Karel S. Kits

A glia-derived acetylcholine-binding protein that modulates synaptic transmission

There is accumulating evidence that glial cells actively modulate neuronal synaptic transmission. We identified a glia-derived soluble acetylcholine-binding protein (AChBP), which is a naturally occurring analogue of the ligand-binding domains of the nicotinic acetylcholine receptors (nAChRs). Like the nAChRs, it assembles into a homopentamer with ligand-binding characteristics that are typical for a nicotinic receptor; unlike the nAChRs, however, it lacks the domains to form a transmembrane ion channel. Presynaptic release of acetylcholine induces the secretion of AChBP through the glial secretory pathway. We describe a molecular and cellular mechanism by which glial cells release AChBP in the synaptic cleft, and propose a model for how they actively regulate cholinergic transmission between neurons in the central nervous system.

Plasticity in Fast Synaptic Inhibition of Adult Oxytocin Neurons Caused by Switch in GABAA Receptor Subunit Expression

We found that magnocellular oxytocin neurons in adult female rats exhibit an endogenous GABA(A) receptor subunit switch around parturition: a decrease in alpha1:alpha2 subunit mRNA ratio correlated with a decrease in allopregnanolone potentiation and increase in decay time constant of the GABA(A) receptor-mediated IPSCs in these cells. The causal relationship between changes in alpha1:alpha2 mRNA ratio and the ion channel kinetics was confirmed using in vitro antisense deletion. Further, GABA(A) receptors exhibited a tonic inhibitory influence upon oxytocin release in vivo, and allopregnanolone helped to restrain oxytocin neuron in vitro firing only before parturition, when the alpha1:alpha2 subunit mRNA ratio was still high. Such observations provide evidence for the physiological significance of GABA(A) receptor subunit heterogeneity and plasticity in the adult brain.

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 (3α‐OH‐DHP) was suppressed. 4 Throughout a substantial part of the lactation period the decay of synaptic currents remained attenuated, whilst the potentiation by 3α‐OH‐DHP remained suppressed. 5 Several weeks after the end of lactation sIPSC intervals, their current decay velocity as well as the potentiation by 3α‐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 α1 and α2 GABAA receptor subunit mRNA at a relative ratio of 2 : 1 compared with β‐actin. After pregnancy both α1 and α2 subunit mRNA levels were transiently increased, although at a relative ratio of 1 : 4, in line with the hypothesis that α2 plays a large role in postsynaptic receptor functioning. During post‐lactation both α 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 3α‐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.

Postsynaptic mechanism of depression of GABAergic synapses by oxytocin in the supraoptic nucleus of immature rat.

1. Oxytocin is known to act on autoreceptors of oxytocin neurones in the supraoptic nucleus (SON). We investigated whether oxytocin modulates putative oxytocin neurones by suppressing the GABAA receptor‐mediated synaptic inputs on these cells. 2. GABAergic inhibitory postsynaptic currents (IPSCs) were recorded from SON neurones in hypothalamic slices from young rats. Oxytocin specifically reduced the amplitude of both spontaneous and evoked IPSCs, without altering their current kinetics. 3. The effect of oxytocin was observed in 70% of the magnocellular neurones recorded from the dorsomedial part of the SON. d(CH2)5OVT, a specific antagonist of oxytocin receptors, blocked the effect of oxytocin on the IPSCs. Vasopressin had no effect on oxytocin‐sensitive SON neurones. 4. The intervals between spontaneous IPSCs were not affected by oxytocin. This suggested that oxytocin had a postsynaptic effect on SON neurones. 5. This postsynaptic origin was further substantiated by application of TTX, which blocked all evoked release but did not prevent the suppressive effect of oxytocin on the amplitude of the spontaneous IPSCs still present in the recording. The selective effect of oxytocin on IPSC amplitude was also maintained in nominally zero extracellular calcium. 6. Intracellular perfusion of SON neurones with GTP gamma S mimicked the effect of oxytocin on IPSCs, while GDP beta S, similarly applied, abolished the effect of oxytocin. 7. Application of calcium mobilizers such as thapsigargin and caffeine also reduced the amplitude of spontaneous IPSCs without significantly altering the frequency at which IPSCs occurred. 8. Thus, oxytocin depresses GABAergic synapses in the SON via modulation of the postsynaptic GABAA receptors. This would lead to disinhibition of SON neurones sensitive to oxytocin and could, therefore, be a powerful means of controlling the firing of oxytocin neurones.

Cholinergic modulation of nucleus accumbens medium spiny neurons

The rat nucleus accumbens contains acetylcholine‐releasing interneurons, presumed to play a regulatory role in the electrical activity of medium spiny output neurons. In order to examine this issue in detail, we made electrophysiological recordings in rat nucleus accumbens slices. These experiments showed that γ‐aminobutyric acid‐mediated inhibition of the output neurons might be facilitated by activation of nicotinic acetylcholine receptors, in addition to being suppressed via activation of muscarinic acetylcholine receptors. In contrast, glutamatergic excitation of output neurons appeared to be inhibited by activation of muscarinic acetylcholine receptors and to be insensitive to activation of nicotinic acetylcholine receptors. The spontaneous firing frequency of cholinergic neurons appeared to be under control of both a muscarinic and a nicotinic pathway in a bi‐directional manner. Finally, we made paired recordings in which the functional connection between cholinergic neurons and output neurons was monitored. Driving the cholinergic neurons at physiological firing frequencies stimulated γ‐aminobutyric acid‐mediated inhibition of the output neurons, via activation of nicotinic acetylcholine receptors. The onset of this effect was slow and lacked a fixed delay. These data indicate that activation of nicotinic acetylcholine receptors in rat nucleus accumbens may mediate the facilitation of γ‐aminobutyric acid‐mediated inhibition of medium spiny output neurons. Possible mechanisms of neurotransmission, mediating this cholinergic modulation are discussed.

A NOVEL G PROTEIN-COUPLED RECEPTOR MEDIATING BOTH VASOPRESSIN- AND OXYTOCIN-LIKE FUNCTIONS OF LYS-CONOPRESSIN IN LYMNAEA STAGNALIS

We have cloned a receptor, named LSCPR, for vasopressin-related Lys-conopressin in Lymnaea stagnalis. Lys-conopressin evokes Ca(2+)-dependent Cl- currents in Xenopus oocytes injected with LSCPR cRNA. Expression of LSCPR mRNA was detected in central neurons and peripheral muscles associated with reproduction. Upon application of Lys-conopressin, both neurons and muscle cells depolarize owing to an enhancement of voltage-dependent Ca2+ currents and start firing action potentials. Some neurons coexpress LSCPR and Lys-conopressin, suggesting an autotransmitter-like function for this peptide. Lys-conopressin also induces a depolarizing response in LSCPR-expressing neuroendocrine cells that control carbohydrate metabolism. Thus, in addition to oxytocin-like reproductive functions, LSCPR mediates vasopressin-like metabolic functions of Lys-conopressin as well.

Regulation of exocytosis in neuroendocrine cells: spatial organization of channels and vesicles, stimulus-secretion coupling, calcium buffers and modulation.

Neuroendocrine cells display a similar calcium dependence of release as synapses but a strongly different organization of channels and vesicles. Biophysical and biochemical properties of large dense core vesicle release in neuroendocrine cells suggest that vesicles and channels are dissociated by a distance of 100-300 nm. This distinctive organization relates to the sensitivity of the release process to mobile calcium buffers, the resulting relationship between calcium influx and release and the modulatory mechanisms regulating the efficiency of excitation-release coupling. At distances of 100-300 nm, calcium buffers determine the calcium concentration close to the vesicle. Notably, the concentration and diffusion rate of mobile buffers affect the efficacy of release, but local saturation of buffers, possibly enhanced by diffusion barriers, may limit their effects. Buffer conditions may result in a linear relationship between calcium influx and exocytosis, in spite of the third or fourth power relation between intracellular calcium concentration and release. Modulation of excitation-secretion coupling not only concerns the calcium channels, but also the secretory process. Transmitter regulation mediated by cAMP and PKA, as well as use-dependent regulation involving calcium, primarily stimulates filling of the releasable pool. In addition, direct effects of cAMP on the probability of release have been reported. One mechanism to achieve increased release probability is to decrease the distance between channels and vesicles. GTP may stimulate release independently from calcium. Thus, while in most cases primary inputs triggering these pathways await identification, it is evident that large dense core vesicle release is a highly controlled and flexible process.

Cloning and Expression of a Complementary DNA Encoding a Molluscan Octopamine Receptor That Couples to Chloride Channels in HEK293 Cells

A cDNA encoding a G-protein-coupled receptor was cloned from the central nervous system of the pond snail Lymnaea stagnalis The predicted amino acid sequence of this cDNA most closely resembles the Drosophila tyramine/octopamine receptor, the Locusta tyramine receptor, and an octopamine receptor (Lym oa1) that we recently cloned from Lymnaea After stable expression of the cDNA in HEK293 cells, we found that [3H]rauwolscine binds with high affinity to the receptor (KD = 6.2·10−9 M). Octopamine appears to be the most potent naturally occurring agonist to displace the [3H]rauwolscine binding (Ki = 3.0·10−7 M). Therefore, the receptor is considered to be an octopamine receptor and is consequently designated Lym oa2. The novel receptor shares little pharmacological resemblance with Lym oa1, indicating that the two receptors represent different octopamine receptor subfamilies. Octopaminergic stimulation of Lym oa2 does not induce changes in intracellular concentrations of cAMP or inositol phosphates. However, electrophysiological experiments indicate that octopamine is able to activate a voltage-independent Cl− current in HEK293 cells stably expressing Lym oa2. Although opening of this chloride channel most probably does not require the activation of either protein kinase A or C, it can be blocked by inhibition of protein phosphorylation.

Single channel kinetics of a glutamate receptor.

The glutamate receptor-channel of locust muscle membrane was studied using the patch-clamp technique. Muscles were pretreated with concanavalin A to block receptor-channel desensitization, thus facilitating analysis of receptor-channel gating kinetics. Single channel kinetics were analyzed to aid in identification of the molecular basis of channel gating. Channel dwell-time distributions and dwell-time autocorrelation functions were calculated from single channel data recorded in the precence of 10-4M glutamate. Analysis of the dwell time distributions in terms of mixtures of exponential functions revealed there to be at least three open states of the receptor-channel and at least four closed states. Autocorrelation function analysis showed there to be at least three pathways linking the open states with the closed. This results in a minimal scheme for gating of the glutamate receptor-channel, which is suggestive of allosteric models of receptor-channel gating.

Dopamine D2 receptor stimulation differentially affects voltage‐activated calcium channels in rat pituitary melanotropic cells.

1. Whole‐cell voltage clamp recordings were made from 141 rat pituitary melanotropic cells in short‐term, serum‐free, primary culture. The effects of the dopamine D2 receptor agonist, LY 171555, on sodium, potassium and barium currents were investigated. 2. Application of 1 microM‐LY 171555 did not affect the inward sodium and outward potassium currents. 3. Application of LY 171555 reversibly inhibited barium currents, with the strongest inhibition on the early inward current. The effect was dose dependent (IC50 = 4 x 10(‐8) M), maximal inhibition of the total current was 30% and the LY 171555‐induced block (1 microM) was reversibly antagonized by (+/‐)sulpiride (4 microM). 4. Using barium‐selective saline solutions, different types of barium current (T, N, and two L components) were identified on the basis of their voltage‐dependent kinetics. Their relative amplitudes differed between cells. 5. The T‐type current activated at potentials positive to ‐60 mV, reaching peak amplitude between ‐20 and ‐10 mV. At ‐30 mV, this current was inhibited up to 30% by 1 microM‐LY 171555. The time constants of activation (10‐3 ms) and inactivation (50‐20 ms) as well as the voltage dependence of inactivation (potential of half‐maximal inactivation (H), ‐61 mV; slope factor (S), 4.9 mV) were not affected by LY 171555 application. 6. A rapidly inactivating (time constants 100‐50 ms), high threshold current component was identified as an N‐type current. This current activated at command potentials positive to ‐30 mV and reached a maximal amplitude at +10 mV. The steady‐state inactivation was described by a single Boltzmann equation with H = ‐65 mV and S = 11.7 mV. Application of 1 microM‐LY 171555 completely suppressed this current. 7. The slowly inactivating (time constants > 1500 ms), high‐threshold, L‐type current displayed the same voltage dependence of activation as the N current. The voltage dependence of inactivation was modelled by the sum of two Boltzmann equations (L1: H1 = ‐45 mV, S1 = 13.0 mV; L2:H2 = ‐11 mV, S2 = 6.0 mV), indicating the existence of two L channel populations. Neither time course, nor voltage dependence of inactivation were influenced by LY 171555. However, LY 171555 induced a slow‐down in the time course of activation, which necessitated the use of two time constants to model the activation kinetics. One of these (approximately 2 ms) was also observed under control conditions.(ABSTRACT TRUNCATED AT 400 WORDS)