Trevor Batten

Neuronal P2X7 Receptors Are Targeted to Presynaptic Terminals in the Central and Peripheral Nervous Systems

The ionotropic ATP receptor subunits P2X1–6 receptors play important roles in synaptic transmission, yet the P2X7receptor has been reported as absent from neurons in the normal adult brain. Here we use RT-PCR to demonstrate that transcripts for the P2X7 receptor are present in extracts from the medulla oblongata, spinal cord, and nodose ganglion. Using in situ hybridization mRNA encoding, the P2X7 receptor was detected in numerous neurons throughout the medulla oblongata and spinal cord. Localizing the P2X7 receptor protein with immunohistochemistry and electron microscopy revealed that it is targeted to presynaptic terminals in the CNS. Anterograde labeling of vagal afferent terminals before immunohistochemistry confirmed the presence of the receptor in excitatory terminals. Pharmacological activation of the receptor in spinal cord slices by addition of 2′- and 3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP; 30 μm) resulted in glutamate mediated excitation of recorded neurons, blocked by P2X7 receptor antagonists oxidized ATP (100 μm) and Brilliant Blue G (2 μm). At the neuromuscular junction (NMJ) immunohistochemistry revealed that the P2X7 receptor was present in motor nerve terminals. Furthermore, motor nerve terminals loaded with the vital dye FM1–43 in isolated NMJ preparations destained after application of BzATP (30 μm). This BzATP evoked destaining is blocked by oxidized ATP (100 μm) and Brilliant Blue G (1 μm). This indicates that activation of the P2X7 receptor promotes release of vesicular contents from presynaptic terminals. Such a widespread distribution and functional role suggests that the receptor may be involved in the fundamental regulation of synaptic transmission at the presynaptic site.

A GABAergic projection from the central nucleus of the amygdala to the nucleus of the solitary tract: a combined anterograde tracing and electron microscopic immunohistochemical study

The central nucleus of the amygdala is involved in the modulation of autonomic, somatic and endocrine functions, as well as behavioural responses to stressful stimuli. Anatomical and physiological studies have suggested that this nucleus sends projections to the nucleus of the solitary tract, the primary site of termination of vagal and glossopharyngeal afferent fibres in the brain stem. To determine the neurochemical nature of the amygdaloid input to the nucleus of the solitary tract, anterograde tracing with biotinylated dextran amine was combined with post-embedding immunogold labelling for GABA and glutamate immunoreactivities and with pre-embedding labelling for the vesicular GABA transporter. Following injection of biotin dextran amine into the central nucleus of the amygdala, anterogradely labelled axons and varicosities were found throughout the rostrocaudal extent of the nucleus of the solitary tract, particularly in the medial, ventral and ventrolateral subnuclei. The anterogradely labelled terminals were found to make predominantly symmetrical synaptic contacts with dendrites, and occasionally onto cell bodies and dendritic spines, and to contain immunoreactivity for GABA and for the vesicular GABA transporter. Immunolabelling of serial sections with antibodies to glutamate showed that none of these axon terminals contained high enough densities of gold particle labelling to suggest that they contained other than low metabolic levels of glutamate immunoreactivity. These results provide conclusive evidence for a GABAergic pathway from the central nucleus of the amygdala to the nucleus of the solitary tract. This GABAergic projection may provide a substrate for inhibition of lower brain stem visceral reflexes, including baroreflex inhibition, through which the central nucleus of the amygdala could participate in cardiovascular regulation related to emotional behaviour and the defence reaction.

ZMYND10 Is Mutated in Primary Ciliary Dyskinesia and Interacts with LRRC6

Defects of motile cilia cause primary ciliary dyskinesia (PCD), characterized by recurrent respiratory infections and male infertility. Using whole-exome resequencing and high-throughput mutation analysis, we identified recessive biallelic mutations in ZMYND10 in 14 families and mutations in the recently identified LRRC6 in 13 families. We show that ZMYND10 and LRRC6 interact and that certain ZMYND10 and LRRC6 mutations abrogate the interaction between the LRRC6 CS domain and the ZMYND10 C-terminal domain. Additionally, ZMYND10 and LRRC6 colocalize with the centriole markers SAS6 and PCM1. Mutations in ZMYND10 result in the absence of the axonemal protein components DNAH5 and DNALI1 from respiratory cilia. Animal models support the association between ZMYND10 and human PCD, given that zmynd10 knockdown in zebrafish caused ciliary paralysis leading to cystic kidneys and otolith defects and that knockdown in Xenopus interfered with ciliogenesis. Our findings suggest that a cytoplasmic protein complex containing ZMYND10 and LRRC6 is necessary for motile ciliary function.

Glutamate, γ-aminobutyric acid and tachykin-inimmunoreactive synapses in the cat nucleus tractus solitarii

SummaryNeurophysiological and pharmacological evidence suggests that glutamate, γ-aminobutyric acid and tachykinins (substance P and neurokinin A) each have a role in cardiovascular regulation in the nucleus tractus solitarii. This study describes the ultrastructural relationships between nerve terminals immunoreactive for these substances in the nucleus tractus solitarii of the cat using post-embedding immunogold (single and double) labelling techniques on sections of tissue embedded in LR White resin. The technique combines a high specificity of labelling with good ultrastructural and antigenic preservation. Glutamate-immunoreactive terminals, recognized by their high density of gold particle labelling compared to the mean tissue level of labelling, accounted for about 40% of all synaptic terminals in the region of the nucleus tractus solitarii analysed (medial, dorsal, interstitial, gelatinosus and dorsolateral subnuclei). They appeared to comprise several morphological types, but formed mainly asymmetrical synapses, most often with dendrites of varying size, and contained spherical clear vesicles together with fewer dense-cored vesicles. Substance P- and neurokinin A-immunoreactive terminals were fewer in number (9% of all terminals) but similar in appearance, with the immunoreaction restricted to the dense-cored vesicles. Analysis of serial- and double-labelled sections showed a co-existence of substance P and neurokinin A-immunoreactivity in 21% of glutamate-immunoreactive terminals. Immunoreactivity for γ-aminobutyric acid was found in 33% of all terminals in the nucleus tractus solitarii. These predominantly contained pleomorphic vesicles and formed symmetrical synapses on dendrites and somata. Possible sites of axo-axonic contact by γ-aminobutyric acid-immunoreactive terminals onto glutamateor tachykinin-immunoreactive terminals were rare, but examples of adjacent glutamate and γ-aminobutyric acid-immunoreactive terminals synapsing on the same dendritic profile were frequent. These results provide an anatomical basis for a γ-aminobutyric acid mediated inhibition of glutamatergic excitatory inputs to the nucleus tractus solitarii at a post-synaptic level.

Glutamate‐immunoreactivity in identified vagal afferent terminals of the cat: a study combining horseradish peroxidase tracing and postembedding electron microscopic immunogold staining

Using electron microscopic immunohistochemistry we have shown that strong glutamate‐immunoreactivity (glutamate‐ir) is present in neuronal cell bodies of the nodose ganglion, axons in the tractus solitarius and afferent terminals in the nucleus tractus solitarii. Vagal afferent fibres were specifically labelled by transganglionic retrograde transport of horseradish peroxidase (HRP). Fifty‐seven per cent of the HRP‐labelled terminals in the dorsomedial medulla were found to contain a high level of glutamate‐ir, suggesting that a population of vagal afferent fibres uses glutamate as a neurotransmitter substance. There were no apparent ultrastructural differences between glutamate‐ir and non‐glutamate‐ir vagal afferent terminals, both classes mainly containing rounded vesicles and forming asymmetric synapses. However, some difference in their preference for postsynaptic target was noted. The great majority (83%) of non‐glutamate‐ir vagal afferent terminals made axodendritic synapses, but only just over half (57%) of the glutamate‐ir vagal terminals made synaptic contact with dendrites. Approximately 13% of the HRP‐labelled terminals were found to make synaptic contact with HRP‐labelled dendrites or soma of motoneurones of the dorsal vagal motor nucleus, confirming the existence of monosynaptic connections between vagal afferent fibres and vagal motoneurones.

Differential co-localisation of the P2X7 receptor subunit with vesicular glutamate transporters VGLUT1 and VGLUT2 in rat CNS

Presynaptic P2X(7) receptors are thought to play a role in the modulation of transmitter release and have been localised to terminals with the location and morphology typical of excitatory boutons. To test the hypothesis that this receptor is preferentially associated with excitatory terminals we combined immunohistochemistry for the P2X(7) receptor subunit (P2X(7)R) with that for two vesicular glutamate transporters (VGLUT1 and VGLUT2) in the rat CNS. This confirmed that P2X(7)R immunoreactivity (IR) is present in glutamatergic terminals; however, whether it was co-localised with VGLUT1-IR or VGLUT2-IR depended on the CNS region examined. In the spinal cord, P2X(7)R-IR co-localised with VGLUT2-IR. In the brainstem, co-localisation of P2X(7)R-IR with VGLUT2-IR was widespread, but co-localisation with VGLUT1-IR was seen only in the external cuneate nucleus and spinocerebellar tract region of the ventral medulla. In the cerebellum, P2X(7)R-IR co-localised with both VGLUT1 and VGLUT2-IR in the granular layer. In the hippocampus it was co-localised only with VGLUT1-IR, including in the polymorphic layer of the dentate gyrus and the substantia radiatum of the CA3 region. In other forebrain areas, P2X(7)R-IR co-localised with VGLUT1-IR throughout the amygdala, caudate putamen, striatum, reticular thalamic nucleus and cortex and with VGLUT2-IR in the dorsal lateral geniculate nucleus, amygdala and hypothalamus. Dual labelling studies performed using markers for cholinergic, monoaminergic, GABAergic and glycinergic terminals indicated that in certain brainstem and spinal cord nuclei the P2X(7)R is also expressed by subpopulations of cholinergic and GABAergic/glycinergic terminals. These data support our previous hypothesis that the P2X(7)R may play a role in modulating glutamate release in functionally different systems throughout the CNS but further suggest a role in modulating release of inhibitory transmitters in some regions.

Ultrastructural Relationships Between GABAergic Terminals and Cardiac Vagal Preganglionic Motoneurons and Vagal Afferents in the Cat: A Combined HRP Tracing and Immunogold Labelling Study

The ultrastructural relationships between γ‐aminobutyric acid‐immunoreactive (GABA‐ir) neurons and other neurons of the nucleus tractus solitarius (NTS) and motoneurons of the nucleus ambiguus (NA) and dorsal motor vagal nucleus (DMVN), were examined by electron microscopic (EM) immunogold labelling with an anti‐GABA antiserum on brain stem sections in which vagal motoneurons and vagal afferent fibres were labelled with horseradish peroxidase (HRP). HRP was applied to the cervical vagus or the cardiac vagal branch of anaesthetized cats. After 24–48 h survival, brains were glutaraldehyde‐fixed and a stable HRP‐tetramethylbenzidine reaction product compatible with EM processing was revealed on 250 μm vibratome sections. Following osmium postfixation, dehydration and resin embedding, GABA‐ir was localized on ultrathin sections by an immunogold technique. GABA‐ir axon terminals, heavily and specifically labelled with gold particles, were very numerous within NTS, DMVN and NA. All terminals contained small, clear, pleomorphic vesicles and a few also contained larger dense cored vesicles. The density of gold particles over clear vesicles, dense cored vesicles and mitochondria was significantly greater than over the cytoplasm of these terminals. GABA‐ir synapses were found on the soma and dendrites of neurons, but rarely on other axon terminals within NTS, where GABA‐ir cell bodies and dendrites were also seen. These received synaptic contacts from both GABA‐ir terminals and from HRP‐labelled vagal afferents. In both the DMVN and NA, similar GABA‐ir synapses were present on both the soma and dendrites of HRP‐labelled motoneurons. GABA synapses were also present on other cell types in DMVN. These observations provide an anatomical basis for a GABAergic inhibition of neurons forming the central pathways of cardiovascular and other autonomic reflexes.

Electrical stimulation of the vagus increases extracellular glutamate recovered from the nucleus tractus solitarii of the cat by in vivo microdialysis

Release of glutamate into the extracellular space of the cat nucleus tractus solitarii (NTS) was measured by in vivo microdialysis and high performance liquid chromatography. Perfusion of the probe with 100 mM potassium increased glutamate release by 211% (P < 0.001), while electrical stimulation of the cervical vagus increased release by 53% (P < 0.01). These results are compatible with the hypothesis that glutamate is a neurotransmitter released by vagal afferent nerve terminals in the NTS.

Co-localization of neurotransmitter immunoreactivities in putative nitric oxide synthesizing neurones of the cat brain stem

The distribution of nitric oxide producing neurones in the medulla oblongata of the cat was investigated using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, and nitric oxide synthase (NOS) immunohistochemistry. The pattern of staining obtained with both methods was found to be similar. Strongly diaphorase and NOS reactive neurones were present in the paramedian and lateral tegmental fields, including the regions occupied by the A1/C1 catecholamine cell groups, the nucleus ambiguus and lateral reticular nucleus, and in a number of sensory nuclei including the nucleus of the tractus solitarius and the dorsal column nuclei. The extent of co-localization of NADPH-diaphorase with a number of neuropeptides and neurotransmitters was investigated by combining NADPH-diaphorase histochemistry with immunocytochemistry for neuropeptide Y, somatostatin, glutamate, cholecystokinin and tyrosine hydroxylase. NADPH-diaphorase reaction product was observed in neurones immunoreactive for glutamate and somatostatin. These double-labelled cells were found in the paramedian region, lateral reticular field, the nucleus prepositus hypoglossi and in the rostral nucleus of the tractus solitarius. In the rostral ventrolateral medulla NADPH-diaphorase/somatostatin immunoreactive cells were found in the paragigantocellular nucleus. NADPH-diaphorase/glutamate immunoreactive cells overlapped the nucleus ambiguus, the lateral reticular nucleus and the A1/C1 catecholaminergic cell groups. In addition, a few NADPH-diaphorase/glutamate immunoreactive cells were found in the paraolivary area and gigantocellular tegmental field, in the external cuneate and infratrigeminal nuclei. The functional implications of the co-localization of nitric oxide with these neurotransmitters in areas of the medulla concerned with cardiovascular regulation is discussed.

Anatomical distribution of galanin-like immunoreactivity in the brain and pituitary of teleost fishes

Immunohistochemical study of brains of five teleost fishes (molly, sea bass, killifish, flounder, tilapia) revealed similar extensive systems of galanin immunoreactive (GAL-ir) neurons. Cell bodies were located in the anterior preoptic recess (where coexistence with corticotrophin-releasing factor-like-ir was found), posterior tuberal hypothalamus and vagal lobe of the medulla oblongata. Fibres in the fingers of neurohypophysial tissue penetrating the pituitary pars distalis suggested an anatomical relationship between GAL-ir terminals and the hormone secreting cells. Electron microscopic studies on sea bass pituitary revealed contacts of GAL-ir fibres with growth hormone cells and gonadotrophs. Thus a GAL-like peptide may be released from nerve terminals in the teleost pituitary, where it may act locally to modulate the secretion of one or more pituitary hormones.