Functional and neurochemical characterization of angiotensin type 1A receptor-expressing neurons in the nucleus of the solitary tract of the mouse.

Angiotensin II acts via two main receptors within the central nervous system, with the type 1A receptor (AT1AR) most widely expressed in adult neurons. Activation of the AT1R in the nucleus of the solitary tract (NTS), the principal nucleus receiving central synapses of viscerosensory afferents, modulates cardiovascular reflexes. Expression of the AT1R occurs in high density within the NTS of most mammals, including humans, but the fundamental electrophysiological and neurochemical characteristics of the AT1AR-expressing NTS neurons are not known. To address this, we have used a transgenic mouse, in which the AT1AR promoter drives expression of green fluorescent protein (GFP). Approximately one-third of AT1AR-expressing neurons express the catecholamine-synthetic enzyme tyrosine hydroxylase (TH), and a subpopulation of these stained for the transcription factor paired-like homeobox 2b (Phox2b). A third group, comprising approximately two-thirds of the AT1AR-expressing NTS neurons, showed Phox2b immunoreactivity alone. A fourth group in the ventral subnucleus expressed neither TH nor Phox2b. In whole cell recordings from slices in vitro, AT1AR-GFP neurons exhibited voltage-activated potassium currents, including the transient outward current and the M-type potassium current. In two different mouse strains, both AT1AR-GFP neurons and TH-GFP neurons showed similar AT1AR-mediated depolarizing responses to superfusion with angiotensin II. These data provide a comprehensive description of AT1AR-expressing neurons in the NTS and increase our understanding of the complex actions of this neuropeptide in the modulation of viscerosensory processing.

Insulin-responsive autonomic neurons in rat medulla oblongata.

Low blood glucose activates brainstem adrenergic and cholinergic neurons, driving adrenaline secretion from the adrenal medulla and glucagon release from the pancreas. Despite their roles in maintaining glucose homeostasis, the distributions of insulin-responsive adrenergic and cholinergic neurons in the medulla are unknown. We fasted rats overnight and gave them insulin (10 U/kg i.p.) or saline after 2 weeks of handling. Blood samples were collected before injection and before perfusion at 90 min. We immunoperoxidase-stained transverse sections of perfused medulla to show Fos plus either phenylethanolamine N-methyltransferase (PNMT) or choline acetyltransferase (ChAT). Insulin injection lowered blood glucose from 4.9 ± 0.3 mmol/L to 1.7 ± 0.2 mmol/L (mean ± SEM; n = 6); saline injection had no effect. In insulin-treated rats, many PNMT-immunoreactive C1 neurons had Fos-immunoreactive nuclei, with the proportion of activated neurons being highest in the caudal part of the C1 column. In the rostral ventrolateral medulla, 33.3% ± 1.4% (n = 8) of C1 neurons were Fos-positive. Insulin also induced Fos in 47.2% ± 2.0% (n = 5) of dorsal medullary C3 neurons and in some C2 neurons. In the dorsal motor nucleus of the vagus (DMV), insulin evoked Fos in many ChAT-positive neurons. Activated neurons were concentrated in the medial and middle regions of the DMV beneath and just rostral to the area postrema. In control rats, very few C1, C2, or C3 neurons and no DMV neurons were Fos-positive. The high numbers of PNMT-immunoreactive and ChAT-immunoreactive neurons that express Fos after insulin treatment reinforce the importance of these neurons in the central response to a decrease in glucose bioavailability.

Adrenaline: insights into its metabolic roles in hypoglycaemia and diabetes.

Adrenaline is a hormone that has profound actions on the cardiovascular system and is also a mediator of the fight-or-flight response. Adrenaline is now increasingly recognized as an important metabolic hormone that helps mobilize energy stores in the form of glucose and free fatty acids in preparation for physical activity or for recovery from hypoglycaemia. Recovery from hypoglycaemia is termed counter-regulation and involves the suppression of endogenous insulin secretion, activation of glucagon secretion from pancreatic α-cells and activation of adrenaline secretion. Secretion of adrenaline is controlled by presympathetic neurons in the rostroventrolateral medulla, which are, in turn, under the control of central and/or peripheral glucose-sensing neurons. Adrenaline is particularly important for counter-regulation in individuals with type 1 (insulin-dependent) diabetes because these patients do not produce endogenous insulin and also lose their ability to secrete glucagon soon after diagnosis. Type 1 diabetic patients are therefore critically dependent on adrenaline for restoration of normoglycaemia and attenuation or loss of this response in the hypoglycaemia unawareness condition can have serious, sometimes fatal, consequences. Understanding the neural control of hypoglycaemia-induced adrenaline secretion is likely to identify new therapeutic targets for treating this potentially life-threatening condition.

The origin of citrulline-containing proteins in the hair follicle and the chemical nature of trichohyalin, an intracellular precursor.

The present studies have demonstrated that the medulla and inner root sheath cells develop within their cytoplasm a protein that is unique in composition and is present in the trichohyalin granules. The protein is rich in arginine residues, some of which undergo a side-chain conversion in situ into citrulline residues. An unusual Ca2+-dependent enzyme activity distinguishable from cross-linking transamidase has been detected in the hair follicle and will act in vitro on trichohyalin protein as the natural substrate. The conversion in vivo must occur during the time that the medullary and inner root sheath cells move up the follicle and their cytoplasm fills with cross-linked protein containing citrulline. The function of citrulline in these proteins is not understood but its formation is a major process during hair growth.

Spinally projecting preproglucagon axons preferentially innervate sympathetic preganglionic neurons.

Glucagon-like peptide-1 (GLP-1) affects central autonomic neurons, including those controlling the cardiovascular system, thermogenesis, and energy balance. Preproglucagon (PPG) neurons, located mainly in the nucleus tractus solitarius (NTS) and medullary reticular formation, produce GLP-1. In transgenic mice expressing glucagon promoter-driven yellow fluorescent protein (YFP), these brainstem PPG neurons project to many central autonomic regions where GLP-1 receptors are expressed. The spinal cord also contains GLP-1 receptor mRNA but the distribution of spinal PPG axons is unknown. Here, we used two-color immunoperoxidase labeling to examine PPG innervation of spinal segments T1-S4 in YFP-PPG mice. Immunoreactivity for YFP identified spinal PPG axons and perikarya. We classified spinal neurons receiving PPG input by immunoreactivity for choline acetyltransferase (ChAT), nitric oxide synthase (NOS) and/or Fluorogold (FG) retrogradely transported from the peritoneal cavity. FG microinjected at T9 defined cell bodies that supplied spinal PPG innervation. The deep dorsal horn of lower lumbar cord contained YFP-immunoreactive neurons. Non-varicose, YFP-immunoreactive axons were prominent in the lateral funiculus, ventral white commissure and around the ventral median fissure. In T1-L2, varicose, YFP-containing axons closely apposed many ChAT-immunoreactive sympathetic preganglionic neurons (SPN) in the intermediolateral cell column (IML) and dorsal lamina X. In the sacral parasympathetic nucleus, about 10% of ChAT-immunoreactive preganglionic neurons received YFP appositions, as did occasional ChAT-positive motor neurons throughout the rostrocaudal extent of the ventral horn. YFP appositions also occurred on NOS-immunoreactive spinal interneurons and on spinal YFP-immunoreactive neurons. Injecting FG at T9 retrogradely labeled many YFP-PPG cell bodies in the medulla but none of the spinal YFP-immunoreactive neurons. These results show that brainstem PPG neurons innervate spinal autonomic and somatic motor neurons. The distributions of spinal PPG axons and spinal GLP-1 receptors correlate well. SPN receive the densest PPG innervation. Brainstem PPG neurons could directly modulate sympathetic outflow through their spinal inputs to SPN or interneurons.

Changes in synaptic inputs to sympathetic preganglionic neurons after spinal cord injury.

Spinal cord injury (SCI) leads to plastic changes in organization that impact significantly on central nervous control of arterial pressure. SCI causes hypotension and autonomic dysreflexia, an episodic hypertension induced by spinal reflexes. Sympathetic preganglionic neurons (SPNs) respond to SCI by retracting and then regrowing their dendrites within 2 weeks of injury. We examined changes in synaptic input to SPNs during this time by comparing the density and amino acid content of synaptic input to choline acetyltransferase (ChAT)-immunoreactive SPNs in the eighth thoracic spinal cord segment (T8) in unoperated rats and in rats at 3 days or at 14 days after spinal cord transection at T4. Postembedding immunogold labeling demonstrated immunoreactivity for glutamate or gamma-aminobutyric acid (GABA) within presynaptic profiles. We counted the number of presynaptic inputs to measured lengths of SPN somatic and dendritic membrane and identified the amino acid in each input. We also assessed gross changes in the morphology of SPNs using retrograde labeling with cholera toxin B and light microscopy to determine the structural changes that were present at the time of evaluation of synaptic density and amino acid content. At 3 days after SCI, we found that retrogradely labeled SPNs had shrunken somata and greatly shortened dendrites. Synaptic density (inputs per 10-microm membrane) decreased on ChAT-immunoreactive somata by 34% but increased on dendrites by 66%. Almost half of the inputs to SPNs lacked amino acids. By 14 days, the density of synaptic inputs to dendrites and somata decreased by 50% and 70%, respectively, concurrent with dendrite regrowth. The proportion of glutamatergic inputs to SPNs in spinal cord-transected rats ( approximately 40%) was less than that in unoperated rats, whereas the GABAergic proportion (60-68%) increased. In summary, SPNs participate in vasomotor control after SCI despite profound denervation. An altered balance of excitatory and inhibitory inputs may explain injury-induced hypotension.

Synapses on axons of sympathetic preganglionic neurons in rat and rabbit thoracic spinal cord.

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.

Thyrotropin-releasing hormone inputs are preferentially directed towards respiratory motoneurons in rat nucleus ambiguus.

In the present study, we assessed the extent of the thyrotropin-releasing hormone (TRH) input to motoneurons in the ambigual, facial, and hypoglossal nuclei of the rat using a combination of intracellular recording, dye filling, and immunohistochemistry. Twelve motoneurons in the rostral nucleus ambiguus were labelled by intracellular injection in vivo of Neurobiotin (Vector). Seven out of 12 ambigual motoneurons displayed rhythmic fluctuations of their membrane potential in phase with phrenic nerve discharge, whereas the other five had no modulations of any kind. Seven facial motoneurons and seven hypoglossal motoneurons were also filled with Neurobiotin. All three motor nuclei contained TRH-immunoreactive varicosities, with the largest numbers found in the nucleus ambiguus. Close appositions were seen between TRH-immunoreactive boutons and every labelled motoneuron. Respiratory-related motoneurons in the nucleus ambiguus received the largest number of TRH appositions with 74 +/- 38 appositions/neuron (mean +/- S.D.; n = 7). In contrast, nonrespiratory ambigual motoneurons received significantly fewer TRH appositions (11 +/- 5; n = 5; P < 0.05; Mann-Whitney U test). Facial motoneurons received about the same number of TRH appositions as nonrespiratory ambigual motoneurons, with 13 +/- 4 (n = 7). Hypoglossal motoneurons received the fewest appositions from TRH-containing boutons, with 8 +/- 2 (n = 7). There were no differences in the TRH inputs to respiratory and nonrespiratory motoneurons in the facial and hypoglossal nuclei. These results demonstrate that, among motoneurons in the medulla, respiratory motoneurons in the rostral nucleus ambiguus are preferentially innervated by the TRH-immunoreactive boutons.

Substance P immunoreactive boutons form synapses with feline sympathetic preganglionic neurons.

In this study, the relationship between substance P-immunoreactive boutons and antidromically activated sympathetic preganglionic neurons was examined by light and electron microscopy. Sympathetic preganglionic neurons in the T2-T4 spinal segments of the cat were identified by intracellular recording and antidromic activation from the corresponding white ramus. Neurons were filled with lucifer yellow and then stained to reveal, simultaneously, substance P and lucifer yellow immunoreactivity. All of the neurons examined with the light microscope (n = 13) received appositions from substance P-immunoreactive boutons. Appositions were found on all parts of the neuron, including the somata, dendrites, and axon initial segment. In most cases (11/13) few close appositions were seen; however, two neurons received large numbers of appositions from substance P-immunoreactive boutons. On one neuron, 16 substance P-immunoreactive varicosities that were identified as being closely apposed at the light microscope level were serially sectioned and examined with the electron microscope. Of these 16 varicosities, eight either directly contacted the neuron or formed morphologically identifiable synapses. The remaining eight varicosities were separated from the neuron by thin glial processes. Two other sympathetic preganglionic neurons that were examined ultrastructurally also received substance P-immunoreactive synapses and close contacts. These findings suggest that substance P-containing nerve fibres could affect all sympathetic preganglionic neurons but are likely to be important in regulating the activity of only a small proportion of these neurons.

Vesicle shape and amino acids in synaptic inputs to phrenic motoneurons: do all inputs contain either glutamate or GABA?

Varicosities that made synapses or direct contacts with retrogradely labelled rat phrenic motoneurons were examined for their content of immunoreactivity for either glutamate or glutamate decarboxylase, the enzyme involved in synthesis of gamma-aminobutyric acid (GABA). Phrenic motoneurons were identified by retrograde tracing from the diaphragm with cholera toxin B subunit conjugated to horseradish peroxidase. Cell bodies and medium-sized to large dendrites were labelled. Preembedding immunocytochemistry identified glutamate decarboxylase-immunoreactive nerve fibres; glutamate-immunoreactive nerve terminals were identified using postembedding immunogold labelling of ultrathin sections. The presence of glutamate- or glutamate decarboxylase immunoreactivity in nerve terminals was correlated with the morphology of the synaptic vesicles. Two major classes of nerve terminals were identified. Nerve terminals with round (presumably spherical) synaptic vesicles (S terminals) comprised 55% of synapses and contacts on phrenic motoneuron somata and 58% of synapses and direct contacts with dendrites. Nerve terminals with flattened synaptic vesicles (F terminals) comprised 42% of synapses direct contacts with somata and 41% of synapses and direct contacts with dendrites. Analysis of immunogold-labelled sections showed that S terminals contained statistically higher levels of glutamate immunoreactivity than F terminals. At the light microscope level, many glutamate decarboxylase-immunoreactive nerve terminals surrounded retrogradely labelled motoneurons. Varicosities with glutamate decarboxylase immunoreactivity made 33% of all synapses and direct contacts on somata, and 33% of synapses and direct contacts with dendrites of the retrogradely labelled phrenic motoneurons. Flattened synaptic vesicles were present in those glutamate decarboxylase-immunoreactive nerve terminals in which synaptic vesicle morphology could be judged. An additional 10% of all nerve terminals were of the F type, but were not glutamate decarboxylase-immunoreactive. Three percent of terminals on somata and 1% of nerve terminals on dendrites could not be classified as S or F types. These findings suggest that more than 90% of all inputs to phrenic motoneuron cell bodies and proximal dendrites could contain either GABA or glutamate. Some of these glutamatergic and GABAergic nerve fibres undoubtedly represent the source of inspiratory drive to, or expiratory inhibition of, phrenic motoneurons.

Serotonin inputs to rabbit sympathetic preganglionic neurons projecting to the superior cervical ganglion or adrenal medulla.

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.

Thyrotropin-releasing hormone-immunoreactive varicosities synapse on rat phrenic motoneurons.

The relationship between retrogradely labelled or intracellularly filled phrenic motoneurons and varicosities containing thyrotropin-releasing hormone immunoreactivity was investigated in rats by light and electron microscopy. Phrenic motoneurons were identified via retrograde tracing from the diaphragm with cholera toxin B subunit, which was followed by immunocytochemistry to visualise retrogradely labelled motoneurons and thyrotropin-releasing hormone-immunoreactive nerve fibres in their vicinity. At the light microscopic level, varicose thyrotropin-releasing hormone-immunoreactive nerve fibres were distributed sparsely in the phrenic motor nucleus, with some axons surrounding retrogradely labelled motoneurons. In separate intracellular experiments, four phrenic motoneurons identified by antidromic activation from the C5 phrenic nerve root were subsequently filled with Neurobiotin, and nerve fibres that contained thyrotropin-releasing hormone immunoreactivity were identified by immunocytochemistry. The numbers and locations of thyrotropin-releasing hormone-immunoreactive varicosities that were closely appeared to the intracellularly labelled motoneurons were mapped using a camera lucida technique. Close appositions by thyrotropin-releasing hormone-immunoreactive varicosities were seen on somata as well as on proximal and distal dendrites. The closely apposed varicosities were usually present in tight clusters, which were formed by single varicose axons. However, the distribution was nonuniform, in that some dendrites did not receive any close appositions. Ultrastructural analysis of random ultrathin sections through retrogradely labelled neurons showed that varicosities with thyrotropin-releasing hormone immunoreactivity made 1.8% of all synapses and direct contacts on somata and 2.3% of synapses and contacts with dendrites of the retrogradely labelled phrenic motoneurons. The results of these experiments suggest that thyrotropin-releasing hormone-immunoreactive varicosities provide similar numbers of inputs to both the somata and dendrites of phrenic motoneurons. These thyrotropin-releasing hormone-containing inputs seen via light and electron microscopy could modulate the excitability of phrenic motoneurons.

Quantitative ultrastructural analysis of enkephalin-, substance P-, and VIP-immunoreactive nerve fibers in the circular muscle of the guinea pig small intestine.

The present work was undertaken to determine what proportion of all nerve fibers in the circular muscle of the guinea pig small intestine contain the neuropeptides enkephalin, substance P, and vasoactive intestinal peptide and in which combinations these peptides occur in the fibers. It was envisaged that such an analysis would provide insights into the chemical identity of excitatory and inhibitory nerve fibers that innervate the muscle. Whole-mount preparations from normal and extrinsically denervated gut were labelled with antiserum to the individual peptides or with combinations of antipeptide antisera and processed for electron microscopy. Reactive and nonreactive vesicle-containing nerve fiber profiles were examined and counted in ultrathin sections. Vesicle-containing nerve fiber profiles immunoreactive for enkephalin, substance P, or vasoactive intestinal peptide had similar morphologies in that they all contained variable proportions of small clear and large granular vesicles. In all samples stained for single peptides or combinations of peptides, a small proportion of immunoreactive profiles approached smooth muscle cells to within 15-20 nm with no intervening basal lamina. A total of 14,694 vesiculated nerve fiber profiles from three control and three extrinsically denervated animals were scored for the presence of immunoreactivity to enkephalin, substance P, vasoactive intestinal peptide, or combinations of these peptides. Analysis of variance showed that the number of profiles labelled for substance P was not different from the number of profiles labelled for vasoactive intestinal peptide and that the number labelled with the substance P and vasoactive intestinal peptide antisera simultaneously were not different from the sum of the numbers obtained with each alone. The number of profiles labelled for substance P plus enkephalin was greater than the number labelled for substance P alone and the number labelled with vasoactive intestinal peptide plus enkephalin was greater than that with vasoactive intestinal peptide alone. Simultaneous labelling for substance P and vasoactive intestinal peptide resulted in immunoreactivity in the same number of profiles as did reaction for all three peptides at the same time. In both cases, about 95% of the profiles were labelled. The results from extrinsically denervated muscle were not different from control circular muscle. These results indicate that nearly all the intrinsic nerve fibers supplying the circular muscle of the guinea pig small intestine contain either substance P or vasoactive intestinal peptide but not both.(ABSTRACT TRUNCATED AT 400 WORDS)

Close appositions between tyrosine hydroxylase immunoreactive boutons and respiratory neurons in the rat ventrolateral 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)

Bötzinger neurons project towards bulbospinal neurons in the rostral ventrolateral medulla of the rat.

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.

Cholera toxin B-gold, a retrograde tracer that can be used in light and electron microscopic immunocytochemical studies.

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.

Calbindin-immunoreactive neurons in the reticular formation of the rat brainstem: catecholamine content and spinal projections.

Calbindin-D28k (calbindin) is a calcium-binding protein that is distributed widely in the rat brain. The localisation of calbindin immunoreactivity in the medulla oblongata and its colocalisation with adrenaline-synthesising neurons [phenylethanolamine-N-methyltransferase-immunoreactive (PNMT-IR)] was examined (Granata and Chang [1994] Brain Res. 645:265-277). However, detailed information about the distribution of calbindin-IR neurons in the reticular formation of the medulla oblongata in particular is lacking. In this report, the authors address this issue with an emphasis on the quantitation of calbindin-IR neurons, catecholamine neurons [tyrosine hydroxylase (TH)-IR, or PNMT-IR], and spinally projecting neurons in the ventral brainstem. Rats received injections of the retrograde tracing agent cholera toxin B (CTB) into the thoracic spinal cord or into the superior cervical ganglion. Immunocytochemistry was used to reveal calbindin, TH, PNMT, and CTB immunoreactivity. Ten calbindin-IR cell groups were identified within the pontomedullary reticular formation. Seven previously undescribed but distinct clusters of calbindin-IR neurons were found. Within the ventral pons, a population of calbindin-IR neurons occurred dorsal but adjacent to the A5 cell group. These calbindin-IR neurons did not contain either TH or PNMT immunoreactivity, and few if any of these neurons projected to the spinal cord. A distinct group of calbindin-IR neurons was present in the ventral medulla. Seventy-five percent of these calbindin-IR neurons contained TH immunoreactivity, 45% contained PNMT immunoreactivity, and 21% were spinally projecting neurons. Spinally projecting, calbindin-IR neurons were a subpopulation of PNMT-IR cells. In the caudal ventral medulla, no TH-IR or PNMT-IR cells were calbindin-IR. In the intermediolateral cell column, close appositions of calbindin-IR terminals on identified sympathetic preganglionic neurons as well as calbindin-IR synapses indicated that these neurons may affect directly the sympathetic outflow. The results demonstrate for the first time the existence of a new subpopulation of spinally projecting, PNMT-IR neurons in the rostral ventrolateral medulla.

Bulbospinal sympatho-excitatory neurons in the rat caudal raphe.

OBJECTIVES: To explore the rat caudal raphe nuclei for neurons that respond to activation of baroreceptor nerves and that have a spinal axon, and to compare the behavioural properties of barosensitive bulbospinal neurons in the rat caudal raphe with the properties of barosensitive bulbospinal neurons in the rostral ventrolateral medulla. DESIGN: Extracellular unit recordings were obtained from an area extending up to 1.0 mm caudally from the caudal edge of the facial nucleus. Two sites were explored: the rostral ventrolateral medulla and the midline. MATERIALS AND METHODS: Single-unit recordings were made in anaesthetized (75 mg/kg chloral hydrate and 30 mg/kg sodium pentobarbitone then 3-6 mg intravenously as required), immobilized (2 mg pancuronium as required) Sprague-Dawley rats. Central respiratory drive was recorded from phrenic nerve discharge. The barosensitivity of single units was assessed by R-wave triggered histograms and by histograms of their responses to aortic nerve stimulation or to intravenous injection of phenylephrine. Nociceptors were activated by a brief pinch of the tail. RESULTS: Eleven spontaneously active units in the midline that were inhibited by baroreceptor stimulation and had a spinal axon were studied. Respiratory modulation was present and was predominantly inspiratory. Barosensitive neurons in the rostral ventrolateral medulla were activated by nociceptive inputs; midline barosensitive neurons were not. CONCLUSIONS: The behavioural characteristics of midline neurons differ from those of the bulbospinal barosensitive neurons in the rostral ventrolateral medulla, indicating that raphe spinal neurons have different sets of afferent inputs and may subserve to a distinct physiological role. The present paper is the first report of bulbospinal neurons in the rat caudal raphe that are inhibited by activation of arterial baroreceptors.

Catecholamine enzymes and neuropeptides are expressed in fibres and somata in the intermediate gray matter in chronic spinal rats.

Spinal cord injury disrupts control of sympathetic preganglionic neurons because bulbospinal input has been lost and the remaining regulation is accomplished by spinal circuits consisting of dorsal root afferent and spinal neurons. Moreover, an initial retraction and regrowth of dendrites of preganglionic neurons in response to deafferentation creates the potential for remodelling of spinal circuits that control them. Although catecholamines and neuropeptide Y are found in descending inputs to the preganglionic neurons, their presence in spinal circuits has not been established. Spinal circuits controlling preganglionic neurons contain substance P but participation of these peptidergic neurons in remodelling responses has not been examined. Therefore, we compared immunoreactivity for the catecholamine-synthesizing enzyme dopamine beta-hydroxylase, for neuropeptide Y and for substance P in the intermediate gray matter of the spinal cord in control rats and in rats seven or fourteen days after transection at the fourth thoracic cord segment. Sympathetic preganglionic neurons were retrogradely labelled by intraperitoneal injection of the tracer FluoroGold. These experiments yielded three original findings. 1) At one and two weeks after cord transection, fibres and terminals immunoreactive for dopamine beta-hydroxylase and neuropeptide Y were consistently found in the intermediolateral cell column in segments caudal to the transection. The area of fibres and terminals containing these immunoreactivities was markedly reduced compared to control rats or to segments rostral to the transection in the spinal rats. 2) Immunoreactivity for substance P was increased after cord transection and the distribution of fibres immunoreactive for this peptide in segments caudal to the transection extended more widely through the intermediate gray matter. These reactions demonstrated a plastic reaction to cord transection by spinal neurons expressing substance P. 3) Dopamine beta-hydroxylase expression was up-regulated in somata within the intermediate gray matter of spinal segments caudal to the transection. The numbers of somata immunoreactive for this enzyme increased six-fold by 14 days after cord transection, compared to the few somata counted in control rats. In conclusion, the presence of a catecholamine synthesizing enzyme and neuropeptides in fibres surrounding sympathetic preganglionic neurons caudal to a cord transection suggests a source of catecholamines and these peptides within spinal circuits in the chronic spinal rat. The presence of dopamine beta-hydroxylase in a markedly greater number of neuronal somata after cord transection reflects significant up-regulation of gene expression and may indicate a switch by these neurons to an adrenergic phenotype, revealing a plastic response to injury within the spinal cord.

Distribution and amino acid content of enkephalin-immunoreactive inputs onto juxtacellularly labelled bulbospinal barosensitive neurons in rat rostral ventrolateral medulla.

The activity of bulbospinal (presympathetic) vasomotor neurons of the rostral ventrolateral medulla is modulated pre- and postsynaptically by exogenously applied opioid agonists. To determine whether these neurons receive direct opioid inputs, we examined the relationship between bulbospinal barosensitive neurons and nerve terminals immunoreactive for enkephalin in the rostral ventrolateral medulla of rats. By light microscopy, we mapped the distribution of close appositions by enkephalin-immunoreactive varicosities on 10 bulbospinal barosensitive neurons labelled in vivo with biotinamide. We also examined four labelled neurons ultrastructurally for synapses by enkephalin-immunoreactive terminals and determined with post-embedding immunogold labelling whether these enkephalin-positive terminals contained amino acids. Enkephalin-immunoreactive varicosities closely apposed all bulbospinal barosensitive neurons. Maps of the dendritic distribution of appositions indicated that fast-conducting bulbospinal barosensitive neurons with myelinated axons (conduction velocity >3 m/s; n=3) received many appositions (up to 470/neuron); and slowly conducting neurons with unmyelinated axons (conduction velocity <0.90 m/s; n=3), substantially fewer. Ultrastructural analysis of three fast- and one slowly conducting bulbospinal barosensitive neurons revealed numerous synapses from enkephalin-immunoreactive terminals on cell bodies and dendrites. Enkephalin-positive terminals synapsing on bulbospinal barosensitive neurons contained one or more amino acid: GABA+glycine, glutamate alone or GABA+glutamate. Enkephalin-immunoreactive terminals located near biotinamide-labelled cells contained a similar variety of amino acids. In summary, enkephalin-immunoreactive terminals in the rostral ventrolateral medulla densely innervate lightly myelinated presympathetic neurons and more sparsely those with unmyelinated axons. Enkephalin is present in both excitatory (glutamate-immunoreactive) and inhibitory (GABA- and/or glycine-immunoreactive) terminals. The data suggest that endogenous enkephalin inhibits amino acid release from terminals that innervate bulbospinal barosensitive neurons of the rostral ventrolateral medulla.