Phillip Jobling

Orally projecting interneurones in the guinea-pig small intestine

1 Orally projecting, cholinergic interneurones are important in mediating ascending excitatory reflexes in the small intestine. We have shown that there is just one major class of orally projecting interneurone, which we have characterized using retrograde labelling in organ culture, combined with immunohistochemistry, intracellular recording and dye filling. 2 Orally projecting interneurones, previously shown to be immunoreactive for choline acetyltransferase, tachykinins, enkephalin, calretinin and neurofilament protein triplet, have axons up to 14 mm long and are the only class of cells with orally directed axons more than 8.5 mm long. 3 They are all small Dogiel type I neurones with short dendrites, usually lamellar in form, and a single axon which sometimes bifurcates. Their axons give rise to short varicose collaterals in myenteric ganglia more than 3 mm oral to their cell bodies. 4 Orally projecting interneurones receive prominent fast excitatory post synaptic potentials (fast EPSPs). A major source of fast EPSPs is other ascending interneurones located further aborally. They also receive fast EPSPs from circumferential pathways. 5 In the stretched preparations used in this study, orally projecting interneurones were highly excitable, firing repeatedly to depolarizing current pulses and had negligible long after ‐hyperpolarizations following their action potentials. They did not receive measurable non‐cholinergic slow excitatory synaptic inputs. 6 Ascending interneurones had a characteristic inflection in their membrane responses to depolarizing current pulses and their first action potential was typically delayed by approximately 30 ms. Under single electrode voltage clamp, ascending interneurones had a transient outward current when depolarized above –70 mV from more hyperpolarized holding potentials. Ascending interneurones also consistently showed marked inward rectification under both current clamp and voltage clamp conditions. 7 This class of cells has consistent morphological, neurochemical and electrophysiological characteristics and are important in mediating orally directed enteric reflexes.

Most peptide-containing sensory neurons lack proteins for exocytotic release and vesicular transport of glutamate

We used multiple‐labeling immunohistochemistry and confocal microscopy to examine co‐expression of immunoreactivity for vesicular glutamate transporters (VGluTs), synaptic vesicle proteins, and soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins in peptide‐containing sensory neurons of guinea pigs, mice, and toads. Axon terminals in the superficial layers of the dorsal horn of the spinal cord with immunoreactivity (IR) for both substance P (SP) and calcitonin gene‐related peptide (CGRP) lacked IR for synaptosome‐associated protein of 25 kDa (SNAP‐25), syntaxin, synaptotagmin, synaptophysin, and synapsin, although adjacent varicosities without neuropeptides had IR for these synaptic proteins. Similarly, peptide‐containing axon terminals in the superficial dorsal horn lacked IR for VGluT1 and VGluT2, despite the presence of VGluT2‐IR in nearby nonpeptide varicosities. VGluT3‐IR was sparse in the dorsal horn of the mouse spinal cord and was not present in peptide‐containing axons. Most peripheral terminals of sensory neurons with both SP‐IR and CGRP‐IR in the skin, viscera, and autonomic ganglia of guinea pigs and mice also lacked IR for synaptic vesicle proteins, SNARE proteins, VGluT1, and VGluT2. In dorsal root ganglia from guinea pigs and mice, most small neurons with IR for both SP and CGRP lacked IR for SNAP‐25, VGluT1, and VGluT2. Thus, proteins considered essential for vesicular uptake and exocytotic release of glutamate are not expressed at detectable levels by most sensory neurons containing SP and CGRP in rodents and toads. These data raise the possibility that most peptide‐containing sensory neurons may not normally release glutamate as a transmitter. J. Comp. Neurol. 483:1–16, 2005.

Calcium induced calcium release is involved in the afterhyperpolarization in one class of guinea pig sympathetic neurone

The mechanisms underlying two potassium conductances which are activated by Ca2+ influx during the action potential in sympathetic prevertebral neurones of guinea pigs have been investigated pharmacologically. One Ca-activated K+ conductance, which is present in all mammalian sympathetic postganglionic neurones, is maximal after the action potential and decays exponentially with a time constant of about 130 ms; this conductance was inhibited by apamin (50-100 nM) consistent with the involvement of SK channels. A second Ca-activated K+ conductance with much slower kinetics is present in a large subpopulation of coeliac neurones. This conductance was resistant to apamin but markedly inhibited by application of ryanodine (5-20 microM), suggesting that Ca2+ influx during the action potential triggers release of Ca2+ from intracellular stores which in turn activates a different class of K+ channel. Noradrenaline (100 microM) depressed the second K+ conductance selectively.

Nerve–Cancer Cell Cross-talk: A Novel Promoter of Tumor Progression

Recent studies have revealed the essential role played by nerves in tumor progression. Nerves have been shown to infiltrate the tumor microenvironment and actively stimulate cancer cell growth and dissemination. This mechanism involves the release of neurotransmitters, such as catecholamines and acetylcholine, directly into the vicinity of cancer and stromal cells to activate corresponding membrane receptors. Conversely, the secretion of neurotrophic growth factors by cancer cells drives the outgrowth of nerves in solid tumors. This reciprocal interaction between nerves and cancer cells provides new insights into the cellular and molecular bases of tumorigenesis and points to the potential utility of antineurogenic therapies. This review will discuss our evolving understanding of the cross-talk between nerves and cancer cells.

Electrophysiological responses in the rat tail artery during reinnervation following lesions of the sympathetic supply.

1. Responses to perivascular stimuli have been recorded with intracellular microelectrodes from the smooth muscle of isolated segments of the main caudal artery of rats at various times between 7 and 128 days after all four collector nerve trunks had been lesioned near the base of the tail at 21 days of age. 2. In proximal segments (< 40 mm distal to the lesions), excitatory junction potentials (EJPs) and neurogenic alpha‐depolarizations (NADs) evoked by stimuli presented via a proximally located suction electrode were similar to those in the same segments of unoperated control animals of the same age. Supramaximal EJPs in these segments decreased in amplitude with age. 3. Stimuli just supramaximal for EJPs in innervated preparations failed to evoke responses in segments farther than 30‐40 mm distal to the lesions at any time after the nerves had been cut and 1 cm excised. Higher voltages evoked slow depolarizing potentials (SDPs) which were of longer time course than EJPs. Similar responses occurred in segments over 60 mm distal to the lesions at 20‐50 days after the nerves had been frozen, and in all segments sampled over 100 mm distal to nerve lesions. 4. Spontaneous transient depolarizations (STDs) were recorded at all depths of the media in denervated segments. These occurred at frequencies similar to those of spontaneous events (including attenuated spontaneous EJPs) in innervated segments. 5. The earliest signs of reinnervation (24‐42 days after freeze lesions) consisted of very small amplitude EJPs of normal time course which facilitated markedly during a short train of stimuli (5‐10 Hz); these were followed by NADs which were large relative to the amplitudes of the EJPs. Less commonly, small focal EJPs of brief time course (resembling spontaneous EJPs in superficial cells of innervated arteries) were evoked in very restricted regions of the vessel wall. 6. At later times (57‐128 days postoperative), six of eight segments located 40‐70 mm distal to freeze lesions showed EJPs of nearly control amplitude, but NADs that were larger than in equivalent segments from control animals. In the remaining two cases, reinnervation at this level was similar to that seen at the earliest postoperative times. High stimulus voltages prolonged the decay of EJPs in both control and reinnervated arteries. 7. Sensitivity to exogenous noradrenaline, assessed in terms of membrane depolarization, was increased in both denervated and reinnervated segments. 8. Catecholamine fluorescence disappeared from the arteries at a distance greater than 30‐40 mm distal to the site of the nerve lesions.(ABSTRACT TRUNCATED AT 400 WORDS)

Cocaine potentiates excitatory drive in the perifornical/lateral hypothalamus

•  Drugs of addiction are well‐established in their capacity to alter brain reward pathways. •  The perifornical/lateral hypothalamus has previously been shown to be drug responsive, participate in relapse to drug taking, and project to key reward pathway structures. •  This study demonstrates that cocaine enhances excitatory drive to perifornical/lateral hypothalamic neurones, and these changes involve altered presynaptic function. Orexin‐positive neurones were among the populations that underwent these presynaptic changes. •  The results indicate that a greater understanding of the drug‐induced synaptic changes in perifornical/lateral hypothalamus may instruct future pharmacotherapies aimed at preventing drug relapse.

Neuronal morphology and the synaptic organisation of sympathetic ganglia

In this article, we provide a short review of the structure and synaptic organisation of the final motor neurons in the sympathetic ganglia of mammals. Combinations of pathway tracing, multiple-labelling immunofluorescence and intracellular dye injection have shown that neurons in different functional pathways differ not only in their patterns of neuropeptide expression, but also in the size of their cell bodies and dendritic fields. Thus, vasoconstrictor neurons consistently are smaller than any other major functional class of neurons. Serial section ultrastructural analysis of dye filled neurons, together with electron microscopic and confocal microscopic analysis of immunolabelled synaptic inputs to sympathetic final motor neurons indicate that synapses are rare and randomly distributed over the surface of the neurons. The total number of synapses is simply proportional to the total surface area of the neurons. Many terminal boutons of peptide-containing preganglionic neurons do not make conventional synapses with target neurons. Furthermore, there is a spatial mismatch in the distribution of peptide-containing terminals and neurons expressing receptors for the corresponding peptides. Together, these results suggest that there are likely to be significant differences in the ways that the final sympathetic motor neurons in distinct functional pathways integrate their synaptic inputs. In at least some pathways, heterosynaptic actions of neuropeptides probably contribute to subtle modulation of ganglionic transmission.

Synaptic density, convergence, and dendritic complexity of prevertebral sympathetic neurons

Prevertebral sympathetic ganglia contain a unique population of final motor neurons receiving convergent synaptic inputs not only from spinal preganglionic neurons, but also from peripheral intestinofugal neurons projecting from the gut. We used quantitative confocal and ultrastructural immunohistochemistry to determine how this increased synaptic convergence is accommodated by sympathetic final motor neurons in the celiac ganglion of guinea pigs. Terminals of intestinofugal neurons were identified by their immunoreactivity to vasoactive intestinal peptide. Stereologic analyses were based on transects and point counts at confocal and ultrastructural levels. The relative amount of dendritic neuropil in the medial regions of the ganglion was approximately 2.5 times greater than in the lateral regions of the ganglion, consistent with the 2 to 3 times difference in average dendritic field size of neurons in these regions. The total numbers of boutons and synaptic profiles showed significant positive correlations with the relative amount of neuropil in a region. However, the overall density of synaptic boutons was twice as high in the medial region of the ganglion compared with the lateral regions. Because the relative density of preganglionic synapses was similar in each region, this difference was due to the selective projection of intestinofugal inputs to neurons in the medial celiac ganglion, where they provided 45% of synaptic contacts. These results show that, compared with vasoconstrictor neurons, sympathetic neurons regulating gastrointestinal activity support a higher number of convergent inputs in two ways: in addition to having larger dendritic fields, they also have a twofold higher density of synapses. J. Comp. Neurol. 455:285–298, 2003.

An electrophysiological study of responses evoked in isolated segments of rat tail artery during growth and maturation.

1. Intracellular recordings from the smooth muscle of isolated segments of the main caudal artery of rats at various ages between 45 and 150 days postnatal were made in order to relate the spontaneous depolarizations and responses to perivascular stimulation at different levels along the artery to the differences in vessel structure and innervation density during growth of the animals. 2. In the outermost smooth muscle cells close to the neuromuscular junctions, spontaneous depolarizations with fast time courses (spontaneous excitatory junction potentials or SEJPs) were recorded. In cells lying deeper in the media, spontaneous depolarizations had a wide range of time courses and amplitudes, but only a few of those could be attributed to electrotonic attenuation of SEJPs. 3. In arterial segments taken from animals of all ages, stimuli which evoked maximal amplitude excitatory junction potentials (EJPs) 1‐2 mm caudal to a suction electrode also evoked neurogenic alpha‐depolarizations (NADs) with time to peak of 15 s and duration nearly 1 min. Both responses decreased progressively in amplitude along the length of the artery. NADs were blocked by phentolamine (10(‐6) M) or idazoxan (10(‐7) M) which were without effects on EJPs. 4. During short trains of stimuli (5 at 1 or 10 Hz), EJPs facilitated but to a lesser extent with distance along the tail. Such trains evoked NADs of greater amplitude than those following a single stimulus; these were often preceded by contractions of the artery which were restricted to the region close to the stimulating electrode. 5. Increasing stimulus voltage led to progressive prolongation of the decay phase of the EJP. After the addition of tetrodotoxin (10(‐7) M), or in the presence of reduced Ca2+ and raised Mg2+ concentration, slow depolarizing potentials (SDPs) (with time to peak of 150‐300 ms and decay lasting > 2 s) were recorded which were graded in amplitude with stimulus voltage. SDPs were attenuated by increasing Ca2+ concentration to 5 mM. These responses often added to the EJP at supramaximal stimulus voltages. 6. The mean amplitudes of the EJP and NAD declined significantly with age, the former to a greater degree than the latter. These changes may be explained by changes in the electrical properties of the media related to hypertrophy of smooth muscle cells as the animals grew, and emphasize the need to allow for such growth effects in studies of young rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Heterogeneous expression of SNAP-25 and synaptic vesicle proteins by central and peripheral inputs to sympathetic neurons

Neurons in prevertebral sympathetic ganglia receive convergent synaptic inputs from peripheral enteric neurons in addition to inputs from spinal preganglionic neurons. Although all inputs are functionally cholinergic, inputs from these two sources have distinctive neurochemical and functional profiles. We used multiple‐labeling immunofluorescence, quantitative confocal microscopy, ultrastructural immunocytochemistry, and intracellular electrophysiologic recordings to examine whether populations of inputs to the guinea pig coeliac ganglion express different levels of synaptic proteins that could influence synaptic strength. Boutons of enteric intestinofugal inputs, identified by immunoreactivity to vasoactive intestinal peptide, showed considerable heterogeneity in their immunoreactivity to synaptosome‐associated protein of 25 kDa (SNAP‐25), synapsin, synaptophysin, choline acetyltransferase, and vesicular acetylcholine transporter. Mean levels of immunoreactivity to these proteins were significantly lower in terminals of intestinofugal inputs compared with terminals of spinal preganglionic inputs. Nevertheless, many boutons with undetectable levels of SNAP‐25 immunoreactivity formed morphologically normal synapses with target neurons. Treatment with botulinum neurotoxin type A (20–50 nM for 2 hours in vitro) generated significant cleavage of SNAP‐25 and produced similar dose‐ and time‐dependent inhibitions of synaptic transmission from all classes of inputs, regardless of their mean level of SNAP‐25 expression. The simplest interpretation of these results is that only synaptic boutons with detectable levels of SNAP‐25 immunoreactivity contribute significantly to fast cholinergic transmission. Consequently, the low synaptic strength of intestinofugal inputs to final motor neurons in sympathetic pathways may be due in part to the low proportion of their boutons that express SNAP‐25 and other synaptic proteins. J. Comp. Neurol. 459:25–43, 2003.