell bodies, dendrites, and occasionally axons of sympathetic preganglionic neurons. Rare neurons in both groups of retrogradely labelled cells received no appositions from serotonin-containing nerve fibres. At the ultrastructural level, synapses were found between serotonin-positive boutons and sympathetic preganglionic neurons projecting either to the superior cervical ganglion or to the adrenal medulla. These results indicate that, through direct synaptic contacts, serotonin-immunoreactive, presumably bulbospinal, nerve fibres affect the activity of the vast majority of sympathetic preganglionic neurons that send axons either to the superior cervical ganglion or to the adrenal medulla. This serotonin input may be sympathoexcitatory and could mediate increases in sympathetic nerve activity and in the release of catecholamines from the adrenal medulla.

The extent of the adrenergic input to respiratory neurons in the ventrolateral medulla oblongata of rats was assessed by using a combination of intracellular recording, dye filling, and immunohistochemistry. Twenty-two neurons that displayed a pronounced respiration-related modulation of their membrane potential, and could not be antidromically activated by electrical stimulation of the superior laryngeal, vagus, or facial nerves, were labelled by intracellular injection of biocytin. Three types of respiration-related neurons were labelled: small neurons located in the Bötzinger complex between 0.5 and 1.0 mm caudal to the facial nucleus; medium-sized neurons located in the ventral respiratory group 1.0 to 2.0 mm caudal to the facial nucleus; and large motoneurons located within the nucleus ambiguus 0.5 to 2.0 mm caudal to the facial nucleus. Small Bötzinger neurons [length = 22 +/- 5 microns, width = 13 +/- 3 microns, area = 222 +/- 79 microns2; (mean +/- SD, n = 5)] had membrane potentials of -15 to -27 mV during the recording period. Four of five of these cells had profuse axonal terminations between 50 microns caudal and 450 microns rostral to their somata, suggesting that they may form part of local networks responsible for generating respiratory activity. Medium-sized ventral respiratory group neurons (length = 26 +/- 5 microns, width = 18 +/- 4 microns, area = 377 +/- 141 microns2; n = 5) were found in the vicinity of the nucleus ambiguus dorsal to the lateral reticular nucleus. Three of five of these neurons had an axon that crossed the midline and travelled caudally. One axon had a collateral with varicosities close to its soma. The somata of motoneurons (length = 29 +/- 6 microns, width = 21 +/- 4 microns, area = 485 +/- 142 microns2; n = 12) were located within the nucleus ambiguus, and had axons that could be traced to exist points from the medulla. Tyrosine hydroxylase immunoreactive cells and their terminal fibres within the medulla were localised by immunocytochemistry. Small Bötzinger neurons received the largest number of close appositions from tyrosine hydroxylase immunoreactive boutons (13 +/- 2 appositions/neuron; n = 5). Medium-sized ventral respiratory group neurons received fewer appositions (8 +/- 4 appositions/neuron; n = 5). Most motoneurons (n = 10) received few appositions from tyrosine hydroxylase immunoreactive boutons, while two received none. The average number was 3 +/- 3 appositions/neuron (n = 12).(ABSTRACT TRUNCATED AT 400 WORDS)

The colocalization of parvalbumin (PV), calbindin-D28k (CaBP), GABA immunoreactivities, and the ability to accumulate 3H-D-aspartate selectively were investigated in neurons of laminae I-IV of the dorsal horn of the rat spinal cord. Following injection of 3H-D-aspartate into the basal dorsal horn (laminae IV-VI), perikarya selectively accumulating 3H-D-aspartate were detected in araldite embedded semithin sections by autoradiography, and consecutive semithin sections were treated to reveal PV, CaBP and GABA by postembedding immunocytochemistry. Perikarya accumulating 3H-D-aspartate were found exclusively in laminae I-III, and no labelled somata were found in deeper layers or in the intermediolateral column although the labelled amino acid clearly spread to these regions. More than half of the labelled cells were localized in lamina II. In this layer, 16.4% of 3H-D-aspartate-labelled perikarya were also stained for CaBP. In contrast to CaBP, PV or GABA was never detected in neurons accumulating 3H-D-aspartate. A high proportion of PV-immunoreactive perikarya were also stained for GABA in laminae II and III (70.0% and 61.2% respectively). However, the majority of CaBP-immunoreactive perikarya were GABA-negative. GABA-immunoreactivity was found in less than 2% of the total population of cells stained for CaBP in laminae I-IV. A significant proportion of the GABA-negative but PV-immunoreactive neurons also showed CaBP-immunoreactivity in laminae II and IV. These results show that out of the two calcium-binding proteins, CaBP is a characteristic protein of a small subpopulation of neurons using excitatory amino acids and PV is a characteristic protein of a subpopulation of neurons utilizing GABA as a transmitter. However, both proteins are present in additional subgroups of neurons, and neuronal populations using inhibitory or excitatory amino acid transmitters are heterogeneous with regard to their content of calcium-binding proteins in the dorsal horn of the rat spinal cord.

The purpose of this study was to test whether a new retrograde tracer, the B subunit of cholera toxin conjugated to colloidal gold particles (CTB-gold), was taken up and transported by neurons in the central nervous system of the rat. Retrograde transport of CTB-gold was assessed from axon terminals, from damaged nerve fibers, and from axons of passage. For light microscopy, CTB-gold was visualized by silver intensification; for electron microscopy, sections were silver-intensified with or without subsequent gold toning. Retrogradely transported CTB-gold was detected in neurons after survival times of 12 hours to 42 days and appeared as black punctate deposits in perikarya and proximal dendrites at the light microscope level. Ultrastructurally, the deposits were usually associated with lysosomes. Injections of CTB-gold into the caudal ventrolateral medulla or into the lateral horn of the spinal cord gave small well-defined injection sites and resulted in retrograde labelling in medullary neurons in the same locations as similarly placed injections of wheat germ agglutinin-horseradish peroxidase. When injected into the superior cervical ganglion, CTB-gold was transported to nerve cell bodies in the spinal cord, but application of CTB-gold to the cut cervical sympathetic trunk did not label neurons in the spinal cord. Injection of CTB-gold into the nodose ganglion retrogradely labelled neurons in the dorsal motor nucleus of the vagus and the nucleus ambiguus. CTB-gold was not transported anterogradely from injections sites within the medulla. Nerve fibers and cell bodies containing neuropeptides, monoamines, or neurotransmitter-synthesizing enzymes were readily immunostained after silver intensification of retrogradely transported CTB-gold. Immunoreactivity for neuropeptides and enzymes was also demonstrated ultrastructurally after silver intensification and gold toning. These results show that CTB-gold is retrogradely transported from nerve terminals and fibers of passage but not from damaged axons. CTB-gold gives well-localized injection sites and persists in neurons for weeks. Transported CTB-gold is easily visualized and its detection is compatible with light and electron microscopic immunocytochemistry. These properties make CTB-gold a valuable tool for studying the connectivity and neurochemistry of pathways in the central nervous system.