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.

Synaptic assembly of the brain in the absence of neurotransmitter secretion.

Brain function requires precisely orchestrated connectivity between neurons. Establishment of these connections is believed to require signals secreted from outgrowing axons, followed by synapse formation between selected neurons. Deletion of a single protein, Munc18-1, in mice leads to a complete loss of neurotransmitter secretion from synaptic vesicles throughout development. However, this does not prevent normal brain assembly, including formation of layered structures, fiber pathways, and morphologically defined synapses. After assembly is completed, neurons undergo apoptosis, leading to widespread neurodegeneration. Thus, synaptic connectivity does not depend on neurotransmitter secretion, but its maintenance does. Neurotransmitter secretion probably functions to validate already established synaptic connections.

Plasticity in fast synaptic inhibition of adult oxytocin neurons caused by switch in GABA(A) 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.

Long-term plasticity of postsynaptic GABAA-receptor function in the adult brain: insights from the oxytocin neurone.

The subunit switching of ligand-gated receptors is a potentially important mechanism through which synaptic plasticity can be achieved in the nervous system. Although established in an activity-dependent manner for neurotransmission that is mediated by excitatory amino acids, there is much less direct evidence for a role of subunit switching in long-term plasticity of GABAA receptors in the adult. We argue that the hypothalamic oxytocin neurones, which exhibit marked plasticity through each reproductive cycle, provide an excellent model of both presynaptic and postsynaptic long-term plasticity of GABA-mediated transmission in the mature nervous system. The postsynaptic plasticity involves GABAA-receptor-subunit switching in an activity-independent manner. It also has profound effects on the electrical behaviour of the oxytocin neurones and, thus, the neural control of pregnancy and lactation.

Impaired dendritic spine maturation in GABAA receptor alpha1 subunit knock out mice.

In this study we investigated the functional implications of GABAA receptor alpha1 subunit deletion on dendritic arborization and spine maturation in the visual cortex. This subunit is normally strongly upregulated during early postnatal development. Our main finding is that mice lacking the GABAA receptor alpha1 subunit displayed an increased density of dendritic filopodia during the second and third postnatal weeks. However, there was a concomitant decreased density of mature mushroom-shaped spines, which became more pronounced in adults. In contrast, dendritic arborization was not altered in these mice. We propose that an increased efficacy of the inhibitory synaptic transmission in the alpha1 knock out mice may lead to an enhancement of the outgrowth of filopodia around eye opening, but to a failure in spine maturation at later stages.

Discharge induction in molluscan peptidergic cells requires a specific set of autoexcitatory neuropeptides.

The peptidergic caudodorsal cells of the pond snail Lymnaea stagnalis generate long lasting discharges of synchronous spiking activity to release their products. During caudodorsal cell discharges a peptide factor is released which induces similar discharges in silent caudodorsal cells [Ter Maat A. et al. (1988) Brain Res. 438, 77-82]. To identify this factor, the electrophysiological effects of putative caudodorsal cell gene products, calfluxin, caudodorsal cell hormone, four alpha caudodorsal cell peptides and three beta caudodorsal cell peptides, were tested individually and in various combinations. Calfluxin, alpha caudodorsal cell peptide and beta 1 caudodorsal cell peptide each had no effect on membrane potential or excitability of the caudodorsal cells. All other caudodorsal cell peptides caused excitatory responses, but did not induce discharges. Instead, only a specific combination of four caudodorsal cell peptides, caudodorsal cell hormone and alpha caudodorsal cell peptide (1-11, 3-11 and 3-10), evoked caudodorsal cell discharges with similar characteristics to electrically evoked discharges. Incomplete versions of this combination failed to cause a discharge. In addition, antibodies to caudodorsal cell hormone or alpha caudodorsal cell peptide reduced caudodorsal cell excitability and prevented the generation of discharges by electrical stimulation. These results suggest that excitatory autotransmission caused by four caudodorsal cell peptides provides a means to amplify excitatory inputs, thus leading to the generation of the all-or-nothing caudodorsal cell discharge.

GABAA receptor maturation in relation to eye opening in the rat visual cortex.

Changes in subunit composition of N-methyl-D-aspartate (NMDA) receptors have been reported to be affected by visual experience and may therefore form a major aspect of neuronal plasticity in the CNS during development. In contrast, putative alterations in the expression and functioning of the inhibitory GABAA receptor around eye opening have not been well defined yet. Here we describe the timing of changes in GABAA receptor subunit expression and the related synaptic functioning in the neonatal rat visual cortex and the influence of visual experience on this process. Quantitative analysis of all GABAA receptor subunit transcripts revealed a marked alpha3 to alpha1 subunit switch, in addition to a change in alpha4 and alpha5 expression. The changes were correlated with an acceleration of the decay of spontaneous inhibitory postsynaptic currents (sIPSCs). Both changes in receptor expression and synaptic functioning were initiated well before eye opening. Moreover, dark rearing could not prevent the robust upregulation of alpha1 or the change in sIPSC kinetics, indicating that this is not dependent of sensory (visual) input. Upon eye opening a positive correlation was observed between a faster decay of the sIPSCs and an increase in sIPSC frequency, which was absent in dark-reared animals. Thus, lack of extrinsic input to the cortex does not affect overall developmental regulation of synaptic functioning of GABAA receptors. However, we cannot exclude the possibility that visual experience is involved in proper shaping of the inhibitory network of the primary visual cortex.

alpha-MSH-induced changes in protein phosphorylation of Cloudman S91 mouse melanoma cells.

The role of protein phosphorylation in MSH-induced melanogenesis was investigated with an in vivo phosphorylation assay using intact cultured Cloudman S91 mouse melanoma cells preincubated with [32P]orthophosphate. Exposure of the cells to alpha-MSH increased the extent of labelling of two protein bands on SDS gel electrophoresis with estimated molecular weights of 43 and 34 kDa, respectively. The 32P incorporation was concentration-dependent and reached a maximal value at 10(-8) M alpha-MSH for the 43 kDa band (156% of controls) and at 10(-5) M alpha-MSH for the 34 kDa band (250% of controls). The corresponding ED50s were 5 X 10(-10) M (43 kDa) and 3 X 10(-8) M (34 kDa). The 32P incorporation into the 34 kDa band reached a maximum after a 5 min exposure to alpha-MSH whereas 43 kDa phosphorylation was maximal after a 30-60 min incubation with hormone. The effect was completely reversible after removal of the hormone and specific for melanotropic peptides. Dibutyryl cAMP (10(-3) M) and forskolin (10(-4) M) together with isobutylmethylxanthine (10(-4) M) mimicked the effect of alpha-MSH, pointing to an involvement of adenylate cyclase activation in the phosphorylation of both the 34 kDa and the 43 kDa protein. Preliminary observations showed that the 34 kDa protein is membrane-bound whereas the 43 kDa protein is of mitochondrial or melanosomal origin.

One receptor type mediates two independent effects of FMRFa on neurosecretory cells of Lymnaea.

Structure activity relations (SAR) of FMRFa on the transient hyperpolarizing response and long lasting depression of excitability of neurosecretory caudo dorsal cells (CDCs) of the pond snail Lymnaea stagnalis were examined. Although these effects to FMRFa occur independently, the SARs for the induction of both responses were identical suggesting that CDCs possess a single type of FMRFa receptors. Native GDPFLRFa and SDPFLRFa were equipotent to FMRFa receptors. It is concluded that activation of the receptor requires [Arg3-Phe4]-NH2, whereas N-terminal amino acids are involved in binding.

Peripheral injection of DNS-RFa, a FMRFa agonist, suppresses morphine-induced analgesia in rats.

The present results demonstrate an antagonistic effect of DNS-RFa on morphine-induced analgesia in rats. This confirms previous evidence presented by others on the effects of FMRFa-related peptides when applied centrally. Unlike these peptides, however, it is shown here that DNS-RFa is effective upon peripheral injection. The effects of DNS-RFa on morphine-induced analgesia were dose-dependent (ED50 = 0.5 mg/kg). DNS-RFa alone (5 mg/kg) did not affect the control level of nociception. Peripheral injection of FMRFa (5 mg/kg) did not affect morphine-induced analgesia. DNS-RFa defines the minimal configuration to activate neuronal FMRFa receptors in the pond snail. The present report suggests also that in vertebrates the Arg-Phe-NH2 sequence is essential and that DNS-RFa readily penetrates the blood-brain barrier.

Antisense oligonucleotides induce functional deletion of ligand gated ion channels in cultured neurons and brain explants.

The in situ application of the antisense technique for the study of ligand gated channels is discussed here. Using antisense oligodeoxynucleotides to downregulate a gene of interest means being confronted with a number of choices that will determine the success. These include choosing a target sequence, considering chemical modifications of the oligo as well as its length and estimation of the turnover of the target protein in order to set up the treatment schedule. In this paper a short overview of technical aspects of the antisense approach on primary cultured neurons and brain slice cultures is presented. In addition, the effects of antisense oligos on the expression of neuronal nicotinic acetylcholine receptors and GABA(A) receptors are discussed: Patch-clamp recordings of neurons treated with specific antisense oligos targeted at individual subunits showed a clear downregulation of the expression of native ligand gated channels. Moreover, in a number of experiments novel channel types with altered properties were observed following antisense treatment. Thus, non-targeted channel subunits that remain expressed after antisense deletion, may aggregate to form novel channel types that are normally not present. Alternatively, the translational arrest of a protein may be accompanied by compensatory changes in the synthesis and/or targeting of other channel subunits to the cell surface. The antisense technique enables identification of the functional contribution of individual channel subunits to endogenous channel activity in the central nervous system. As such it paves the way to the elucidation of in vivo channel-subunit composition and channel functions, of post- as well as pre-synaptic ligand gated channel receptors.

4-Aminopyridine affects synaptosomal protein phosphorylation in rat hippocampal slices.

Rat brain hippocampal slices were incubated with or without the convulsant 4-aminopyridine (4-AP). From these slices a crude mitochondrial/synaptosomal membrane fraction was prepared and analyzed for endogenous protein phosphorylation. 4-AP (10(-5) M) stimulated the phosphorylation of a 50 kDa protein by 86%. The phosphorylation of this 50 kDa protein is Ca2+/calmodulin-dependent and we suggest that this protein is the lower molecular weight subunit of Ca2+/calmodulin-dependent protein kinase II (CaMK II).

Dual inhibitory action of FMRFamide on neurosecretory cells controlling egg laying behavior in the pond snail.

We describe here the electrophysiological characterization of a dual inhibitory action of FMRFamide (FMRFa, Phe-Met-Arg-Phe-NH2) on the caudodorsal cells (CDCs) of the pond snail Lymnaea stagnalis: (i) a transient hyperpolarizing response (H-response) and (ii) a suppression of the excitability of the cells, which lasted as long as the peptide was present. Both effects of FMRFa occurred in silent, excitable cells as well as discharging cells. The effects were reversible and dose-dependent in the range of 10(-9) to 10(-5) M. The H-response was not blocked by any of the antagonists to classical neurotransmitters that were tested. The reversal potential of the H-response was dependent on the [K+]o, which suggests that K+ is the major charge carrier in this response. 4-Aminopyridine (4-AP) blocked the H-response but did not affect the suppression of the excitability by FMRFa. This indicates that the effects of the peptide on these cells are independent. Experiments on the mechanism of the inhibition of the excitability indicated that FMRFa blocks the cAMP-dependent activation of the pacemaking mechanism of the CDCs. In experiments with isolated cells it was demonstrated that the actions of FMRFa are mediated directly through receptors on CDCs (H-response: ED50 = 10(-8) M). Finally, anti-FMRFa-positive varicosities and axons close to the somata, the axons and the neurohaemal endings of the CDCs were demonstrated immunocytochemically. The duality of the action of FMRFa on the neural activity of CDCs indicates its role of high priority in the regulation of egg laying behavior.

Antisense oligonucleotide-induced block of individual GABAA receptor alpha subunits in cultured visual cortex slices reduces amplitude of evoked inhibitory postsynaptic currents.

Whole cell patch clamp recordings were made in layer II-IV from organotypic slices of rat primary visual cortex, explanted at postnatal day 6 and maintained in a serum-free medium. Neurons evinced current clamp characteristics typical for stellate cells. Between 7 and 21 days in culture, both glutamate- and GABA-mediated postsynaptic currents were observed. Long-term culturing in the presence of a degenerate 15-mer antisense oligonucleotide directed against the transcripts of all alpha subunits genes of the GABAA receptor resulted in a dose dependent reduction of evoked GABA synaptic currents. This reduction was maximal (80%) at 20 microM. A randomized control oligo had no effect. Evoked glutamatergic excitatory postsynaptic currents were unaffected following oligo treatment. A 15-mer antisense oligo directed against the alpha 1 subunit gave variable effects: in some cells the amplitude of evoked GABAergic inhibitory postsynaptic currents (IPSCs) was reduced by 50-75%, while in other cells recorded from the same slices, there was little or no effect. An antisense oligo, directed against the alpha 2 subunit, however, gave a consistent and robust 80% reduction of the amplitude of evoked IPSCs. A 15-mer 3-base mismatch oligo against alpha 2 had no effect. We conclude that the alpha 2 subunit functions in postsynaptic GABAA receptors located on or close to the cell bodies of stellate cells. The role of the alpha 1 subunit is less clear, but this subunit seems spatially differentiated. The in situ antisense oligo technique should provide further insight into the biophysical and pharmacological consequences of the subunit composition of ligand gated channels at functional synapses.

Inhibitory modulation of neuronal voltage-dependent sodium current by Phe-Met-Arg-Phe-amide.

The tetrodotoxin (TTX)-sensitive, voltage-gated Na(+)-current (INa) in a cluster of peptidergic neurons, involved in egg laying, in the CNS of the mollusc Lymnaea stagnalis, is modulated by the neuropeptide FMRFa (Phe-Met-Arg-Phe-NH2). Application of FMRFa reversibly reduced the isolated INa in a dose-dependent fashion. The physiological consequence is that the threshold for action potential generation is increased, causing an arrest of ongoing firing activity. The inhibitory action of FMRFa reported here is the first known example of modulation of the voltage-gated INa by a putative neurotransmitter in intact nerve cells. This finding underlines the importance of modulation of ionic currents as a mechanism of regulation of neuronal excitability and includes the voltage dependent Na current in the range of currents subject to transmitter modulation.

Oxytocin suppresses the GABAergic synaptic input in supraoptic neurones from the rat.

Oxytocin, acting as an autotransmitter, gives rise to release of calcium from intracellular store(s) within magnocellular neurons in the supraoptic nucleus (SON). A possible target for a rise in intracellular calcium is the GABAA receptor, since it is known that the functioning of this receptor may depend (directly) on the intracellular free calcium concentration. Therefore the effect of oxytocin on the GABAergic synaptic input in the SON was analyzed. In situ patch clamp recordings from individual neurons of the SON were performed. Spontaneously occurring, bicuculline sensitive GABAergic inhibitory synaptic currents (IPSCs) were pharmacologically isolated from the excitatory glutamatergic synaptic input. This isolated GABAergic synaptic input was spontaneously and tonically active, arising from both somatic as well as from dendritic synaptic contacts and operated a chloride conductance. Application of oxytocin during such recordings, strongly reduced the amplitude of the IPSCs in 73% of the recordings. This reduction was (i) completely reversed by washing, (ii) blocked by a specific oxytocin receptor antagonist, and (iii) observed in slices from both female and from male animals. Thus autotransmission involving disinhibition of magnocellular neurons may explain why oxytocin facilitates it own release.

Reduced 5-HT1A- and GABAB receptor function in dorsal raphé neurons upon chronic fluoxetine treatment of socially stressed rats.

Autoinhibitory serotonin 1A receptors (5-HT(1A)) in dorsal raphé nucleus (DRN) have been implicated in chronic depression and in actions of selective serotonin reuptake inhibitors (SSRI). Due to experimental limitations, it was never studied at single-cell level whether changes in 5-HT(1A) receptor functionality occur in depression and during SSRI treatment. Here we address this question in a social stress paradigm in rats that mimics anhedonia, a core symptom of depression. We used whole cell patch-clamp recordings of 5-HT- and baclophen-induced G-protein-coupled inwardly rectifying potassium (GIRK) currents as a measure of 5-HT(1A)- and GABA(B) receptor functionality. 5-HT(1A)- and GABA(B) receptor-mediated GIRK-currents were not affected in socially stressed rats, suggesting that there was no abnormal (auto)inhibition in the DRN on social stress. However, chronic fluoxetine treatment of socially stressed rats restored anticipatory behavior and reduced the responsiveness of 5-HT(1A) receptor-mediated GIRK currents. Because GABA(B) receptor-induced GIRK responses were also suppressed, fluoxetine does not appear to desensitize 5-HT(1A) receptors but rather one of the downstream components shared with GABA(B) receptors. This fluoxetine effect on GIRK currents was also present in healthy animals and was independent of the animal's "depressed" state. Thus our data show that symptoms of depression after social stress are not paralleled by changes in 5-HT(1A) receptor signaling in DRN neurons, but SSRI treatment can alleviate these behavioral symptoms while acting strongly on the 5-HT(1A) receptor signaling pathway.

NMDA receptors trigger neurosecretion of 5-HT within dorsal raphe nucleus of the rat in the absence of action potential firing.

Activity and calcium-dependent release of neurotransmitters from the somatodendritic compartment is an important signalling mechanism between neurones throughout the brain. NMDA receptors and vesicles filled with neurotransmitters occur in close proximity in many brain areas. It is unknown whether calcium influx through these receptors can trigger the release of somatodendritic vesicles directly, or whether postsynaptic action potential firing is necessary for release of these vesicles. Here we addressed this question by studying local release of serotonin (5-HT) from dorsal raphé nucleus (DRN) neurones. We performed capacitance measurements to monitor the secretion of vesicles in giant soma patches, in response to short depolarizations and action potential waveforms. Amperometric measurements confirmed that secreted vesicles contained 5-HT. Surprisingly, two-photon imaging of DRN neurones in slices revealed that dendritic calcium concentration changes in response to somatic firing were restricted to proximal dendritic areas. This implied that alternative calcium entry pathways may dominate the induction of vesicle secretion from distal dendrites. In line with this, transient NMDA receptor activation, in the absence of action potential firing, was sufficient to induce capacitance changes. By monitoring GABAergic transmission onto DRN 5-HT neurones in slices, we show that endogenous NMDA receptor activation, in the absence of postsynaptic firing, induced release of 5-HT, which in turn increased the frequency of GABAergic inputs through activation of 5-HT(2) receptors. We propose here that calcium influx through NMDA receptors can directly induce postsynaptic 5-HT release from DRN neurones, which in turn may facilitate GABAergic input onto these cells.

Developmental changes in transmitter sensitivity and synaptic transmission in embryonic chicken sympathetic neurons innervated in vitro.

Dispersed neurons from embryonic chicken sympathetic ganglia were innervated in vitro by explants of spinal cord containing the autonomic preganglionic nucleus or somatic motor nucleus. The maturation of postsynaptic acetylcholine (ACh) sensitivity and synaptic activity was evaluated from ACh and synaptically evoked currents in voltage-clamped neurons at several stages of innervation. All innervated cells are more sensitive to ACh than uninnervated neurons regardless of the source of cholinergic input. Similarly, medium conditioned by either dorsal or ventral explants mimics innervation by enhancing neuronal ACh sensitivity. This increase is due to changes in the rate of appearance of ACh receptors on the cell surface. There are also several changes in the nature of synaptic transmission with development in vitro, including an increased frequency of synaptic events and the appearance of larger amplitude synaptic currents. In addition, the mean amplitude of the unit synaptic current mode increases, as predicted from the observed changes in postsynaptic sensitivity. Although spontaneous synaptic current amplitude histograms with multimodal distributions are seen at all stages of development, histograms from early synapses are typically unimodal. Changes in the synaptic currents and ACh sensitivity between 1 and 4 days of innervation were paralleled by an increase in the number of synaptic events that evoked suprathreshold activity in the postsynaptic neurons. The early pre- and postsynaptic differentiation described here for interneuronal synapses formed in vitro may be responsible for increased efficacy of synaptic transmission during development in vivo.