Heparan Sulfates Regulate Axonal Excitability and Context Generalization through Ca2+/Calmodulin-Dependent Protein Kinase II.

Our previous studies demonstrated that enzymatic removal of highly sulfated heparan sulfates with heparinase 1 impaired axonal excitability and reduced expression of ankyrin G at the axon initial segments in the CA1 region of the hippocampus ex vivo, impaired context discrimination in vivo, and increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity in vitro. Here, we show that in vivo delivery of heparinase 1 in the CA1 region of the hippocampus elevated autophosphorylation of CaMKII 24 h after injection in mice. Patch clamp recording in CA1 neurons revealed no significant heparinase effects on the amplitude or frequency of miniature excitatory and inhibitory postsynaptic currents, while the threshold for action potential generation was increased and fewer spikes were generated in response to current injection. Delivery of heparinase on the next day after contextual fear conditioning induced context overgeneralization 24 h after injection. Co-administration of heparinase with the CaMKII inhibitor (autocamtide-2-related inhibitory peptide) rescued neuronal excitability and expression of ankyrin G at the axon initial segment. It also restored context discrimination, suggesting the key role of CaMKII in neuronal signaling downstream of heparan sulfate proteoglycans and highlighting a link between impaired CA1 pyramidal cell excitability and context generalization during recall of contextual memories.

Medroxyprogesterone acetate positively modulates specific GABAA-receptor subtypes - affecting memory and cognition.

Medroxyprogesterone acetate (MPA) is a progestin widely used in humans as hormone replacement therapy and at other indications. Many progestin metabolites, as the progesterone metabolite allopregnanolone, have GABAA-receptor modulatory effects and are known to affect memory, learning, appetite, and mood. In women, 4 years chronic treatment with MPA doubles the frequency of dementia and in rats, MPA causes cognitive impairment related to the GABAergic system. Activation of the membrane bound GABAA receptor results in a chloride ion flux that can be studied by whole-cell patch-clamp electrophysiological recordings. The purpose of this study was to clarify the modulatory effects of MPA and specific MPA metabolites, with structures like known GABAA-receptor modulators, on different GABAA-receptor subtypes. An additional aim was to verify the results as steroid effects on GABA response in single cells taken from rat hypothalamus. HEK-293 cell-lines permanently expressing the recombinant human GABAA-receptor subtype α1ß2γ2L or α5ß3γ2L or α2ß3γ2S were created. The MPA metabolites 3α5α-MPA,3ß5α-MPA and 3ß5ß-MPA were synthesised and purified for electrophysiological patch-clamp measurements with a Dynaflow system. The effects of MPA and tetrahydrodeoxycorticosterone were also studied. None of the studied MPA metabolites affected the responses mediated by α1ß2γ2L or α5ß3γ2L GABAA receptors. Contrary, MPA clearly acted both as a positive modulator and as a direct activator of the α5ß3γ2L and α2ß3γ2S GABAA receptors. However, in concentrations up to 10 µM, MPA was inactive at the α1ß2γ2L GABAA receptor. In the patch-clamp recordings from dissociated cells of the preoptic area in rats, MPA increased the amplitude of responses to GABA. In addition, MPA alone without added GABA, evoked a current response. In conclusion, MPA acts as a positive modulator of specific GABAA receptor subtypes expressed in HEK cells and at native GABA receptors in single cells from the hypothalamic preoptic area.

Contribution of Resting Conductance, GABAA-Receptor Mediated Miniature Synaptic Currents and Neurosteroid to Chloride Homeostasis in Central Neurons.

Maintenance of a low intraneuronal Cl- concentration, [Cl-]i, is critical for inhibition in the CNS. Here, the contribution of passive, conductive Cl- flux to recovery of [Cl-]i after a high load was analyzed in mature central neurons from rat. A novel method for quantifying the resting Cl- conductance, important for [Cl-]i recovery, was developed and the possible contribution of GABAA and glycine receptors and of ClC-2 channels to this conductance was analyzed. The hypothesis that spontaneous, action potential-independent release of GABA is important for [Cl-]i recovery was tested. [Cl-]i was examined by gramicidin-perforated patch recordings in medial preoptic neurons. Cells were loaded with Cl- by combining GABA or glycine application with a depolarized voltage, and the time course of [Cl-]i was followed by measurements of the Cl- equilibrium potential, as obtained from the current recorded during voltage ramps combined with GABA or glycine application. The results show that passive Cl- flux contributes significantly, in the same order of magnitude as does K+-Cl- cotransporter 2 (KCC2), to [Cl-]i recovery and that Cl- conductance accounts for ∼ 6% of the total resting conductance. A major fraction of this resting Cl- conductance is picrotoxin (PTX)-sensitive and likely due to open GABAA receptors, but ClC-2 channels do not contribute. The results also show that when the decay of GABAA receptor-mediated miniature postsynaptic currents (minis) is slowed by the neurosteroid allopregnanolone, such minis may significantly quicken [Cl-]i recovery, suggesting a possible steroid-regulated role for minis in the control of Cl- homeostasis.

How to Properly Measure a Current-Voltage Relation?-Interpolation vs. Ramp Methods Applied to Studies of GABAA Receptors.

The relation between current and voltage, I-V relation, is central to functional analysis of membrane ion channels. A commonly used method, since the introduction of the voltage-clamp technique, to establish the I-V relation depends on the interpolation of current amplitudes recorded at different steady voltages. By a theoretical computational approach as well as by experimental recordings from GABAA-receptor mediated currents in mammalian central neurons, we here show that this interpolation method may give reversal potentials and conductances that do not reflect the properties of the channels studied under conditions when ion flux may give rise to concentration changes. Therefore, changes in ion concentrations may remain undetected and conclusions on changes in conductance, such as during desensitization, may be mistaken. In contrast, an alternative experimental approach, using rapid voltage ramps, enable I-V relations that much better reflect the properties of the studied ion channels.

Firing clamp: a novel method for single-trial estimation of excitatory and inhibitory synaptic neuronal conductances.

Understanding non-stationary neuronal activity as seen in vivo requires estimation of both excitatory and inhibitory synaptic conductances from a single trial of recording. For this purpose, we propose a new intracellular recording method, called "firing clamp." Synaptic conductances are estimated from the characteristics of artificially evoked probe spikes, namely the spike amplitude and the mean subthreshold potential, which are sensitive to both excitatory and inhibitory synaptic input signals. The probe spikes, timed at a fixed rate, are evoked in the dynamic-clamp mode by injected meander-like current steps, with the step duration depending on neuronal membrane voltage. We test the method with perforated-patch recordings from isolated cells stimulated by external application or synaptic release of transmitter, and validate the method with simulations of a biophysically-detailed neuron model. The results are compared with the conductance estimates based on conventional current-clamp recordings.

Cl(-) concentration changes and desensitization of GABA(A) and glycine receptors.

Desensitization of ligand-gated ion channels plays a critical role for the information transfer between neurons. The current view on γ-aminobutyric acid (GABA)(A) and glycine receptors includes significant rapid components of desensitization as well as cross-desensitization between the two receptor types. Here, we analyze the mechanism of apparent cross-desensitization between native GABA(A) and glycine receptors in rat central neurons and quantify to what extent the current decay in the presence of ligand is a result of desensitization versus changes in intracellular Cl(-) concentration ([Cl(-)](i)). We show that apparent cross-desensitization of currents evoked by GABA and by glycine is caused by changes in [Cl(-)](i). We also show that changes in [Cl(-)](i) are critical for the decay of current in the presence of either GABA or glycine, whereas changes in conductance often play a minor role only. Thus, the currents decayed significantly quicker than the conductances, which decayed with time constants of several seconds and in some cells did not decay below the value at peak current during 20-s agonist application. By taking the cytosolic volume into account and numerically computing the membrane currents and expected changes in [Cl(-)](i), we provide a theoretical framework for the observed effects. Modeling diffusional exchange of Cl(-) between cytosol and patch pipettes, we also show that considerable changes in [Cl(-)](i) may be expected and cause rapidly decaying current components in conventional whole cell or outside-out patch recordings. The findings imply that a reevaluation of the desensitization properties of GABA(A) and glycine receptors is needed.

Mechanism of estradiol-induced block of voltage-gated K+ currents in rat medial preoptic neurons.

The present study was conducted to characterize possible rapid effects of 17-ß-estradiol on voltage-gated K(+) channels in preoptic neurons and, in particular, to identify the mechanisms by which 17-ß-estradiol affects the K(+) channels. Whole-cell currents from dissociated rat preoptic neurons were studied by perforated-patch recording. 17-ß-Estradiol rapidly (within seconds) and reversibly reduced the K(+) currents, showing an EC(50) value of 9.7 µM. The effect was slightly voltage dependent, but independent of external Ca(2+), and not sensitive to an estrogen-receptor blocker. Although 17-α-estradiol also significantly reduced the K(+) currents, membrane-impermeant forms of estradiol did not reduce the K(+) currents and other estrogens, testosterone and cholesterol were considerably less effective. The reduction induced by estradiol was overlapping with that of the K(V)-2-channel blocker r-stromatoxin-1. The time course of K(+) current in 17-ß-estradiol, with a time-dependent inhibition and a slight dependence on external K(+), suggested an open-channel block mechanism. The properties of block were predicted from a computational model where 17-ß-estradiol binds to open K(+) channels. It was concluded that 17-ß-estradiol rapidly reduces voltage-gated K(+) currents in a way consistent with an open-channel block mechanism. This suggests a new mechanism for steroid action on ion channels.

Spontaneous ryanodine-receptor-dependent Ca2+-activated K+ currents and hyperpolarizations in rat medial preoptic neurons.

The aim of the present study was to clarify the identity of slow spontaneous currents, the underlying mechanism and possible role for impulse generation in neurons of the rat medial preoptic nucleus (MPN). Acutely dissociated neurons were studied with the perforated patch-clamp technique. Spontaneous outward currents, at a frequency of approximately 0.5 Hz and with a decay time constant of approximately 200 ms, were frequently detected in neurons when voltage-clamped between approximately -70 and -30 mV. The dependence on extracellular K(+) concentration was consistent with K(+) as the main charge carrier. We concluded that the main characteristics were similar to those of spontaneous miniature outward currents (SMOCs), previously reported mainly for muscle fibers and peripheral nerve. From the dependence on voltage and from a pharmacological analysis, we concluded that the currents were carried through small-conductance Ca(2+)-activated (SK) channels, of the SK3 subtype. From experiments with ryanodine, xestospongin C, and caffeine, we concluded that the spontaneous currents were triggered by Ca(2+) release from intracellular stores via ryanodine receptor channels. An apparent voltage dependence was explained by masking of the spontaneous currents as a consequence of steady SK-channel activation at membrane potentials > -30 mV. Under current-clamp conditions, corresponding transient hyperpolarizations occasionally exceeded 10 mV in amplitude and reduced the frequency of spontaneous impulses. In conclusion, MPN neurons display spontaneous hyperpolarizations triggered by Ca(2+) release via ryanodine receptors and SK3-channel activation. Thus such events may affect impulse firing of MPN neurons.

Differential control of spontaneous and evoked GABA release by presynaptic L-type Ca(2+) channels in the rat medial preoptic nucleus.

To clarify the role of presynaptic L-type Ca(2+) channels in GABA-mediated transmission in the medial preoptic nucleus (MPN), spontaneous, miniature, and impulse-evoked inhibitory postsynaptic currents (sIPSCs, mIPSCs, and eIPSCs, respectively) were recorded from MPN neurons in a slice preparation from rat brain. The effects of different stimulus protocols and pharmacological tools to detect contributions of L-type Ca(2+) channels and of Ca(2+)-activated K(+) (K(Ca)) channels were analyzed. Block of L-type channels did not affect the sIPSC and mIPSC properties (frequency, amplitude, decay time course) in the absence of external stimulation but unexpectedly potentiated the eIPSCs evoked at low stimulus frequency (0.1-2.0 Hz). This effect was similar to and overlapping with the effect of K(Ca)-channel blockers. High-frequency stimulation (50 Hz for 10 s) induced a substantial posttetanic potentiation (PTP) of the eIPSC amplitude and of the sIPSC frequency. Block of L-type channels still potentiated the eIPSC during PTP, but in contrast, reduced the sIPSC frequency during PTP. It was concluded that L-type channels provide a means for differential control of spontaneous and impulse-evoked GABA release and that this differential control is prominent during short-term synaptic plasticity. Functional coupling of the presynaptic L-type channels to K(Ca) channels explains the observed effects on eIPSCs.

Short-term plasticity in excitatory synapses of the rat medial preoptic nucleus.

The medial preoptic nucleus (MPN) regulates sexual behavior which is subject to experience-dependent modifications. Such modifications must depend on functional plasticity in the controlling neural circuits. Thus, MPN synapses are likely candidates for the site of alterations. The present work is a first systematic study of functional synaptic plasticity at glutamatergic synapses in the MPN. Short-term activity-dependent plasticity was investigated using a slice preparation from young male rats. The average efficacy of AMPA/kainate-receptor-mediated synaptic transmission was activity-dependent, showing a peak at a steady stimulation rate of 2 Hz. The variation in efficacy was attributed to mainly presynaptic factors since the average response amplitude was roughly paralleled by the response probability. Upon paired-pulse stimulation, paired-pulse facilitation as well as paired-pulse depression was observed. In some cases, paired-pulse facilitation as well as paired-pulse depression was recorded from an individual neuron depending on the interval between the paired stimuli. On average, paired-pulse facilitation was observed at intervals <500 ms, and paired-pulse depression at intervals in the range 1-4 s. The findings thus reveal complex activity-dependent short-term plasticity of the functional synaptic properties in the medial preoptic nucleus.

Fast neurotransmission in the rat medial preoptic nucleus.

The functional properties of neurotransmission in the medial preoptic nucleus (MPN) were studied in a brain slice preparation from young male rats. The aims were to evaluate the thin slice preparation for studying evoked synaptic responses in MPN neurons, to characterize the fast responses triggered by activation of presynaptic nerve fibers in the MPN, and to identify the involved receptor types. Presynaptic stimulation within the MPN evoked postsynaptic voltage and current responses that were blocked by 200 microM Cd2+ or by 2.0 microM tetrodotoxin and were attributed to action potential-evoked transmitter release. The relation to stimulus strength and comparison with spontaneous synaptic currents suggested that in many cases only one presynaptic nerve fiber was excited by the stimulus. Furthermore, the transmission was probabilistic in nature, with frequent failures. Thus, response probability, most likely reflecting transmitter release probability, could be evaluated in the thin slice preparation. Evoked excitatory postsynaptic currents recorded under voltage-clamp conditions were, due to kinetics, I-V relation, and pharmacological properties, attributed to AMPA/kainate receptors and NMDA receptors, whereas inhibitory currents were attributed to GABAA receptors. No responses that could be attributed to glycine or other types of primary transmitters were detected. Although serotonin (5-HT) did not appear to function as a primary transmitter, glutamate- as well as GABA-mediated transmission was suppressed by 500 microM 5-HT, with a clear reduction in response probability observed. 5-HT also reduced the frequency, but not the amplitude, of spontaneous postsynaptic currents and was therefore ascribed a presynaptic site of action.

Bicuculline free base blocks voltage-activated K+ currents in rat medial preoptic neurons.

The effects of the well-known GABA(A)-receptor blocker bicuculline on voltage-gated K(+) currents were studied in neurons from the medial preoptic nucleus (MPN) of rat. Whole-cell currents were recorded using the perforated-patch technique. Voltage steps from -54 to +6 mV resulted in tetraethylammonium-sensitive K(+) currents of delayed rectifier type. The total K(+) current (at 300 ms), including Ca(2+)-dependent and Ca(2+)-independent components, was reversibly reduced (17 +/- 4%) by 100 microM bicuculline methiodide and (37 +/- 5%) by 100 microM bicuculline as free base. The Ca(2+)-independent fraction (77 +/- 2%) of K(+) current evoked by a voltage step was, however, reduced (54 +/- 6%) only by bicuculline free base, but was not affected by bicuculline methiodide. The half-saturating concentration of bicuculline free base for blocking this purely voltage-gated K(+) current was 113 microM, whereas for blocking a steady Ca(2+)-dependent K(+) current it was 36 microM. The bicuculline-sensitive voltage-gated K(+) current was composed of 4-AP-sensitive and 4-AP-resistant components with different kinetic properties. No component of the purely voltage-gated K(+) current was affected neither by 100 nM alpha-dendrotoxin nor by 100 nM I-dendrotoxin. The possible K(+)-channel subtypes mediating the bicuculline-sensitive current in MPN neurons are discussed.

Allopregnanolone modulates spontaneous GABA release via presynaptic Cl- permeability in rat preoptic nerve terminals.

The endogenous neurosteroid 3alpha-hydroxy-5alpha-pregnane-20-one (allopregnanolone) affects presynaptic nerve terminals and thereby increases the frequency of spontaneous GABA release. The present study aimed at clarifying the mechanisms underlying this presynaptic neurosteroid action, by recording the frequency of spontaneous GABA-mediated inhibitory postsynaptic currents (sIPSCs) in neurons from the medial preoptic nucleus (MPN) of rat. Acutely dissociated neurons with functional adhering nerve terminals were studied by perforated-patch recording under voltage-clamp conditions. It was shown that the sIPSC frequency increased with the external K(+) concentration ([K(+)](o)). Further, the effect of allopregnanolone on the sIPSC frequency was strongly dependent on [K(+)](o). In a [K(+)](o) of 5 mM, 2.0 microM allopregnanolone caused a clear increase in sIPSC frequency. However, the effect declined rapidly with increased [K(+)](o) and at high [K(+)](o) allopregnanolone reduced the sIPSC frequency. The effect of allopregnanolone was also strongly dependent on the external Cl(-) concentration ([Cl(-)](o)). In a reduced [Cl(-)](o) (40 mM, but with a standard [K(+)](o) of 5 mM), the effect on sIPSC frequency was larger than that in the standard [Cl(-)](o) of 146 mM. The dependence of the effect of allopregnanolone on [K(+)](o) and on estimated presynaptic membrane potential was also altered by the reduction in [Cl(-)](o). As in standard [Cl(-)](o), the effect in low [Cl(-)](o) declined when [K(+)](o) was raised, but reversed at a higher [K(+)](o). The GABA(A) receptor agonist muscimol also potentiated the sIPSC frequency. Altogether, the results suggest that allopregnanolone exerts its presynaptic effect by increasing the presynaptic Cl(-) permeability, most likely via GABA(A) receptors.

Dual and opposing roles of presynaptic Ca2+ influx for spontaneous GABA release from rat medial preoptic nerve terminals.

Calcium influx into the presynaptic nerve terminal is well established as a trigger signal for transmitter release by exocytosis. By studying dissociated preoptic neurons with functional adhering nerve terminals, we here show that presynaptic Ca2+ influx plays dual and opposing roles in the control of spontaneous transmitter release. Thus, application of various Ca2+ channel blockers paradoxically increased the frequency of spontaneous (miniature) inhibitory GABA-mediated postsynaptic currents (mIPSCs). Similar effects on mIPSC frequency were recorded upon washout of Cd2+ or EGTA from the external solution. The results are explained by a model with parallel Ca2+ influx through channels coupled to the exocytotic machinery and through channels coupled to Ca2+-activated K+ channels at a distance from the release site.