Target-specific control of nicotinic receptor expression at developing interneuronal synapses in chick.

Neuronal differentiation and development of synaptic specializations are strongly influenced by cellular interactions. We compared the effects of interaction with distinct autonomic targets on the molecular and biophysical differentiation of 'upstream' neuron-neuron synapses. Contact with cardiac tissue induced expression of nicotinic receptor channels (nAChRs) distinct from those induced by renal tissue in presynaptic autonomic neurons. The kinetics of cholinergic currents at interneuronal synapses are dictated by the peripheral target contacted. Analysis of the nAChR channel subtypes and subunits in individual neurons demonstrated that the profile of transmitter receptors expressed at mature neuron-neuron synapses develops from the convergent influences of input-derived (anterograde) and target-specific (retrograde) signals.

Changes in properties and neurosteroid regulation of GABAergic synapses in the supraoptic nucleus during the mammalian female reproductive cycle.

1. GABAA receptor-mediated synaptic innervation of oxytocin neurones in the supraoptic nucleus (SON) was analysed in adult female rats going through their first reproductive cycle by recording the spontaneous inhibitory postsynaptic currents (sIPSCs) at six stages of female reproduction. 2. During pregnancy we observed a reduction in the interval between monoquantal sIPSCs. The synaptic current amplitude, current decay and neurosteroid sensitivity of postsynaptic GABAA receptors observed at this stage were not distinguishable from those measured in virgin stage SON. 3. Upon parturition an increase in monoquantal synaptic current decay occurred, whereas potentiation by the progesterone metabolite allopregnanolone (3alpha-OH-DHP) was suppressed. 4. Throughout a substantial part of the lactation period the decay of synaptic currents remained attenuated, whilst the potentiation by 3alpha-OH-DHP remained suppressed. 5. Several weeks after the end of lactation sIPSC intervals, their current decay velocity as well as the potentiation by 3alpha-OH-DHP were restored to pre-pregnancy levels, which is indicative of the cyclical nature of synaptic plasticity in the adult SON. 6. Competitive polymerase chain reaction (PCR) analysis showed that virgin animals expressed alpha1 and alpha2 GABAA receptor subunit mRNA at a relative ratio of 2 : 1 compared with beta-actin. After pregnancy both alpha1 and alpha2 subunit mRNA levels were transiently increased, although at a relative ratio of 1 : 4, in line with the hypothesis that alpha2 plays a large role in postsynaptic receptor functioning. During post-lactation both alpha subunits were downregulated. 7. We propose that synaptic remodelling in the SON during pregnancy includes changes in the putative number of GABA release sites per neurone. At parturition, and during the two consecutive weeks of lactation, a subtype of postsynaptic GABAA receptors was observed, distinct from the one being expressed before and during pregnancy. Synaptic current densities, calculated in order to compare the impact of synaptic inhibition, showed that, in particular, the differences in 3alpha-OH-DHP potentiation of these two distinct GABAA receptor subtypes produce robust shifts in the impact of synaptic inhibition of oxytocin neurones at the different stages of female reproduction.

A cysteine-rich isoform of neuregulin controls the level of expression of neuronal nicotinic receptor channels during synaptogenesis.

We report here that neuregulin (NRG) isoforms with a conserved cysteine-rich domain (CRD) in their N terminus regulate expression of nicotinic acetylcholine receptors (nAChRs) at developing interneuronal synapses and report the isolation of transmembrane NRG isoforms with this CRD within the N-terminal portion. CRD-NRG mRNA and immunoreactive protein are detected early in developing presynaptic (visceral motor) neurons. The levels of expression of CRD-NRG peak prior to the formation of synapses with their postsynaptic partners, the ganglionic sympathetic neurons. Recombinant CRD-NRG mimics the effects of presynaptic input on target neurons. Functional deletion of CRD-NRG from presynaptic neurons abolishes the upregulation of nAChR expression induced by input-derived soluble material. Thus, CRD-NRG appears to be both a necessary and a sufficient signal for the control of neuronal nAChR expression during synaptogenesis.

Nicotine enhancement of fast excitatory synaptic transmission in CNS by presynaptic receptors.

The behavioral and cognitive effects of nicotine suggest that nicotinic acetylcholine receptors (nAChRs) participate in central nervous system (CNS) function. Although nAChR subunit messenger RNA (mRNA) and nicotine binding sites are common in the brain, there is little evidence for synapses mediated by nAChRs in the CNS. To test whether, CNS nAChRs might modify rather than mediate transmission, the regulation of excitatory synaptic transmission by these receptors was examined. Nanomolar concentrations of nicotine enhanced both glutamatergic and cholinergic synaptic transmission by activation of presynaptic nAChRs that increased presynaptic [Ca2]i. Pharmacological and subunit deletion experiments reveal that these presynaptic nAChRs include the alpha 7 subunit. These findings reveal that CNS nAChRs enhance fast excitatory transmission, providing a likely mechanism for the complex behavioral effects of nicotine.

Regulation of nAChR subunit gene expression relative to the development of pre- and postsynaptic projections of embryonic chick sympathetic neurons.

Previous work has established that synaptic input influences the differentiation of muscle and glandular targets and that these targets reciprocally influence the differentiation of the innervating neurons. In contrast, there is little information on the impact of pre- vs postsynaptic contact on the differentiation of neuronal targets. We have delineated the timing of presynaptic projections to lumbar sympathetic neurons as well as the timing of the projections from these neurons to their targets during normal embryonic development. A combination of anterograde and retrograde labeling techniques and immunohistochemical approaches reveal that the establishment of significant synaptic input to chick sympathetic ganglia is a rather protracted process spanning late embryonic development. Although target contact in the periphery also occurs over mid to late embryogenesis, significant target projections appear to be established prior to the evolution of a comparable level of presynaptic input. Analysis of concurrent changes in the pattern and levels of expression of genes encoding neuronal nicotinic receptor (nAChR) subunits are consistent with both presynaptic input and target contact regulating nAChR expression during embryogenesis. These studies also suggest that retrograde influences of target contact on receptor expression may be more substantial than previously appreciated.

Functional contribution of neuronal AChR subunits revealed by antisense oligonucleotides.

Although multiple related genes encoding nicotinic acetylcholine receptor (AChR) subunits have been identified, how each of these subunits contributes to AChRs in neurons is not known. Sympathetic neurons express four classes of AChR channels and six AChR subunit genes (alpha 3, alpha 4, alpha 5, alpha 7, beta 2, and beta 4). The contribution of individual subunits to AChR channel subtypes in these neurons was examined by selective deletion with antisense oligonucleotides. An alpha 3 antisense oligonucleotide decreased the number and altered the properties of the normally expressed ACh-activated channels. The remaining AChR channels have distinct biophysical and pharmacological properties that indicate an important functional contribution of the alpha 7 subunit.

Protein kinase C in larval brain of wild-type and dunce memory-mutant Drosophila.

Protein kinase C activity has been measured in extracts of larval brain of Drosophila melanogaster, with the synthetic nonapeptide substrate Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide. Protein kinase C activity in such extracts is abolished in a Ca2+-dependent manner at 18 degrees C, and partly converted to a form independent of effectors. The decay of protein kinase C activity can be prevented by leupeptin or a crude calpastatin preparation isolated from fly heads, indicating the presence of the Ca2+-dependent neutral protease, calpain, in larval brains. The total protein kinase C levels were nearly the same in wild type and three different dunce "memory-mutant" strains. In contrast, the soluble/particulate activity ratios were different: wild-type, 0.91; dunce M11, 0.46; dunce M11/Df(1)dm75e19, 1.23; dunce2, 0.88. These data suggest that the membrane adherence of protein kinase C in larval brain is governed by the actor of genes other than dunce.

Changes in the distribution of the neuron-specific B-50, neurofilament protein and glial fibrillary acidic proteins following an unilateral mesencephalic lesion in the rat.

Following a unilateral electrolytic lesion in the ventral rat mesencephalon, changes in the immunocytochemical distribution of the neuron-specific B-50, neurofilament (NF) protein and glial fibrillary acidic (GFAP) proteins were studied around the lesion after 0, 3, 10 and 28 days. At all recovery times, the controls displayed on immunostaining with anti-B-50 and anti-neurofilament antibodies, a characteristic pattern of synaptic and neuritic localization of these antigens, whereas anti-GFAP staining revealed a distribution typical for astrocytes. The lesion was characterized by a center of coagulated material that exhibited immunoreactivity to B-50 (BIR) and NF (NFIR), but never GFAP-immunoreactivity. From 3 days on, the center became surrounded by disintegrating cells which were unreactive to the antibodies. The antigen distribution changed temporally, predominantly at the lesion rim. By 10 and 28 days postlesion, additional BIR was observed as punctuate dots in fibers and membranes of neurons. Enhanced NFIR was detected in fibers and cell bodies. Many astrocytes were detected around the lesion rim, forming by 28 days postsurgery a barrier between the lesion cavity and the uninjured tissue. Our study shows that distribution changes in B-50, NF and GFAP around the lesion may indicate local degenerative and adaptative processes as a temporal response to brain trauma.

Cyclic AMP-induced phosphorylation of 27.5-kDa protein(s) in larval brains of normal and memory-mutant Drosophila melanogaster.

Whole brains dissected from third-instar larvae of Drosophila melanogaster increase their cAMP content when incubated with octopamine, dopamine and serotonin, especially in the presence of theophylline, a phosphodiesterase inhibitor. The most pronounced rise in cAMP was produced by forskolin. When brains prelabelled with [32P]orthophosphate were subjected to these treatments, the increased labelling of 27.5-kDa protein(s) of pI 6.3-6.6 (substrate A) was observed in all cases, as revealed by two-dimensional gel electrophoresis and autoradiography. Labelling of substrate A was enhanced without any drug addition in brains of the dunceM11 mutant strain, which has abnormally high basal cAMP levels. The labelling of substrate A was also promoted by the potassium channel blocker 4-aminopyridine. The data suggest that in Drosophila larval brain the 27.5-kDa protein is a target of cAMP-triggered phosphorylation and may be related to potassium channel(s).

Altered autophosphorylation of adenosine 3',5'-phosphate-dependent protein kinase in the dunce memory mutant of Drosophila melanogaster.

The phosphorylation-dephosphorylation, in the presence of adenosine 5'-[gamma-32P]triphosphate, of a polypeptide of apparent molecular mass 53,000 has been compared in head homogenates of wild type and memory mutant dunceM11 strains of Drosophila melanogaster. In both strains, labelling of the 53 kilodalton protein required exogenous adenosine 3',5'-phosphate (cAMP), but in dunceM11 cAMP at higher concentration (above approximately 3 microM) caused the rapid disappearance of the label. This differential dephosphorylation can be attributed to the lack of a cAMP-specific phosphodiesterase isoenzyme in the mutant. Several lines of evidence indicate that the 53 kilodalton protein is identical with the regulatory subunit of cAMP-dependent protein kinase. The findings suggest that in the mutant's nerve cells the state of phosphorylation of the regulatory subunit of cAMP-dependent protein kinase is altered, which may contribute to the biochemical disorder leading to the memory deficit.

Differences in protein phosphorylation in vivo and in vitro between wild type and dunce mutant strains of Drosophila melanogaster.

The protein phosphorylation patterns of wild type and dunce mutant strains of Drosophila melanogaster, as detected by sodium dodecylsulfate-gel electrophoresis and autoradiography, have been compared. After labelling in vivo with 32Pi or in vitro in homogenates with [gamma-32P]ATP, radioactive bands at and above apparent polypeptide mol. wt approximately 110,000 were more pronounced in dunce fly heads than in wild type heads. When labelling in vitro, in dunceM11 there appeared a radioactive band at apparent mol. wt approximately equal to 53,000 that was faintly visible in the wild strain. The same band could be intensified in both strains by adding cyclic AMP to the homogenate or by performing homogenization in the presence of theophylline. The data suggest that the mol. wt approximately equal to 53,000 protein is a substrate for cyclic AMP-dependent protein kinase.

Cyclic nucleotide phosphodiesterases in larval brain of wild type and dunce mutant strains of Drosophila melanogaster: isoenzyme pattern and activation by Ca2+/calmodulin.

Like adult heads and whole flies, larval brains of wild type Drosophila melanogaster contain two major soluble cyclic nucleotide phosphodiesterases, forms I and II. Larval brains of the learning-defective mutant strain, dunceM11, contain only the form I enzyme. In both wild type and dunce strains the form I enzyme is activated by Ca2+/calmodulin. A time-dependent loss of this Ca2+ activation was observed.