Cntnap4 differentially contributes to GABAergic and dopaminergic synaptic transmission.

Although considerable evidence suggests that the chemical synapse is a lynchpin underlying affective disorders, how molecular insults differentially affect specific synaptic connections remains poorly understood. For instance, Neurexin 1a and 2 (NRXN1 and NRXN2) and CNTNAP2 (also known as CASPR2), all members of the neurexin superfamily of transmembrane molecules, have been implicated in neuropsychiatric disorders. However, their loss leads to deficits that have been best characterized with regard to their effect on excitatory cells. Notably, other disease-associated genes such as BDNF and ERBB4 implicate specific interneuron synapses in psychiatric disorders. Consistent with this, cortical interneuron dysfunction has been linked to epilepsy, schizophrenia and autism. Using a microarray screen that focused upon synapse-associated molecules, we identified Cntnap4 (contactin associated protein-like 4, also known as Caspr4) as highly enriched in developing murine interneurons. In this study we show that Cntnap4 is localized presynaptically and its loss leads to a reduction in the output of cortical parvalbumin (PV)-positive GABAergic (γ-aminobutyric acid producing) basket cells. Paradoxically, the loss of Cntnap4 augments midbrain dopaminergic release in the nucleus accumbens. In Cntnap4 mutant mice, synaptic defects in these disease-relevant neuronal populations are mirrored by sensory-motor gating and grooming endophenotypes; these symptoms could be pharmacologically reversed, providing promise for therapeutic intervention in psychiatric disorders.

Altered prevalence of gating modes in neurotransmitter inhibition of N-type calcium channels.

G protein-mediated inhibition of voltage-activated calcium channels by neurotransmitters has important consequences for the control of synaptic strength. Single-channel recordings of N-type calcium channels in frog sympathetic neurons reveal at least three distinct patterns of gating, designated low-Po, medium-Po, and high-Po modes according to their probability of being open (Po) at -10 millivolts. The high-Po mode is responsible for the bulk of divalent cation entry in the absence of neurotransmitter. Norepinephrine greatly decreased the prevalence of high-Po gating and increased the proportion of time a channel exhibited low-Po behavior or no activity at all, which thereby reduced the overall current. Directly observed patterns of transition between the various modes suggest that activated G protein alters the balance between modal behaviors that freely interconvert even in the absence of modulatory signaling.

Roles of N-type and Q-type Ca2+ channels in supporting hippocampal synaptic transmission.

Several types of calcium channels found in the central nervous system are possible participants in triggering neurotransmitter release. Synaptic transmission between hippocampal CA3 and CA1 neurons was mediated by N-type calcium channels, together with calcium channels whose pharmacology differs from that of L- and P-type channels but resembles that of the Q-type channel encoded by the alpha 1A subunit gene. Blockade of either population of channels strongly increased enhancement of synaptic transmission with repetitive stimuli. Even after complete blockade of N-type channels, transmission was strongly modulated by stimulation of neurotransmitter receptors or protein kinase C. These findings suggest a role for alpha 1A subunits in synaptic transmission and support the idea that neurotransmitter release may depend on multiple types of calcium channels under physiological conditions.

Adrenaline: mechanism of action on the pacemaker potential in cardiac Purkinje fibers.

The pacemaker potential in Purkinje fibers is generated by a slow fall in potassium current which allows the inward background currents to depolarize the membrane. Adrenaline shifts the relation between activation of the potassium current and membrane potential in a depolarizing direction. Consequently, during the pacemaker potential, the potassium current falls more rapidly to lower values and the inward currents then depolarize the membrane more quickly. The shift in the potassium activation curve produced by adrenaline is large compared to that produced by calcium ions. The molecular action of adrenaline may involve either a large change in the surface charge of the membrane or a change in the dependence of the potassium permeability on the local electric field.

Inhibition of postsynaptic PKC or CaMKII blocks induction but not expression of LTP.

Long-term potentiation (LTP) of synaptic transmission is a widely studied cellular example of synaptic plasticity. However, the identity, localization, and interplay among the biochemical signals underlying LTP remain unclear. Intracellular microelectrodes have been used to record synaptic potentials and deliver protein kinase inhibitors to postsynaptic CA1 pyramidal cells. Induction of LTP is blocked by intracellular delivery of H-7, a general protein kinase inhibitor, or PKC(19-31), a selective protein kinase C (PKC) inhibitor, or CaMKII(273-302), a selective inhibitor of the multifunctional Ca2+-calmodulin-dependent protein kinase (CaMKII). After its establishment, LTP appears unresponsive to postsynaptic H-7, although it remains sensitive to externally applied H-7. Thus both postsynaptic PKC and CaMKII are required for the induction of LTP and a presynaptic protein kinase appears to be necessary for the expression of LTP.

Calcium channels in planar lipid bilayers: insights into mechanisms of ion permeation and gating.

Electrophysiological recordings were used to analyze single calcium channels in planar lipid bilayers after membranes from bovine cardiac sarcolemmal vesicles had been incorporated into the bilayer. In these cell-free conditions, channels in the bilayer showed unitary barium or calcium conductances, gating kinetics, and pharmacological responses that were similar to dihydropyridine-sensitive calcium channels in intact cells. The open channel current varied in a nonlinear manner with voltage under asymmetric (that is, physiological) ionic conditions. However, with identical solutions on both sides of the bilayer, the current-voltage relation was linear. In matched experiments, calcium channels from skeletal muscle T-tubules differed significantly from cardiac calcium channels in their conductance properties and gating kinetics.

Presynaptic component of long-term potentiation visualized at individual hippocampal synapses.

Long-term potentiation has previously been studied with electrophysiological techniques that do not readily separate presynaptic and postsynaptic contributions. Changes in exocytotic-endocytotic cycling have now been monitored at synapses between cultured rat hippocampal neurons by measuring the differential uptake of antibodies that recognize the intraluminal domain of the synaptic vesicle protein synaptotagmin. Vesicular cycling increased markedly during glutamate-induced long-term potentiation. The degree of potentiation was heterogeneous, appearing greater at synapses at which the initial extent of vesicular turnover was low. Thus, changes in presynaptic activity were visualized directly and the spatial distribution of potentiation could be determined at the level of single synaptic boutons.

Dominant role of N-type Ca2+ channels in evoked release of norepinephrine from sympathetic neurons.

Multiple types of calcium channels have been found in neurons, but uncertainty remains about which ones are involved in stimulus-secretion coupling. Two types of calcium channels in rat sympathetic neurons were described, and their relative importance in controlling norepinephrine release was analyzed. N-type and L-type calcium channels differed in voltage dependence, unitary barium conductance, and pharmacology. Nitrendipine inhibited activity of L-type channels but not N-type channels. Potassium-evoked norepinephrine release was markedly reduced by cadmium and the conesnail peptide toxin omega-Conus geographus toxin VIA, agents that block both N- and L-type channels, but was little affected by nitrendipine at concentrations that strongly reduce calcium influx, as measured by fura-2. Thus N-type calcium channels play a dominant role in the depolarization-evoked release of norepinephrine.

Enhancement of N- and L-type calcium channel currents by protein kinase C in frog sympathetic neurons.

The effect of protein kinase C (PKC) stimulation on Ca2+ channels was studied in frog sympathetic neurons. 12,13-Phorbol dibutyrate (PDBu) consistently augmented Ca2+ channel currents in whole-cell recordings. This enhancement was blocked by staurosporine and PKC(19-31), but not produced by 4 alpha-phorbol 12,13-didecanoate, indicating that PDBu acts via PKC. Both N- and L-type currents, as isolated pharmacologically, were increased. PKC enhancement was independent of the extent of G protein activation, indicating that it was not caused by removal of tonic G protein inhibition. In unitary recordings PDBu produced dramatic increases in single N- and L-type channel activity by sharply decreasing closed time intervals between adjacent openings, but did not alter the unitary current size or mean open time. This up-modulation by PKC may constitute a positive feedback mechanism in the regulation of neuronal Ca2+ channel activity.

Phase-dependent contributions from Ca2+ entry and Ca2+ release to caffeine-induced [Ca2+]i oscillations in bullfrog sympathetic neurons.

Sympathetic neurons display robust [Ca2+]i oscillations in response to caffeine and mild depolarization. Oscillations occur at constant membrane potential, ruling out voltage-dependent changes in plasma membrane conductance. They are terminated by ryanodine, implicating Ca(2+)-induced Ca2+ release. Ca2+ entry is necessary for sustained oscillatory activity, but its importance varies within the oscillatory cycle: the slow interspike rise in [Ca2+]i requires Ca2+ entry, but the rapid upstroke does not, indicating that it reflects internal Ca2+ release. Sudden alterations in [Ca2+]o, [K+]o, or [caffeine]o produce immediate changes in d[Ca2+]i/dt and provide information about the relative rates of surface membrane Ca2+ transport as well as uptake and release by internal stores. Based on our results, [Ca2+]i oscillations can be explained in terms of coordinated changes in Ca2+ fluxes across surface and store membranes.

Variability of neurotransmitter concentration and nonsaturation of postsynaptic AMPA receptors at synapses in hippocampal cultures and slices.

To understand the elementary unit of synaptic communication between CNS neurons, one must know what causes the variability of quantal postsynaptic currents and whether unitary packets of transmitter saturate postsynaptic receptors. We studied single excitatory synapses between hippocampal neurons in culture. Focal glutamate application at individual postsynaptic sites evoked currents (I(glu)) with little variability compared with quantal excitatory postsynaptic currents (EPSCs). The maximal I(glu) was >2-fold larger than the median EPSC. Thus, variations in [glu]cleft are the main source of variability in EPSC size, and glutamate receptors are generally far from saturation during quantal transmission. This conclusion was verified by molecular antagonism experiments in hippocampal cultures and slices. The general lack of glutamate receptor saturation leaves room for increases in [glu]cleft as a mechanism for synaptic plasticity.

Signaling from synapse to nucleus: postsynaptic CREB phosphorylation during multiple forms of hippocampal synaptic plasticity.

Phosphorylation of the transcription factor CREB is thought to be important in processes underlying long-term memory. It is unclear whether CREB phosphorylation can carry information about the sign of changes in synaptic strength, whether CREB pathways are equally activated in neurons receiving or providing synaptic input, or how synapse-to-nucleus communication is mediated. We found that Ca(2+)-dependent nuclear CREB phosphorylation was rapidly evoked by synaptic stimuli including, but not limited to, those that induced potentiation and depression of synaptic strength. In striking contrast, high frequency action potential firing alone failed to trigger CREB phosphorylation. Activation of a submembranous Ca2+ sensor, just beneath sites of Ca2+ entry, appears critical for triggering nuclear CREB phosphorylation via calmodulin and a Ca2+/calmodulin-dependent protein kinase.

Glutamate-induced increases in intracellular Ca2+ in cultured frog tectal cells mediated by direct activation of NMDA receptor channels.

Influx of Ca2+ through NMDA channels may initiate the stabilization of coactive synapses during development of the retinotectal projection in frogs. Ca2+ imaging techniques were applied to cultured tectal cells to investigate whether excitatory amino acids cause a rise in [Ca2+]i. High [K+], NMDA, and glutamate increase [Ca2+]i in about 75% of the cells. NMDA and glutamate responses were completely blocked in the absence of extracellular Ca2+ and by the NMDA receptor or channel blockers APV and MK-801. The NMDA response was also blocked by Mg2+. Quisqualate and kainate produced little or no rise in [Ca2+]i. These studies indicate that when tectal cells are exposed to the retinal ganglion cell transmitter glutamate, the predominant means of Ca2+ entry is through NMDA channels.

Inhibition of Ca2+ and K+ channels in sympathetic neurons by neuropeptides and other ganglionic transmitters.

Neuropeptides are known to modulate the excitability of frog sympathetic neurons by inhibiting the M-current and increasing the leak current, but their effects on Ca2+ channels are poorly understood. We compared effects of LHRH, substance P, epinephrine, and muscarine on Ca2+, K+, and leak currents in dissociated frog sympathetic neurons. At concentrations that inhibit M-current, LHRH and substance P strongly reduced N-type Ca2+ current and induced a leak conductance that may contribute to slow EPSPs. In contrast, muscarine produced little reduction of Ca2+ current, even in cells in which it strongly suppressed the M-current. We find that peptidergic inhibition of Ca2+ channels involves G proteins, but does not require protein kinases. In addition, it leads to reductions in Ca2(+)-activated K+ current and catecholamine release.

Dendritic Ca2+ channels characterized by recordings from isolated hippocampal dendritic segments.

Dendritic arbors are critical for the information processing capability of central neurons, but quantitative analysis of their membrane properties has been hampered by their geometrical complexity. Here, we have focused on an important source of Ca2+ entry in dendrites, the voltage-gated Ca2+ channels, by applying the whole-cell voltage-clamp technique to isolated dendritic segments ("dendrosomes") from rat hippocampal neurons. We found that low voltage-activated T-type Ca2+ channels provide a significantly larger fraction of the Ca2+ influx in dendrites than their counterparts in cell bodies. Surprisingly, 60%-70% of the high voltage-activated Ca2+ current in dendrosomes was N and P/Q type, and these channels were susceptible to neurotransmitter inhibition, suggesting a novel physiological role for G protein-regulated Ca2+ channel modulation in controlling dendritic excitability and Ca2+ signaling.

Ca2+ channel selectivity at a single locus for high-affinity Ca2+ interactions.

Ca2+ channels display remarkable selectivity and permeability, traditionally attributed to multiple, discrete Ca2+ binding sites lining the pore. Each of the four pore-forming segments of Ca2+ channel alpha 1 subunits contains a glutamate residue that contributes to high-affinity Ca2+ interactions. Replacement of all four P-region glutamates with glutamine or alanine abolished micromolar Ca2+ block of monovalent current without revealing any additional independent high-affinity Ca2+ binding site. Pairwise replacements of the four glutamates excluded the hypothesis that they form two independent high-affinity sites. Systematic alterations of side-chain length, charge, and polarity by glutamate replacement with aspartate, glutamine, or alanine weakened the Ca2+ interaction, with considerable asymmetry from one repeat to another. The P-region glutamate in repeat I was unusual in its sensitivity to aspartate replacement but not glutamine substitution. While all four glutamates cooperate in supporting high-affinity interactions with single Ca2+ ions, they also influence the interaction between multiple divalent cations.

Multiple structural elements in voltage-dependent Ca2+ channels support their inhibition by G proteins.

Molecular determinants of Ca2+ channel responsiveness to inhibition by receptor-coupled G proteins were investigated in Xenopus oocytes. The inhibitory response of alpha1B (N-type) channels was much larger than alpha1A (P/Q-type) channels, while alpha1C (L-type) channels were unresponsive. Differences in both degree and speed of inhibition were accounted for by variations in inhibitor off-rate. We tested proposals that inhibitory G protein and Ca2+ channel beta subunits compete specifically at the I-II loop. G protein-mediated inhibition remained unaltered in alpha1B subunits containing a point mutation in the I-II loop segment critical for Ca2+ channel beta subunit binding, and in chimeras where the I-II loop of alpha1B was replaced with counterparts from alpha1A or alpha1c. Full interconversion between modulatory behaviors of alpha1B and alpha1A was achieved only by swapping both motif I and the C-terminus in combination. Thus, essential structural elements for G protein modulation reside in multiple Ca2+ channel domains.

Visualization of synaptic activity in hippocampal slices with FM1-43 enabled by fluorescence quenching.

Fluorescence imaging of presynaptic uptake and release of styryl dyes such as FM1-43 has provided valuable insights into synaptic function. However, in studies of CNS neurons, the utility of these dyes has been severely limited by nonsynaptic background fluorescence. This has thwarted the use of FM dyes in systems more intact than dissociated neuronal cultures. Here, we describe an approach to selectively reduce undesired fluorescence through quenching of the surface-bound FM1-43 signal. The introduction of sulforhodamine, a fluorophore that is not taken up by synaptic vesicles, selectively reduced the nonsynaptic fluorescence in FM1-43-labeled hippocampal cultures. When applied to rat hippocampal slices, this procedure allowed us to observe activity-dependent staining and destaining of functional synapses. Extending the usefulness of styryl dyes to slice preparations may help make functional synaptic networks amenable to optical measurements.

Rapid reuse of readily releasable pool vesicles at hippocampal synapses.

Functional presynaptic vesicles have been subdivided into readily releasable (RRP) and reserve (RP) pools. We studied recycling properties of RRP vesicles through differential retention of FM1-43 and FM2-10 and by varying the time window for FM dye uptake. Both approaches indicated that vesicles residing in the RRP underwent rapid endocytosis (tau approximately 1s), whereas newly recruited RP vesicles were recycled slowly (tau approximately 30 s). With repeated challenges (hypertonic or electrical stimuli), the ability to release neurotransmitter recovered 10-fold more rapidly than restoration of FM2-10 destaining. Finding neurotransmission in the absence of destaining implied that rapidly endocytosed RRP vesicles were capable of reuse, a process distinct from repopulation from the RP. Reuse would greatly expand the functional capabilities of a limited number of vesicles in CNS terminals, particularly during intermittent bursts of activity.

The kinetics of synaptic vesicle recycling measured at single presynaptic boutons.

We used the fluorescent membrane probe FM 1-43 to label recycling synaptic vesicles within the presynaptic boutons of dissociated hippocampal neurons in culture. Quantitative time-lapse fluorescence imaging was employed in combination with rapid superfusion techniques to study the dynamics of synaptic vesicles within single boutons. This approach enabled us to measure exocytosis and to analyze the kinetics of endocytosis and the preparation of endocytosed vesicles for re-release (repriming). Our measurements indicate that under sustained membrane depolarization, endocytosis persists much longer than exocytosis, with a t1/2 approximately 60 s (approximately 24 degrees C); once internalized, vesicles become reavailable for exocytosis in approximately 30 s. Furthermore, we have shown that endocytosis is not dependent on membrane potential and, unlike exocytosis, that it is independent of extracellular Ca2+.