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.

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.

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.

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.

Preproglucagon (PPG) neurons innervate neurochemically identified autonomic neurons in the mouse brainstem.

Preproglucagon (PPG) neurons produce glucagon-like peptide-1 (GLP-1) and occur primarily in the nucleus tractus solitarius (NTS). GLP-1 affects a variety of central autonomic circuits, including those controlling the cardiovascular system, thermogenesis, and most notably energy balance. Our immunohistochemical studies in transgenic mice expressing YFP under the control of the PPG promoter showed that PPG neurons project widely to central autonomic regions, including brainstem nuclei. Functional studies have highlighted the importance of hindbrain receptors for the anorexic effects of GLP-1. In this study, we assessed YFP innervation of neurochemically identified brainstem neurons in transgenic YFP-PPG mice. Immunoreactivity for YFP plus choline acetyltransferase (ChAT), tyrosine hydroxylase (TH) and/or serotonin (5-HT) was visualised with two- or three-colour immunoperoxidase labelling using black (YFP), brown and blue-grey reaction products. In the dorsal motor nucleus of the vagus (DMV), terminals from fine YFP-immunoreactive axons closely apposed a small proportion of ChAT-positive and rare TH-positive/ChAT-positive motor neurons, mostly ventral to AP. YFP-immunoreactive innervation was virtually absent from the compact and loose formations of the nucleus ambiguus. In the NTS, some TH-immunoreactive neurons were closely apposed by YFP-containing axons. In the A1/C1 column in the ventrolateral medulla, close appositions on TH-positive neurons were more common, particularly in the caudal portion of the column. A single YFP-immunoreactive axon usually provided 1-3 close appositions on individual ChAT- or TH-positive neurons. Serotonin-immunoreactive neurons were most heavily innervated, with the majority of raphé pallidus, raphé obscurus and parapyramidal neurons receiving several close appositions from large varicosities of YFP-immunoreactive axons. These results indicate that GLP-1 neurons innervate various populations of brainstem autonomic neurons. These include vagal efferent neurons and catecholamine neurons in areas linked with cardiovascular control. Our data also indicate a synaptic connection between GLP-1 neurons and 5-HT neurons, some of which might contribute to the regulation of appetite.

Oxytocin-immunoreactive innervation of identified neurons in the rat dorsal vagal complex.

BACKGROUND: Oxytocin (OXT) has been implicated in reproduction and social interactions and in the control of digestion and blood pressure. OXT-immunoreactive axons occur in the dorsal vagal complex (DVC; nucleus tractus solitarius, NTS, dorsal motor nucleus of the vagus, DMV, and area postrema, AP), which contains neurons that regulate autonomic homeostasis. The aim of the present work is to provide a systematic investigation of the OXT-immunoreactive innervation of dorsal motor nucleus of the vagus (DMV) neurons involved in the control of gastrointestinal (GI) function. METHODS: We studied DMV neurons identified by (i) prior injection of retrograde tracers in the stomach, ileum, or cervical vagus or (ii) induction of c-fos expression by glucoprivation with 2-deoxyglucose. Another subgroup of DMV neurons was identified electrophysiologically by stimulation of the cervical vagus and then juxtacellularly labeled with biotinamide. We used two- or three-color immunoperoxidase labeling for studies at the light microscopic level. KEY RESULTS: Close appositions from OXT-immunoreactive varicosities were found on the cell bodies, dendrites, and axons of DMV neurons that projected to the GI tract and that responded to 2-deoxyglucose and juxtacellularly labeled DMV neurons. Double staining for OXT and choline acetyltransferase revealed that OXT innervation was heavier in the caudal and lateral DMV than in other regions. OXT-immunoreactive varicosities also closely apposed a small subset of tyrosine hydroxylase-immunoreactive NTS and DMV neurons. CONCLUSIONS & INFERENCES: Our results provide the first anatomical evidence for direct OXT-immunoreactive innervation of GI-related neurons in the DMV.

Hypothalamic cocaine- and amphetamine-regulated transcript and corticotrophin releasing factor neurons are stimulated by extracellular volume and osmotic changes.

Several studies suggest that hypothalamic cocaine- and amphetamine-regulated transcript (CART) may interact with the hypothalamic-pituitary-adrenal (HPA) axis in the control of neuroendocrine function and may also participate in cardiovascular regulation. Therefore, this study aimed to evaluate, in experimental models of isotonic (I-EVE) and hypertonic (H-EVE) extracellular volume expansion and water deprivation (WD), the activation of CART- and corticotrophin releasing factor (CRF)-immunoreactive neurons, as well as the relative expression of CART and CRF mRNAs in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Both H-EVE (0.30M NaCl, 2mL/100g of body weight, in 1 minute) and 24 hours of WD significantly increased plasma sodium concentrations, producing, respectively, either an increase or a decrease in extracellular volume. I-EVE (0.15M NaCl, 2mL/100g of body weight, in 1 minute) evoked a significant increase in the circulating volume accompanied by unaltered plasma concentrations of sodium. CART-expressing neurons of both magnocellular and parvocellular hypothalamic divisions were activated to produce Fos in response to H-EVE but not in response to I-EVE. Furthermore, increased expression of CART mRNA was found in the PVN of H-EVE but not I-EVE rats. These data show for the first time that EVE not only activates hypothalamic CRF neurons but also increases CRF mRNA expression in the PVN. In contrast, WD increases the number of CART-immunoreactive neurons activated to produce Fos in the PVN and SON but does not change the number of neurons double labeled for Fos and CRF or expression of CRF mRNA in the PVN. These findings provided new insights into the participation of CART in diverse processes within the PVN and SON, including its possible involvement in activation of the HPA axis and cardiovascular regulation in response to changes in extracellular volume and osmolality.

Preproglucagon neurons project widely to autonomic control areas in the mouse brain.

Glucagon-like peptide 1 (GLP-1) and its analogue exendin-4 inhibit food intake, reduce blood glucose levels and increase blood pressure and heart rate by acting on GLP-1 receptors in many brain regions. Within the CNS, GLP-1 is produced only by preproglucagon (PPG) neurons. We suggest that PPG neurons mediate the central effects of GLP-1 by modulating sympathetic and vagal outflow. We therefore analysed the projections of PPG neurons to brain sites involved in autonomic control. In transgenic mice expressing yellow fluorescent protein (YFP) under the control of the PPG promoter, we assessed YFP-immunoreactive innervation using an anti-GFP antiserum and avidin-biotin-peroxidase. PPG neurons were intensely YFP-immunoreactive and axons could be easily discriminated from dendrites. YFP-immunoreactive cell bodies occurred primarily within the caudal nucleus tractus solitarius (NTS) with additional somata ventral to the hypoglossal nucleus, in raphé obscurus and in the intermediate reticular nucleus. The caudal NTS contained a dense network of dendrites, some of which extended into the area postrema. Immunoreactive axons were widespread throughout NTS, dorsal vagal nucleus and reticular nucleus with few in the hypoglossal nucleus and pyramids. The dorsomedial and paraventricular hypothalamic nuclei, ventrolateral periaqueductal grey and thalamic paraventricular nucleus exhibited heavy innervation. The area postrema, rostral ventrolateral medulla, pontine central grey, locus coeruleus/Barrington's nucleus, arcuate nucleus and the vascular organ of the lamina terminalis were moderately innervated. Only a few axons occurred in the amygdala and subfornical organ. Our results demonstrate that PPG neurons innervate primarily brain regions involved in autonomic control. Thus, central PPG neurons are ideally situated to modulate sympathetic and parasympathetic outflow through input at a variety of central sites. Our data also highlight that immunohistochemistry improves detection of neurons expressing YFP. Hence, animals in which specific populations of neurons have been genetically-modified to express fluorescent proteins are likely to prove ideal for anatomical studies.

Orexin-immunoreactive inputs to rat sympathetic preganglionic neurons.

Orexin increases blood pressure and orexin-immunoreactive (IR) axons robustly innervate the spinal cord. Seeking anatomical evidence for direct effects of orexin on sympathetic preganglionic neurons (SPN), we used immunohistochemistry to study the relationships between orexin-IR axons and SPN identified by immunoreactivity for choline acetyltransferase (ChAT) or for cholera toxin B retrogradely transported from the superior cervical ganglion (SCG). In the intermediolateral cell column (IML), varicose, orexin-positive axons closely apposed almost all SPN in segments T1 and T2, but appositions were rare in T4-L2. Orexin fibers also apposed ChAT-IR cell bodies in the intercalated nucleus and the central autonomic area from T1 to L2. Orexin-IR synapses were identified ultrastructurally on SPN projecting to the SCG. Since SPN involved in cardiovascular control cluster in the IML of mid- and lower thoracic cord, these findings suggest that orexin affects blood pressure by acting on supraspinal neurons rather than SPN.

Physiological, pharmacological, and immunohistochemical characterisation of juxtacellularly labelled neurones in rat nucleus tractus solitarius.

The pharmacology and anatomy of neurones in the nucleus tractus solitarius (NTS) have proved to be difficult to study in vivo because of their generally small size and high packing density. To overcome these problems, we have developed an approach that combines drug application through multibarrelled electrodes with juxtacellular labelling via an attached single-barrelled electrode followed by immunohistochemical processing. This approach has allowed us to assess the responses of individual NTS neurones in vivo to ionotropic glutamate receptor agonists and antagonists and then, to determine whether the neurones expressed the glutamate receptor subunits, GLUR2,3 and NMDAR2a,b. It should also be possible to extend these techniques further and correlate morphology with these features and to examine pharmacologically characterised, dye-filled neurones at the ultrastructural level.

Glutamate and GABA content of calbindin-immunoreactive nerve terminals in the rat intermediolateral cell column.

Immunoreactivity for calbindin-D28K (calbindin) occurs in some bulbospinal vasopressor neurons in the rostral ventrolateral medulla and calbindin-immunoreactive terminals form synapses in the intermediolateral cell column (IML), where the cell bodies of sympathetic preganglionic neurons are located. In this study, we used post-embedding immunogold labelling to determine whether calbindin terminals in the IML contained the excitatory amino acid neurotransmitter glutamate. We also assessed GABA immunoreactivity in semi-serial sections through the same terminals since this inhibitory amino acid transmitter is present in the inputs to sympathetic preganglionic neurons that lack glutamate. Analysis of 42 calbindin-positive terminals whose postsynaptic targets were not identified revealed two major groups on the basis of amino acid content. One group was immunoreactive for glutamate; and the other, for GABA. In addition, about 20% of the calbindin terminals were positive for both glutamate and GABA. Our anatomical methods cannot differentiate whether this third group is a subset of the GABAergic terminals or a separate population capable of co-releasing the two amino acids.

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.

Neuropeptide Y mRNA expression in interneurons in rat spinal cord.

Neuropeptide Y (NPY)-immunoreactive axons are present within the spinal cord. Some of these axons originate from neurons in the brainstem. Other axons arise from within the spinal cord since NPY-immunoreactivity can be detected after complete spinal cord transection. To identify spinal neurons that might express NPY, we localized NPY mRNA in rat spinal cord using in situ hybridization histochemistry. NPY mRNA-containing neurons were localized in the dorsal horn, in medial laminae of the grey matter and in the lateral spinal nucleus in thoracic, lumbar and sacral cord. The location of some of these neurons, and their proximity to sympathetic preganglionic neurons, suggest some NPY-containing interneurons are likely to be involved in spinal as well as supraspinal autonomic reflex pathways.

Neurokinin-1 receptor immunoreactivity in hypotension sensitive sympathetic preganglionic neurons.

Substance P activation of neurokinin-1 (NK1) receptors on spinal sympathetic preganglionic neurons (SPN) influences blood pressure. We identified SPN likely to subserve the baroreceptor reflex and established if these neurons showed NK1 receptor-immunoreactivity. Nitroprusside (NP) infusion or inferior vena cava (IVC) constriction activated similar numbers of SPN. Of these, about 40% were NK1 receptor-immunoreactive after NP infusion, but only about 20% were NK1 receptor-immunoreactive after IVC constriction. The distribution of Fos/NK1 receptor SPN suggested that substance P may preferentially target sympathoadrenal SPN.

Patterns of colocalization of GABA, glutamate and glycine immunoreactivities in terminals that synapse on dendrites of noradrenergic neurons in rat locus coeruleus.

Amino acid transmitters play a key role in regulating the activity of noradrenergic neurons in the locus coeruleus. We investigated the anatomical substrate for this regulation by quantifying immunoreactivity for GABA, glutamate and glycine in terminals that contacted the dendrites of tyrosine hydroxylase-immunoreactive principal neurons in rat locus coeruleus. Pre-embedding peroxidase immunocytochemistry was used to detect tyrosine hydroxylase-immunoreactivity in Vibratome sections of tissue perfused with 2.5% glutaraldehyde. GABA, glutamate and glycine were localized with postembedding immunogold labelling. Gold particle densities over terminals were measured in three semiserial ultrathin sections, each reacted for a different amino acid. More than 90% (range among rats, 89%-95%) of the terminals analyzed (n = 288) were immunoreactive for at least one amino acid. A high proportion (39%-49%) were positive for two or three amino acids. About two-thirds (60%-69%) of the boutons contained GABA, of which more than half (51%-55%) also contained glycine. More than one-third (36%-38%) of the terminals were positive for glycine. Terminals immunoreactive for glycine alone were rare (0%-2%). About one-third of the terminals showed glutamate-immunoreactivity (32%-37%). GABA and/or glycine occurred in one-fifth to one-third of these. These results show that amino acid-immunoreactivity is present in almost all of the terminals that synapse on tyrosine hydroxylase-positive dendrites in locus coeruleus. Glutamate provides a major excitatory input. The almost complete colocalization of glycine with GABA suggests that the inhibitory input to locus coeruleus is predominantly GABAergic with a contribution from glycine in about half of the GABAergic boutons.

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.

Tracer-toxins: cholera toxin B-saporin as a model.

We have shown previously that retrogradely-transported cholera toxin B (CTB)-saporin has eliminated sympathetic preganglionic neurons by 7 days after injection (Llewellyn-Smith, I.J., Martin, C.L., Arnolda, L.F., Minson, J.B., 1999. NeuroReport 10, 307). To ascertain whether this tracer-toxin can kill other types of neurons that transport CTB retrogradely with a similar time course, we injected CTB-saporin into the facial nerves of rats and allowed them to survive for 7 days. Facial motoneurons were counted ipsilateral and contralateral to the injected nerves in sections of perfused medulla processed to reveal immunoreactivity for choline acetyltransferase (ChAT). There was a statistically significant decrease in the number of ChAT-immunoreactive neurons ipsilateral to the injected nerve in three out of nine rats. Inadequate injections were probably the reason that most rats showed no decrease in motoneurons numbers after treatment with CTB-saporin, since the staining intensity and numbers of facial motoneurons that showed CTB-immunoreactivity varied markedly between rats after retrograde tracing with unconjugated CTB. These results show that CTB-saporin can eliminate motoneurons as well as sympathetic preganglionic neurons, indicate that protocols for the injection of tracer-toxins should be optimized to ensure maximum neuronal death and support our contention that CTB-saporin should kill any central neuron that expresses GM1 ganglioside, the membrane component to which CTB binds.

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.

Nitric oxide limits pressor responses to sympathetic activation in rat spinal cord.

N-methyl D-aspartate (NMDA) receptor stimulation is known to activate nitric oxide (NO) synthase, an enzyme present in a high proportion of sympathetic preganglionic neurons. In this study, we have examined the possibility that NO modulates the pressor responses elicited by NMDA receptor stimulation in the spinal cord. In experiments on anesthetized rats, we determined whether intrathecal administration of either 3-morpholinylsydnoneimine chloride (SIN-1), an NO donor, or N:(G)-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, affected the response to stimulation of spinal NMDA receptors by NMDA (1 pmol to 1 micromol in 10-microL intrathecal administration). Intrathecal NMDA resulted in dose-dependent increases in blood pressure. SIN-1 (100 nmol) attenuated the pressor responses to NMDA (F(1,70)=12, P=0.001). Conversely, L-NAME (1 nmol to 1 micromol) augmented the pressor response to NMDA in a dose-dependent manner (F(3,161)=28.3, P<0.001). The effect of L-NAME to amplify the pressor response to NMDA was reversed by L-arginine but not by D-arginine. These results indicate that endogenous synthesis of NO in the spinal cord limits the pressor response to stimulation of spinal NMDA receptors.

Substance P-immunoreactive boutons closely appose inspiratory protruder hypoglossal motoneurons in the cat.

In anesthetized cats, we recorded intracellularly from 26 hypoglossal motoneurons which were antidromically activated following electrical stimulation of either the medial or lateral branches of the hypoglossal nerve. Twenty-one of these neurons were protruder motoneurons 6 of which had inspiratory activity. Three of the protruder motoneurons with inspiratory activity were filled with Neurobiotin and found to be closely apposed to substance P-like immunoreactive nerve terminals.