Maria Vidovic

Bridging the Synaptic Gap: Neuroligins and Neurexin I in Apis mellifera

Vertebrate studies show neuroligins and neurexins are binding partners in a trans-synaptic cell adhesion complex, implicated in human autism and mental retardation disorders. Here we report a genetic analysis of homologous proteins in the honey bee. As in humans, the honeybee has five large (31–246 kb, up to 12 exons each) neuroligin genes, three of which are tightly clustered. RNA analysis of the neuroligin-3 gene reveals five alternatively spliced transcripts, generated through alternative use of exons encoding the cholinesterase-like domain. Whereas vertebrates have three neurexins the bee has just one gene named neurexin I (400 kb, 28 exons). However alternative isoforms of bee neurexin I are generated by differential use of 12 splice sites, mostly located in regions encoding LNS subdomains. Some of the splice variants of bee neurexin I resemble the vertebrate α- and β-neurexins, albeit in vertebrates these forms are generated by alternative promoters. Novel splicing variations in the 3′ region generate transcripts encoding alternative trans-membrane and PDZ domains. Another 3′ splicing variation predicts soluble neurexin I isoforms. Neurexin I and neuroligin expression was found in brain tissue, with expression present throughout development, and in most cases significantly up-regulated in adults. Transcripts of neurexin I and one neuroligin tested were abundant in mushroom bodies, a higher order processing centre in the bee brain. We show neuroligins and neurexins comprise a highly conserved molecular system with likely similar functional roles in insects as vertebrates, and with scope in the honeybee to generate substantial functional diversity through alternative splicing. Our study provides important prerequisite data for using the bee as a model for vertebrate synaptic development.

Variation in mRNA expression of alpha-adrenergic, neurokinin and muscarinic receptors amongst four arteries of the rat

Different mechanisms mediate constriction and dilation in different vascular beds. We have used reverse transcription-polymerase chain reaction to investigate whether specific patterns of receptor gene expression may underlie these variable responses. Total RNA, from the basilar, pulmonary, mesenteric and tail arteries of anaesthetised adult Wistar rats, was reverse transcribed and amplified using primers specific for the molecular subtypes of the alpha 1(A, B, D)- and alpha 2(A, B, C)-adrenergic, neurokinin (NK1-NK3) and muscarinic (m1-m5), receptors. Results showed that the pattern of gene expression was variable with no two arteries having the same receptor profile. Messenger RNA for the alpha 1A, alpha 1B, alpha 2B, NK1, NK3, m3 and m5 receptor subtypes were detected in all vessels studied while the remaining subtypes showed a variable expression amongst the arteries. This is the first description of mRNA for the m5 muscarinic receptor in peripheral tissue. The NK3 receptor was the major neurokinin receptor expressed in all vessels except the pulmonary artery, in which the NK1 receptor was also strongly expressed. We conclude that each artery expressed a specific receptor array which may permit some unique neural and hormonal controls.

Synaptic activation of transient receptor potential channels by metabotropic glutamate receptors in the lateral amygdala

Classical mammalian transient receptor potential channels form non-selective cation channels that open in response to activation of phospholipase C-coupled metabotropic receptors, and are thought to play a key role in calcium homeostasis in non-excitable cells. Within the nervous system transient receptor potential channels are widely distributed but their physiological roles are not well understood. Here we show that in the rat lateral amygdala transient receptor potential channels mediate an excitatory synaptic response to glutamate. Activation of group I metabotropic glutamate receptors on pyramidal neurons in the lateral amygdala with either exogenous or synaptically released glutamate evokes an inward current at negative potentials with a current voltage relationship showing a region of negative slope and steep outward rectification. This current is blocked by inhibiting G protein function with GTP-beta-S, by inhibiting phospholipase C or by infusing transient receptor potential antibodies into lateral amygdala pyramidal neurons. Using RT-PCR and Western blotting we show that transient receptor potential 1, transient receptor potential 4 and transient receptor potential 5 are present in the lateral amygdala. Single cell PCR confirms the presence of transient receptor potential 1 and transient receptor potential 5 in pyramidal neurons and we show by co-immunoprecipitation that transient receptor potential 1 and transient receptor potential 5 co-assemble as a heteromultimers in the amygdala. These results show that in lateral amygdala pyramidal neurons synaptically released glutamate activates transient receptor potential channels, which we propose are likely to be heteromultimeric channels containing transient receptor potential 1 and transient receptor potential 5/transient receptor potential 4.

Expression of mRNA and functional alpha1‐adrenoceptors that suppress the GIRK conductance in adult rat locus coeruleus neurons

Locus coeruleus neurons in adult rats express binding sites and mRNA for α1‐adrenoceptors even though the depolarizing effect of α1‐adrenoceptor agonists on neonatal neurons disappears during development. In this study intracellular microelectrodes were used to record from locus coeruleus neurons in brain slices of adult rats and reverse transcription‐polymerase chain reaction (RT – PCR) was used to investigate the mRNA expression of α1‐ and α2‐adrenoceptors in juvenile and adult rats. The α1‐adrenoceptor agonist phenylephrine had no effect on the membrane conductance of locus coeruleus neurons (Vhold −60 mV) but decreased the G protein coupled, inward rectifier potassium (GIRK) conductance induced by α2‐adrenoceptor or μ‐opioid agonists. The GIRK conductance induced by noradrenaline was increased in amplitude when α1‐adrenoceptors were blocked with prazosin. RT – PCR of total cellular RNA isolated from microdissected locus coeruleus tissue demonstrated strong mRNA expression of α1a‐, α1b‐ and α1d‐adrenoceptors in both juvenile and adult rats. However, only mRNA transcripts for the α1b‐adrenoceptors were consistently detected in cytoplasmic samples taken from single locus coeruleus neurons of juvenile rats, suggesting that this subtype may be responsible for the physiological effects seen in juvenile rats. Juvenile and adult locus coeruleus tissue expressed mRNA for the α2a‐ and α2c‐adrenoceptors while the α2b‐adrenoceptor was only weakly expressed in juveniles and was not detected in adults. The results of this study show that α1‐adrenoceptors expressed in adult locus coeruleus neurons function to suppress the GIRK conductance that is activated by μ‐opioid and α2‐adrenoceptors.

Identification of α2 adrenergic receptor gene expression in sympathetic neurones using polymerase chain reaction and in situ hybridization

Abstract α2 Adrenergic receptors are involved in mediating pre- and postsynaptic responses in the sympathetic nervous system. In this study, the expression of α2 genes was examined by the amplification of mRNA, extracted from adult rat superior cervical ganglion through reverse transcription and subsequent amplification of appropriate target sequence using polymerase chain reaction and sequence specific oligonucleotide primers for the three α2 receptor genes. Results from these studies have shown that the major α2 adrenergic mRNA transcript was the one that encodes the α2A receptor. Nucleotide sequence of the 312 base-pair (bp) α2A cDNA was homologous to the RG20 adrenergic receptor, the rat homologue of the human α2A receptor. The 312 bp α2A cDNA was used as a probe in Northern blot analysis of the mRNA from superior cervical ganglion and brain. A 3.9 kb mRNA transcript was present in these extracts. To confirm that the α2A gene expression was in the sympathetic neurones we have used the 312 bp α2A cDNA, biotinylated, as a probe for in situ hybridization studies and have demonstrated that the α2A mRNA was found only in the cell bodies of sympathetic neurones.

Alpha adrenoceptor gene expression in the rat iris during development and maturity.

We have examined the developmental profile of the alpha-1 and alpha-2 adrenergic receptor mRNA expression in the rat iris. The expression of the six mRNAs was studied using reverse transcription-polymerase chain reaction. At 4 weeks, the dominant mRNA transcripts in the rat iris were alpha-1B, alpha-1C and alpha-2A. This pattern of alpha adrenoceptor expression was seen from birth, throughout development, and maintained into old age.

Withdrawal of collaterals of sympathetic axons to the rat eye during postnatal development: the role of function

Neurones in the superior cervical ganglion (SCG) of the rat can be retrogradely labelled by injection of Fast blue (FB) into the anterior eye chamber with the maximum number being labelled during the second postnatal week. In adult rats, however, many fewer neurones can be so labelled. In the present study, we have investigated whether this reduction may be due to the withdrawal of collaterals of neurones which project to the posterior eye compartments. For these experiments, we have used either one (FB) or two (FB and horseradish peroxidase (HRP)) retrogradely transported markers injected with a micropipette into the anterior or posterior (vitreous) parts of the eye in rats aged 14, 31 and greater than 50 days. Using FB, we have shown that in 14-day-old rats, 40% of the neurones which project to the eye have collaterals in anterior and posterior compartments while only 1% of neurones maintain dual projections in animals aged more than 7 weeks. Furthermore, there is a reduction in the total number of neurones projecting to the eye between 14 and 31 days postbirth. The presence of significantly greater numbers of cells with collaterals to both eye compartments in the young versus the adult rats has been confirmed using both FB and HRP as retrograde markers. The importance of function on the rearrangement of these projections was investigated by either rearing animals in the dark from the time of birth, or by sectioning the preganglionic nerve trunk to the SCG, soon after birth.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental time course of the sympathetic postganglionic innervation of the rat eye

The time course of the innervation of the rat iris by sympathetic postganglionic axons from the superior cervical ganglion (SCG) has been studied in postnatal rats. A retrogradely transported fluorescent dye, Fast blue (FB) was injected bilaterally into the anterior eye chamber of rats within 3 h of birth or at 7 or 14 days postnatal. Littermates were killed at 24 h intervals, both SCG removed, fixed and sectioned. A substantial number of sympathetic neurones were labelled retrogradely from the eye at birth. The number of labelled cells tripled over the first postnatal week to reach proportions equivalent to those labelled in 3-week-old rats. The degree of sympathetic fibre ingrowth to the iris was examined at 3 postnatal ages, using fluorescence histochemistry. This method showed a small proportion of catecholamine-containing fibres in the iris at birth with a more extensive fibre density over the iris by 7 days after birth. A further increase in fibre density had occurred by the end of the 3rd postnatal week even though the number of cells projecting to the iris at these two ages was similar. This difference in fibre density may arise from ramification of fibres which have reached the iris by 7 days postbirth.

Marsupial retinocollicular system shows differential expression of messenger RNA encoding EphA receptors and their ligands during development

The protracted development of the wallaby (Macropus eugenii) has allowed study of messenger RNAs encoding Eph receptors EphA3 and EphA7 and ligands ephrin‐A2 and ‐A5 in the retina and superior colliculus at intervals throughout the development of the retinocollicular projection: from birth, before retinal innervation, to postnatal day 95, when the projection is mature. Reverse transcription‐polymerase chain reaction showed messenger RNAs for both receptors and ligands were expressed at all ages. EphA7 was expressed more highly in the rostral superior colliculus. Ephrin‐A2 and ‐A5 were expressed more highly in the caudal colliculus. EphA3 was expressed in a complementary manner, more highly in temporal than in nasal retina. There are higher levels of expression of the ligands when the projection is only coarsely topographically organised. This suggests a role for them and their receptor EphA3 in this stage, by repulsive interactions which restrict temporal axons to rostral superior colliculus. This is the first account in a marsupial mammal of the appearance of this molecular family, substantiating its ubiquitous role in topographically organised neuronal connections. Nevertheless, expression is not the same as in the mouse, suggesting differences in the details of topographic coding between species. J. Neurosci. Res. 57:244–254, 1999.

Cross talk between receptors mediating contraction and relaxation in the arterioles but not the dilator muscle of the rat iris

1 Sympathetic nerve stimulation causes contraction of the dilator muscle and the large arterioles of the iris via the activation of α1B‐adrenoceptors. We have investigated whether increases in adenosine 3′: 5′‐cyclic monophosphate (cyclic AMP) and the activation of receptors in these tissues can modulate these nerve‐mediated contractions. 2 0Increasing intracellular cyclic AMP with dibutyryl cyclic AMP (1 mM), forskolin (50 μm) or isobutylmethylxanthine (100 μm) produced relaxation of both the dilator and the arterioles, abolished the nerve‐mediated constriction of the arterioles, but potentiated the nerve‐mediated contraction of the iris dilator. 3 Pretreatment of the preparations with cholera toxin, to activate Gs permanently, caused a dilatation of the arterioles and abolished the nerve‐mediated constriction but had no effect on the dilator muscle. 4 The β‐adrenoceptor agonist, isoprenaline (1 μm), the adenosine‐A1,‐A2 agonist, N‐ethylcarboxami‐doadenosine NECA (100 nM), in the presence of the A1selective antagonist, 8‐cyclopentyl‐l, 3‐dipropylxanthine (DPCPX, 10 nM), and calcitonin gene‐related peptide (CGRP, 10 nM) all separately caused a dilatation of the arterioles and abolished the nerve‐mediated constriction, while only isoprenaline (1 μm) produced an effect on the dilator, i.e. a relaxation but a potentiation of the nerve‐mediated contraction. These results suggest the presence of at least 3 types of receptor linked to Gs and an increase in cyclic AMP in the arterioles, i.e. β‐adrenoceptor, adenosine‐A2 and CGRP, but only 1 Gs‐linked receptor, i.e. β‐adrenoceptors, on the dilator muscle cells. 5 Neither the A1‐selective agonist, cyclohexyladenosine (CHA, 10 nM) nor the A1‐selective antagonist, DPCPX (10 nM) had any significant effect on the nerve‐mediated constriction of the arterioles, suggesting that presynaptic A1‐receptors do not play a role in modulating the sympathetic nerve‐mediated constriction. 6 Forskolin (50 μm), in the presence of capsaicin (10 μm) to inactivate unmyelinated CGRP‐containing sensory nerves, still caused a dilatation of the arterioles and abolished nerve‐mediated constriction suggesting that the effects on the blood vessels were due to increases in cyclic AMP in the arteriolar cells and not in the sensory nerves. 7 Using reverse transcription polymerase chain reaction, we have demonstrated expression of the α1B and α1c molecular subtypes in the tissues of the iris. These molecular subtypes most likely correspond to the α1B and α1A pharmacological subtypes, respectively. 8 Preincubation in NG‐nitro‐L‐arginine methyl ester (L‐NAME, 10 μm), but not D‐NAME (10 μm), reduced the efficacy of forskolin in inhibiting the sympathetic nerve‐mediated vasoconstriction. These results suggest that the inhibition of sympathetic nerve‐mediated constriction by cyclic AMP in the arterioles is indirect via the production of nitric oxide. 9 Our results demonstrate that there is ‘cross talk’ between receptors linked to increases in cyclic AMP and α1B‐adrenoceptors in the arterioles of the rat iris, but that no such interaction occurs between cyclic AMP and α1B‐adrenoceptors in the dilator muscle. The interaction in the arterioles occurs as a result of cyclic AMP‐mediated stimulation of nitric oxide synthesis, presumably by the arteriolar endothelial cells.