Biomedical subjects

I J Llewellyn-Smith

Publications and source records attributed to I J Llewellyn-Smith.

At least 19 recordsLinked to original sources

PubMed2003

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.

Acetylcholine↗

PubMed2002

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.

Animals↗

PubMed2002

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.

Animals↗

PubMed2001

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.

Animals↗

PubMed2001

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.

Animals↗

PubMed2001

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.

Amino Acids↗

PubMed2001

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.

Action Potentials↗

PubMed2001

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.

Afferent Pathways↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Anesthesia↗

PubMed1999

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.

Animals↗

PubMed1999

Retrogradely transported CTB-saporin kills sympathetic preganglionic neurons.

Aiming to ablate sympathetic preganglionic neurons (SPN) innervating a defined target, we injected saporin conjugated to cholera toxin B subunit (CTB) unilaterally into the superior cervical ganglion of rats. In spinal cord segments T1-T3, the numbers of cholinergic neurons in the intermediolateral cell column ipsilateral and contralateral to the injected ganglion were significantly different by 3 days post-injection. By day 14, 77% of ipsilateral cholinergic neurons had disappeared. A higher percentage of neurons were killed in T1-T2 than in T3. Comparing SPN counts from CTB-saporin injected rats and counts from rats receiving unconjugated CTB into the superior cervical ganglion indicated that 84% of SPN supplying the ganglion had died by 14 days. Retrogradely transported CTB-saporin kills sympathetic preganglionic neurons and may also eliminate other types of neurons that transport CTB.

Animals↗

PubMed1999

Neurokinin-1 receptors and spinal cord control of blood pressure in spontaneously hypertensive rats.

In this study we examined blood pressure and heart rate responses to intrathecal administration of a synthetic NK1-receptor agonist, H2N-(CH2)4-CO-Phe-Phe-Pro-NmeLeu-Met-NH2 (GR 73,632), in spontaneously hypertensive rats (SHR) and their progenitor strain, the Wistar-Kyoto rat (WKY). Sodium pentobarbitone anaesthetised rats with implanted intrathecal catheters were paralysed (pancuronium dibromide) and artificially ventilated. Injection of GR 73,632 at the T9 spinal level evoked dose-dependent increases in mean arterial pressure (MAP) in WKY and SHR. SHR had a lower MAP response threshold than WKY but increase in response with increasing dose was less in SHR than WKY. Biphasic blood pressure responses at high doses were observed in both strains. Prior administration of the NK1-receptor antagonist (3 aR,7aR)-7,7-diphenyl-2-[1-imino-2(methoxyphenyl)ethyl] perhydroisoindol-4-one (RP 67,580) significantly reduced the pressor response in WKY but not SHR. The depressor response was not attenuated in either strain.

Analgesics↗

PubMed1999

Animal models of heart failure.

Animal models of heart failure present homogenous groups of animals all with heart failure produced by a well defined lesion at a particular stage of evolution, in contrast to humans, who present with heart failure of uncertain duration from a wide variety of causes and with marked variation in age and pre-morbid health and fitness. Animal models of heart failure provide diseased groups of animals in which experimental procedures, not possible in humans, can be evaluated and in which new treatments can be tested before their safety is established in humans. An ideal model should have a common human counterpart and should closely mimic heart failure in humans. Thus the haemodynamic changes should include increased cardiac filling pressures and low cardiac output. There should be evidence of activation of the sympathetic nervous system and increased secretion of hormones such as renin, angiotensin, aldosterone, vasopressin, atrial natriuretic factor and endothelin. The clinical features of the human syndrome such as cardiomegaly, lung and peripheral oedema and decreased exercise tolerance should be present. Lastly, the model should be inexpensive and technically simple to produce and study. This paper reviews some commonly used models of heart failure in relation to the criteria listed above. There is no perfect animal model of heart failure and in practice one should match the model to the purpose of the study.

Animals↗

PubMed1999

Activation of spinal opioid receptors contributes to hypotension after hemorrhage in conscious rats.

Opioid receptors are activated during severe hemorrhage, resulting in sympathoinhibition and a profound fall in blood pressure. This study examined the location and subtypes of opioid receptors that might contribute to hypotension after hemorrhage. Intrathecal naloxone methiodide (100 nmol) abolished the fall in blood pressure after hemorrhage (1.5% of body wt; mean arterial pressure 122 +/- 8 mmHg after naloxone methiodide vs. 46 +/- 5 mmHg in controls, P < 0. 001). Intracisternal naloxone methiodide was less effective than intrathecal naloxone methiodide, whereas intravenous naloxone methiodide, which does not cross the blood-brain barrier, did not alter the fall in blood pressure after hemorrhage. These results demonstrate that spinal opioid receptors contribute to hypotension after hemorrhage but do not exclude supraspinal effects. In separate experiments, the subtype-specific opioid antagonists ICI-174864 (delta-antagonist), norbinaltorphimine (nor-BNI; kappa-antagonist), and H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP; mu-antagonist) were each administered intrathecally to determine the minimum dose that would attenuate hypotension during severe hemorrhage. These antagonists were effective at similar doses (3 nmol for CTOP, 6 nmol for ICI-174864, and 10 nmol for nor-BNI), although the binding affinities of these three different agents for their target receptors varied >1600-fold. Comparisons of the minimum effective doses of these antagonists in relation to their binding affinities provides strong evidence for the participation of delta-receptors in mediating hypotension after hemorrhage. In contrast, the dose at which nor-BNI was effective suggests an effect at delta-receptors but not kappa-receptors. The efficacy of CTOP, albeit at a high dose, also suggests an effect at mu-receptors.

Animals↗

PubMed1998

Ultrastructural localization of P2X3 receptors in rat sensory neurons.

We used isolated IgG antibodies selective for P2X3 receptors to study the ultrastructural distribution of these receptors in rat sensory neurons. In trigeminal ganglia, P2X3 receptor immunoreactivity occurred in small and large nerve cell bodies and their processes. Endoplasmic reticulum and Golgi apparatus were heavily stained; cytoplasmic matrix was faintly to moderately stained. In synaptic glomeruli in lamina II of cervical dorsal horn, P2X3 receptor-immunoreactive core terminals were postsynaptic to unlabelled vesicle-containing dendrites and axons. In the nucleus of the solitary tract, receptor-positive boutons synapsed on dendrites and cell bodies and had complex synaptic relationships with other axon terminals and vesiculated dendrites. These observations identify sites from which ATP could be released to influence sensory signalling within the central nervous system.

Animals↗

PubMed1998

GABA- and glutamate-immunoreactive synapses on sympathetic preganglionic neurons projecting to the superior cervical ganglion.

Our previous work suggests that virtually all of the synapses on sympathetic preganglionic neurons projecting to the rat adrenal medulla are immunoreactive for either the inhibitory amino acid, gamma-aminobutyric acid (GABA) or the excitatory amino acid, L-glutamate. To investigate whether or not this is true for other groups of sympathetic preganglionic neurons, and to determine whether or not the proportion of inputs containing each type of amino acid neurotransmitter is the same for different groups of sympathetic preganglionic neurons, we retrogradely labelled rat and rabbit sympathetic preganglionic neurons projecting to the superior cervical ganglion and used post-embedding immunogold on ultrathin sections to localise GABA- and glutamate-immunoreactivity. The cell bodies and dendrites of both rat and rabbit sympathetic preganglionic neurons projecting to the superior cervical ganglion received synapses and direct contacts from nerve fibres immunoreactive for GABA and from nerve fibres immunoreactive for glutamate. In the rat, GABA was present in 48.9% of the inputs to sympathetic preganglionic neurons projecting to the superior cervical ganglion, and glutamate was present in 51.7% of inputs. Double immunogold labelling for glutamate and GABA on the same section, as well as labelling of consecutive serial sections for the two antigens, indicated that GABA and glutamate occur in separate populations of nerve fibres that provide input to rat sympathetic preganglionic neurons projecting to the superior cervical ganglion. We now have shown that GABA or glutamate is present in virtually all of the inputs to sympathetic preganglionic neurons projecting to the superior cervical ganglion and in essentially all of the inputs to sympathetic preganglionic neurons supplying the adrenal medulla. These findings are consistent with the hypothesis that all fast synaptic transmission in central autonomic pathways may be mediated by either excitatory or inhibitory amino acids. Furthermore, we showed a statistically significant difference in the proportion of glutamate-immunoreactive inputs between sympathetic preganglionic neurons projecting to the superior cervical ganglion and sympathoadrenal neurons (data from Llewellyn-Smith et al. [Llewellyn-Smith, I.J., Phend, K.D., Minson, J.B., Pilowsky, P.M., Chalmers, J.P., 1992. Glutamate immunoreactive synapses on retrogradely labelled sympathetic neurons in rat thoracic spinal cord. Brain Res. 581, 67-80]), with preganglionics supplying the adrenal medulla receiving more excitatory inputs than those supplying the superior cervical ganglion. This increased excitatory input to sympathoadrenal neurons may explain the predominant activation of these neurons following baroreceptor unloading.

Animals↗