Australian Scorpion Hormurus waigiensis Venom Fractions Show Broad Bioactivity Through Modulation of Bio-Impedance and Cytosolic Calcium.

Scorpion venoms are a rich source of bioactive molecules, but characterisation of toxin peptides affecting cytosolic Ca2+, central to cell signalling and cell death, is limited. We undertook a functional screening of the venom of the Australian scorpion Hormurus waigiensis to determine the breadth of Ca2+ mobilisation. A human embryonic kidney (HEK293) cell line stably expressing the genetically encoded Ca2+ reporter GCaMP5G and the rabbit type 1 ryanodine receptor (RyR1) was developed as a biosensor. Size-exclusion Fast Protein Liquid Chromatography separated the venom into 53 fractions, constituting 12 chromatographic peaks. Liquid chromatography mass spectroscopy identified 182 distinct molecules with 3 to 63 components per peak. The molecular weights varied from 258 Da-13.6 kDa, with 53% under 1 kDa. The majority of the venom chromatographic peaks (tested as six venom pools) were found to reversibly modulate cell monolayer bioimpedance, detected using the xCELLigence platform (ACEA Biosciences). Confocal Ca2+ imaging showed 9/14 peak samples, with molecules spanning the molecular size range, increased cytosolic Ca2+ mobilization. H. waigiensis venom Ca2+ activity was correlated with changes in bio-impedance, reflecting multi-modal toxin actions on cell physiology across the venom proteome.

Scorpion toxin peptide action at the ion channel subunit level.

This review categorizes functionally validated actions of defined scorpion toxin (SCTX) neuropeptides across ion channel subclasses, highlighting key trends in this rapidly evolving field. Scorpion envenomation is a common event in many tropical and subtropical countries, with neuropharmacological actions, particularly autonomic nervous system modulation, causing significant mortality. The primary active agents within scorpion venoms are a diverse group of small neuropeptides that elicit specific potent actions across a wide range of ion channel classes. The identification and functional characterisation of these SCTX peptides has tremendous potential for development of novel pharmaceuticals that advance knowledge of ion channels and establish lead compounds for treatment of excitable tissue disorders. This review delineates the unique specificities of 320 individual SCTX peptides that collectively act on 41 ion channel subclasses. Thus the SCTX research field has significant translational implications for pathophysiology spanning neurotransmission, neurohumoral signalling, sensori-motor systems and excitation-contraction coupling. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.'

Prospective signs of cleidocranial dysplasia in Cebpb deficiency.

BACKGROUND: Although runt-related transcription factor 2 (RUNX2) has been considered a determinant of cleidocranial dysplasia (CCD), some CCD patients were free of RUNX2 mutations. CCAAT/enhancer-binding protein beta (Cebpb) is a key factor of Runx2 expression and our previous study has reported two CCD signs including hyperdontia and elongated coronoid process of the mandible in Cebpb deficient mice. Following that, this work aimed to conduct a case-control study of thoracic, zygomatic and masticatory muscular morphology to propose an association between musculoskeletal phenotypes and deficiency of Cebpb, using a sample of Cebpb-/-, Cebpb+/- and Cebpb+/+ adult mice. Somatic skeletons and skulls of mice were inspected with soft x-rays and micro-computed tomography (µCT), respectively. Zygomatic inclination was assessed using methods of coordinate geometry and trigonometric function on anatomic landmarks identified with µCT. Masseter and temporal muscles were collected and weighed. Expression of Cebpb was examined with a reverse transcriptase polymerase chain reaction (RT-PCR) technique. RESULTS: Cebpb-/- mice displayed hypoplastic clavicles, a narrow thoracic cage, and a downward tilted zygomatic arch (p < 0.001). Although Cebpb+/- mice did not show the phenotypes above (p = 0.357), a larger mass percentage of temporal muscles over masseter muscles was seen in Cebpb+/- littermates (p = 0.012). The mRNA expression of Cebpb was detected in the clavicle, the zygoma, the temporal muscle and the masseter muscle, respectively. CONCLUSIONS: Prospective signs of CCD were identified in mice with Cebpb deficiency. These could provide an additional aetiological factor of CCD. Succeeding investigation into interactions among Cebpb, Runx2 and musculoskeletal development is indicated.

N-glycosylation determines ionic permeability and desensitization of the TRPV1 capsaicin receptor.

The balance of glycosylation and deglycosylation of ion channels can markedly influence their function and regulation. However, the functional importance of glycosylation of the TRPV1 receptor, a key sensor of pain-sensing nerves, is not well understood, and whether TRPV1 is glycosylated in neurons is unclear. We report that TRPV1 is N-glycosylated and that N-glycosylation is a major determinant of capsaicin-evoked desensitization and ionic permeability. Both N-glycosylated and unglycosylated TRPV1 was detected in extracts of peripheral sensory nerves by Western blotting. TRPV1 expressed in HEK-293 cells exhibited various degrees of glycosylation. A mutant of asparagine 604 (N604T) was not glycosylated but did not alter plasma membrane expression of TRPV1. Capsaicin-evoked increases in intracellular calcium ([Ca(2+)](i)) were sustained in wild-type TRPV1 HEK-293 cells but were rapidly desensitized in N604T TRPV1 cells. There was marked cell-to-cell variability in capsaicin responses and desensitization between individual cells expressing wild-type TRPV1 but highly uniform responses in cells expressing N604T TRPV1, consistent with variable levels of glycosylation of the wild-type channel. These differences were also apparent when wild-type or N604T TRPV1-GFP fusion proteins were expressed in neurons from trpv1(-/-) mice. Capsaicin evoked a marked, concentration-dependent increase in uptake of the large cationic dye YO-PRO-1 in cells expressing wild-type TRPV1, indicative of loss of ion selectivity, that was completely absent in cells expressing N604T TRPV1. Thus, TRPV1 is variably N-glycosylated and glycosylation is a key determinant of capsaicin regulation of TRPV1 desensitization and permeability. Our findings suggest that physiological or pathological alterations in TRPV1 glycosylation would affect TRPV1 function and pain transmission.

Neurochemical classification and projection targets of CART peptide immunoreactive neurons in sensory and parasympathetic ganglia of the head.

The aims of the present study were to determine if there is neuronal Cocaine and amphetamine regulated transcripts (CART) peptide expression (CART+) in parasympathetic (sphenopalatine (SPG); otic (OG)) and sensory (trigeminal (TG)) ganglia of the head and to examine the neurochemical phenotype (calcitonin gene-related peptide (CGRP), neurofilament 200 (NF200), isolectin B4 (IB4) binding, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY) and enkephalin (ENK) immunoreactivity) and projection targets (lacrimal gland (LG), parotid gland (PG), nasal mucosa (NM), temporomandibular joint (TMJ), middle cerebral artery (MCA) and middle meningeal artery (MMA)) of CART expressing neurons in these ganglia. We found CART+ neurons in both the SPG (5.25±0.07%) and OG (4.32±0.66). A significant proportion of these CART+ neurons contained VIP, NPY or ENK (34%, 26% and 11%, respectively). SPG neurons retrogradely labelled from the lacrimal gland (29%) were CART+, but we were unable to demonstrate CART+ labelling in any of the SPG or OG neurons labelled from other targets. This supports a role for CART peptides in lacrimation or regulation of vascular tone in the lacrimal gland, but not in salivation or nasal congestion. CART+ neurons were also present in the trigeminal ganglion (1.26±0.38%), where their size distribution was confined almost completely to neurons smaller than 800 µm2 (mean=410 µm2; 98%<800 µm2), and were almost always CGRP+, but did not bind IB4. This is consistent with a role for CART peptides in trigeminal pain. However, there were few CART+ neurons amongst any of the trigeminal neurons retrogradely labelled from the targets we investigated and thus we cannot comment on the tissue type where such pain may have originated. Our study shows that some specialization of CART peptide expression (based on neurochemical phenotype and target projection) is evident in sensory and parasympathetic ganglia of the head.

Early emergence of neural activity in the developing mouse enteric nervous system.

Neurons of the enteric nervous system (ENS) arise from neural crest cells that migrate into and along the developing gastrointestinal tract. A subpopulation of these neural-crest derived cells express pan-neuronal markers early in development, shortly after they first enter the gut. However, it is unknown whether these early enteric "neurons" are electrically active. In this study we used live Ca(2+) imaging to examine the activity of enteric neurons from mice at embryonic day 11.5 (E11.5), E12.5, E15.5, and E18.5 that were dissociated and cultured overnight. PGP9.5-immunoreactive neurons from E11.5 gut cultures responded to electrical field stimulation with fast [Ca(2+)](i) transients that were sensitive to TTX and ω-conotoxin GVIA, suggesting roles for voltage-gated Na(+) channels and N-type voltage-gated Ca(2+) channels. E11.5 neurons were also responsive to the nicotinic cholinergic agonist, dimethylphenylpiperazinium, and to ATP. In addition, spontaneous [Ca(2+)](i) transients were present. Similar responses were observed in neurons from older embryonic gut. Whole-cell patch-clamp recordings performed on E12.5 enteric neurons after 2-10 h in culture revealed that these neurons fired both spontaneous and evoked action potentials. Together, our results show that enteric neurons exhibit mature forms of activity at early stages of ENS development. This is the first investigation to directly examine the presence of neural activity during enteric neuron development. Along with the spinal cord and hindbrain, the ENS appears to be one of the earliest parts of the nervous system to exhibit electrical activity.

Postnatal maturation of the hyperpolarization-activated cation current, I(h), in trigeminal sensory neurons.

Hyperpolarization-activated inward currents (I(h)) contribute to neuronal excitability in sensory neurons. Four subtypes of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate I(h), with different activation kinetics and cAMP sensitivities. The aim of the present study was to examine the postnatal development of I(h) and HCN channel subunits in trigeminal ganglion (TG) neurons. I(h) was investigated in acutely dissociated TG neurons from rats aged between postnatal day (P)1 and P35 with whole cell patch-clamp electrophysiology. In voltage-clamp studies, I(h) was activated by a series of hyperpolarizing voltage steps from -40 mV to -120 mV in -10-mV increments. Tail currents from a common voltage step (-100 mV) were used to determine I(h) voltage dependence. I(h) activation was faster in older rats and occurred at more depolarized potentials; the half-maximal activation voltage (V(1/2)) changed from -89.4 mV (P1) to -81.6 mV (P35). In current-clamp studies, blocking I(h) with ZD7288 caused membrane hyperpolarization and increases in action potential half-duration at all postnatal ages examined. ZD7288 also reduced the action potential firing frequency in multiple-firing neurons. Western blot analysis of the TG detected immunoreactive bands corresponding to all HCN subtypes. HCN1 and HCN2 band density increased with postnatal age, whereas the low-intensity HCN3 and moderate-intensity HCN4 bands were not changed. This study suggests that functional I(h) are activated in rat trigeminal sensory neurons from P1 during postnatal development, have an increasing role with age, and modify neuronal excitability.

Peripheral targets of 5-HT(1D) receptor immunoreactive trigeminal ganglion neurons.

OBJECTIVE: The aim of the current study was to determine the proportion of trigeminal primary afferent neurons that innervate the intracranial vasculature, and other craniofacial tissues, that are also 5 hydroxy triptamine (5-HT)(1D) receptor immunoreactive. METHODS: Retrograde tracing and immunohistochemistry was used to identify 5-HT(1D) receptor labeled trigeminal primary afferent neurons that innervate the lacrimal gland (n = 3 animals), nasal mucosa (n = 3 animals), and the intracranial vasculature (middle meningeal artery in the dura [n = 3 animals] and middle cerebral artery [n = 3 animals]). RESULTS: The percentage of neurons that were 5-HT(1D) receptor immunoreactive was greater for primary afferent neurons innervating the middle meningeal artery (41.8 ± 1%) than those innervating the middle cerebral artery (28.4 ± 0.8%), nasal mucosa (25.6 ± 1%), or lacrimal gland (23.5 ± 3%). For each retrograde labeled population, the 5-HT(1D) receptor immunoreactive cells were among the smallest of the retrograde labeled cells. CONCLUSIONS: These findings provide a basis for understanding the role of 5-HT(1D) receptor agonists (eg, triptans) in the treatment of primary vascular headaches and suggest that the selectivity of triptans in the treatment of these headaches does not appear to result from specific localization of the 5-HT(1D) receptor to trigeminovascular neurons alone.

5-HT(1D) receptor immunoreactivity in the sphenopalatine ganglion: implications for the efficacy of triptans in the treatment of autonomic signs associated with cluster headache.

OBJECTIVE: To determine if 5-HT(1D) receptors are located in the sphenopalatine ganglion. BACKGROUND: While the 5-HT(1D) receptor has been described in sensory and sympathetic ganglia in the head, it was not known whether they were also located in parasympathetic ganglia. METHODS: We used retrograde labeling combined with immunohistochemistry to examine 5-HT(1D) receptor immunoreactivity in rat sphenopalatine ganglion neurons that project to the lacrimal gland, nasal mucosa, cerebral vasculature, and trigeminal ganglion. RESULTS: We found 5-HT(1D) receptor immunoreactivity in nerve terminals around postganglionic cell bodies within the sphenopalatine ganglion. All 5-HT(1D) -immunoreactive terminals were also immunoreactive for calcitonin gene-related peptide but not vesicular acetylcholine transporter, suggesting that they were sensory and not preganglionic parasympathetic fibers. Our retrograde labeling studies showed that approximately 30% of sphenopalatine ganglion neurons innervating the lacrimal gland, 23% innervating the nasal mucosa, and 39% innervating the trigeminal ganglion were in apparent contact with 5-HT(1D) receptor containing nerve terminals. CONCLUSION: These data suggest that 5-HT(1D) receptors within primary afferent neurons that innervate the sphenopalatine ganglion are in a position to modulate the excitability of postganglionic parasympathetic neurons that innervate the lacrimal gland and nasal mucosa, as well as the trigeminal ganglion. This has implications for triptan (5-HT(1D) receptor agonist) actions on parasympathetic symptoms in cluster headache.

Hyperpolarization-activated cyclic-nucleotide gated 4 (HCN4) protein is expressed in a subset of rat dorsal root and trigeminal ganglion neurons.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels are active at resting membrane potential and thus are likely to contribute to neuronal excitability. Four HCN channel subunits (HCN1-4) have previously been cloned. The aim of the current study was to investigate the immunoreactivity of HCN4 channel protein in rat trigeminal (TG) and dorsal root ganglion (DRG) sensory neurons. HCN4 was present in 9% of TG neurons and 4.7% of DRG neurons, it was distributed in a discrete population of small-diameter neurons in the TG but was located in cells of all sizes in the DRG. Approximately two thirds of HCN4-containing neurons in each ganglia were labelled with antisera raised against the 200-kDa neurofilament (NF200). The remaining HCN4-containing neurons were NF200-negative, were not labelled with antisera raised against calcitonin-gene related peptide (CGRP), and did not bind the isolectin B4 (IB4). HCN4-containing neurons made up more than half of the population of small-diameter primary afferent neurons that did not contain either NF200 or CGRP or bind IB4 in both TG and DRG. This population was not insignificant, comprising 5% of TG neurons and 2% of DRG neurons.

ATP potentiates neurotransmission in the rat trigeminal subnucleus caudalis.

Ionotropic purine receptors (P2X) have been implicated in nociceptive neurotransmission. In this study, we examine the actions of the P2X receptor agonist alpha,beta methylene adenosine 5'-triphosphate on excitatory neurotransmission in neurons in the deep and superficial laminae of the trigeminal spinal subnucleus caudalis (Vc), which receives nociceptive inputs from the craniofacial region. Alpha, beta methylene adenosine 5'-triphosphate caused an increase in spontaneous excitatory neurotransmission (miniature excitatory postsynaptic currents) in neurons in deep but not superficial laminae of Vc; this effect could be inhibited by the P2X receptor antagonist 2,3-O-2,4,6-trinitrophenyl-ATP. Conversely, the TRPV1 agonist capsaicin caused an increase in miniature excitatory postsynaptic currents in neurons in the superficial but not deep laminae. These data suggest that alpha,beta methylene adenosine 5'-triphosphate acts on presynaptic terminals to increase glutamatergic neurotransmission in deep Vc neurons.