Hyperpolarization-activated Cation Current Ih Research Articles

High dendritic expression of Ih in the proximity of the axon origin controls the integrative properties of nigral dopamine neurons

The hyperpolarization-activated cation current Ih is expressed in dopamine neurons of the substantia nigra, but the subcellular distribution of the current and its role in synaptic integration remain unknown. We used cell-attached patch recordings to determine the localization profile of Ih along the somatodendritic axis of nigral dopamine neurons in slices from young rats. Ih density is higher in axon-bearing dendrites, in a membrane area close to the axon origin, than in the soma and axon-lacking dendrites. Dual current-clamp recordings revealed a similar contribution of Ih to the waveform of single excitatory postsynaptic potentials throughout the somatodendritic domain. The Ih blocker ZD7288 increased the temporal summation in all dendrites with a comparable effect in axon- and non-axon dendrites. The strategic position of Ih in the proximity of the axon may influence importantly transitions between pacemaker and bursting activities and consequently the downstream release of dopamine. Dendrites of most neurons express voltage-gated ion channels in their membrane. In combination with passive properties, active currents confer to dendrites a high computational potential. The hyperpolarization-activated cation current Ih present in the dendrites of some pyramidal neurons affects their membrane and integration properties, synaptic plasticity and higher functions such as memory. A gradient of increasing h-channel density towards distal dendrites has been found to be responsible for the location independence of excitatory postsynaptic potential (EPSP) waveform and temporal summation in cortical and hippocampal pyramidal cells. However, reports on other cell types revealed that smoother gradients or even linear distributions of Ih can achieve homogeneous temporal summation. Although the existence of a robust, slowly activating Ih current has been repeatedly demonstrated in nigral dopamine neurons, its subcellular distribution and precise role in synaptic integration are unknown. Using cell-attached patch-clamp recordings, we find a higher Ih current density in the axon-bearing dendrite than in the soma or in dendrites without axon in nigral dopamine neurons. Ih is mainly concentrated in the dendritic membrane area surrounding the axon origin and decreases with increasing distances from this site. Single EPSPs and temporal summation are similarly affected by blockade of Ih in axon- and non-axon-bearing dendrites. The presence of Ih close to the axon is pivotal to control the integrative functions and the output signal of dopamine neurons and may consequently influence the downstream coding of movement.

Homeostatic regulation of h-conductance controls intrinsic excitability and stabilizes the threshold for synaptic modification in CA1 neurons.

We determined the contribution of the hyperpolarization-activated cationic (h) current (Ih ) to the homeostatic regulation of CA1 pyramidal cells in vitro using chronic treatments (48h) that either increase (picrotoxin) or decrease (kynurenate) neuronal activity. The h-conductance was found to be up- or down-regulated following chronic activity enhancement or activity deprivation, respectively. This bidirectional plasticity of Ih was found to subsequently alter both apparent input resistance and intrinsic neuronal excitability. Bidirectional homeostatic plasticity of Ih also determined EPSP waveform and EPSP summation tested at 5-30Hz. Long-term synaptic modification induced by repetitive stimulation of the Schaffer collaterals was found to be constant across treatments in the presence of Ih but not when Ih was blocked pharmacologically. Thus, bidirectional homeostatic regulation of Ih stabilizes induction of long-term synaptic modification in CA1 pyramidal neurons that depends on EPSP summation. The hyperpolarization-activated cationic (h) current is a voltage-shock absorber, highly expressed in the dendrites of CA1 pyramidal neurons. Up-regulation of Ih has been reported following episodes of intense network activity but the effect of activity deprivation on Ih and the functional consequence of homeostatic regulation of Ih remain unclear. We determined here the contribution of Ih to the homeostatic regulation of CA1 pyramidal cell excitability. Intrinsic neuronal excitability was decreased in neurons treated for 2-3 days with the GABAA channel blocker picrotoxin (PiTx) but increased in neurons treated (2-3days) with the glutamate receptor antagonist kynurenate (Kyn). Membrane capacitance remained unchanged after treatment but the apparent input resistance was reduced for PiTx-treated neurons and enhanced for Kyn-treated neurons. Maximal Ih conductance was up-regulated after chronic hyperactivity but down-regulated following chronic hypoactivity. Up-regulation of Ih in PiTx-treated cultures was found to accelerate EPSP kinetics and reduce temporal summation of EPSPs whereas opposite effects were observed in Kyn-treated cultures, indicating that homeostatic regulation of Ih may control the induction of synaptic modification depending on EPSP summation. In fact, stimulation of the Schaffer collaterals at 3-10Hz induced differential levels of plasticity in PiTx-treated and Kyn-treated neurons when Ih was blocked pharmacologically but not in control conditions. These data indicate that homeostatic regulation of Ih normalizes the threshold for long-term synaptic modification that depends on EPSP summation. In conclusion, bidirectional homeostatic regulation of Ih not only controls spiking activity but also stabilizes the threshold for long-term potentiation induced in CA1 pyramidal neurons by repetitive stimulation.

Presynaptically mediated effects of cholecystokinin-8 on the excitability of area postrema neurons in rat brain slices

Cholecystokinin (CCK) is a well-known gut hormone that shows anorexigenic effects via action at peripheral and central receptors. CCK is also widely distributed throughout the mammalian brain and appears to function as a neurotransmitter and neuromodulator. The area postrema is one of the circumventricular organs, located on the dorsal surface of the medulla oblongata at the caudal end of the fourth ventricle. Blood vessels in the area postrema lack a blood brain barrier, offering specific central neural elements unique access to circulating substances. Immunohistochemical studies show CCK-A receptors in the area postrema, and we reported CCK-sensitive area postrema neurons. However, the receptive mechanism of CCK in area postrema neurons still remains unexplained. We investigated the responses of area postrema neurons to agonists and antagonists of CCK receptors using whole cell and perforated patch-clamp recordings in rat brain slices. The application of CCK-8 elicited excitatory responses, such as increases in the frequency of mEPSCs (miniature excitatory postsynaptic currents), a shift toward larger amplitude mEPSCs, and increases in the frequency of action potentials. These changes were found mostly in cells not displaying the hyperpolarization-activated cation current (Ih), except for small excitatory changes in a minority of Ih-positive neurons. Tonic inward currents or an inhibitory response to CCK-8 were never seen. Analysis of the amplitude of mEPSCs before and after the administration of CCK-8 indicated the responses mediated via the presynaptic receptors. The effect of CCK-8 was abolished in the presence of CNQX (AMPA type glutamate receptor antagonist). In the presence of lorglumide (a selective CCK-A receptor antagonist), CCK-8-induced excitatory responses were inhibited. No cells responded to the administration of non-sulfated CCK-8 (CCK-8NS, a selective CCK-B receptor agonist). We conclude that CCK-8 exerts its action via presynaptic CCK-A receptors to facilitate glutamate release onto Ih-negative area postrema cells.

Upregulation of axonal HCN current by methylglyoxal: Potential association with diabetic polyneuropathy

ObjectiveTo describe functional changes of axonal ion channels by a metabolic derivative of glucose, methylglyoxal (MGO), and its potential contribution to diabetic neuropathy. Methods(1) In wild-type male mice, multiple excitability measurements of sensory nerves were performed at baseline and 1week after serial administration of MGO (50mg/kg).(2) Excitability testing in patients with diabetic neuropathy (N=17) and healthy controls (N=12) were also conducted, and data were interpreted using mathematical modeling. ResultsIn the animal study, there was a decrease in threshold changes by long hyperpolarization and in superexcitability after administration of MGO. In the preliminary human study, the threshold changes by long hyperpolarizing current were decreased in patients with diabetes. Mathematical modeling showed increased hyperpolarization-activated cation current (Ih) in the MGO-treated mice and in patients with diabetes. ConclusionIh was upregulated after MGO administration in normal mice. SignificanceMGO is associated with abnormal axonal excitability. Hyperexcitability in diabetic polyneuropathy may, at least in part, be caused by dysfunctional axonal hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. A future study with a large sample size of the diabetic patients would clarify this hypothesis.

Firing properties of Renshaw cells defined by Chrna2 are modulated by hyperpolarizing and small conductance ion currents Ih and ISK.

Renshaw cells in the spinal cord ventral horn regulate motoneuron output through recurrent inhibition. Renshaw cells can be identified in vitro using anatomical and cellular criteria; however, their functional role in locomotion remains poorly defined because of the difficulty of functionally isolating Renshaw cells from surrounding motor circuits. Here we aimed to investigate whether the cholinergic nicotinic receptor alpha2 (Chrna2) can be used to identify Renshaw cells (RCs(α2)) in the mouse spinal cord. Immunohistochemistry and electrophysiological characterization of passive and active RCs(α2) properties confirmed that neurons genetically marked by the Chrna2-Cre mouse line together with a fluorescent reporter mouse line are Renshaw cells. Whole-cell patch-clamp recordings revealed that RCs(α2) constitute an electrophysiologically stereotyped population with a resting membrane potential of -50.5 ± 0.4 mV and an input resistance of 233.1 ± 11 MΩ. We identified a ZD7288-sensitive hyperpolarization-activated cation current (Ih) in all RCs(α2), contributing to membrane repolarization but not to the resting membrane potential in neonatal mice. Additionally, we found RCs(α2) to express small calcium-activated potassium currents (I(SK)) that, when blocked by apamin, resulted in a complete attenuation of the afterhyperpolarisation potential, increasing cellular firing frequency. We conclude that RCs(α2) can be genetically targeted through their selective Chrna2 expression and that they display currents known to modulate rebound excitation and firing frequency. The genetic identification of Renshaw cells and their electrophysiological profile is required for genetic and pharmacological manipulation as well as computational simulations with the aim to understand their functional role.

The CA3 region of the hippocampus: how is it? What is it for? How does it do it?

The CA3 region of the hippocampus: how is it? What is it for? How does it do it?

Hyperpolarization-activated cation current Ih influences synaptic integration properties in substantia nigra dopamine neurons

Event Abstract Back to Event Hyperpolarization-activated cation current Ih influences synaptic integration properties in substantia nigra dopamine neurons Dominique Engel1* and Vincent Seutin2 1 FNRS, GIGA-Neurosciences, Belgium 2 Université de Liège, GIGA-Neuroscience, Belgium The hyperpolarization-activated cation current Ih is expressed in many pyramidal neurons and in some interneurons. Ih contributes to neuronal signaling via the modulation of passive and active membrane properties, intrinsic resonance, synaptic integration and synaptic plasticity. One of the most characteristic functions of Ih is to influence synaptic integration by inducing a location independence of postsynaptic potentials (EPSPs) waveform and EPSP temporal summation. Herewith, EPSPs arriving at the soma have a similar time course, regardless of their origin. In pyramidal neurons, this effect of Ih is correlated to a specific subcellular distribution of the channel. In the substantia nigra, dopamine (DA) neurons are implicated in the control of movement. Their degeneration is related to motor disturbances as they are described in Parkinson’s disease. Interestingly, the axon of DA neurons generally emerges from a dendrite dividing the dendritic compartment of these neurons in axon- and non-axon bearing dendrites. This morphology contrasts with the typical arrangement of subcellular compartments found in neurons. DA neurons are also well known to display a prominent sag in response to long hyperpolarizing current injections, consecutive to slowly activating h-channels. However, the subcellular distribution of the channel and its role in synaptic integration is unknown for DA neurons. We performed cell-attached patch-clamp recordings along the somatodendritic domain of DA neurons and observed a high Ih current density in the axon-bearing dendrites in a membrane region close to the axon origin. Simultaneous whole-cell somatic and dendritic recordings revealed that Ih reduces EPSP integral and temporal summation. Keywords: Dopamine Neuron, Substantia Nigra, Dendrites, integration properties, hyperpolarization-activated inwardly rectifying cation conductance, Ih Conference: 11th National Congress of the Belgian Society for Neuroscience, Mons, Belgium, 22 May - 22 May, 2015. Presentation Type: Poster presentation Topic: Neuroscience Citation: Engel D and Seutin V (2015). Hyperpolarization-activated cation current Ih influences synaptic integration properties in substantia nigra dopamine neurons. Front. Neurosci. Conference Abstract: 11th National Congress of the Belgian Society for Neuroscience. doi: 10.3389/conf.fnins.2015.89.00016 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 05 May 2015; Published Online: 05 May 2015. * Correspondence: Dr. Dominique Engel, FNRS, GIGA-Neurosciences, Liege, 4000, Belgium, dominique.engel@uliege.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Dominique Engel Vincent Seutin Google Dominique Engel Vincent Seutin Google Scholar Dominique Engel Vincent Seutin PubMed Dominique Engel Vincent Seutin Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Differential effects of static and dynamic inputs on neuronal excitability.

The intrinsic excitability of neurons is known to be dynamically regulated by activity-dependent plasticity and homeostatic mechanisms. Such processes are commonly analyzed in the context of input-output functions that describe how neurons fire in response to constant levels of current. However, it is not well understood how changes of excitability as observed under static inputs translate to the function of the same neurons in their natural synaptic environment. Here we performed a computational study and hybrid experiments on rat bed nucleus of stria terminalis neurons to compare the two scenarios. The inward rectifying Kir current (IKir) and the hyperpolarization-activated cation current (Ih) were found to be considerably more effective in regulating the firing under synaptic inputs than under static stimuli. This prediction was experimentally confirmed by dynamic-clamp insertion of a synthetic inwardly rectifying Kir current into the biological neurons. At the same time, ionic currents that activate with depolarization were more effective regulating the firing under static inputs. When two intrinsic currents are concurrently altered such as those under homeostatic regulation, the effects in firing responses under static vs. dynamic inputs can be even more contrasting. Our results show that plastic or homeostatic changes of intrinsic membrane currents can shape the current step responses of neurons and their firing under synaptic inputs in a differential manner.

Contribution of Ih to the relative facilitation of synaptic responses induced by carbachol in the entorhinal cortex during repetitive stimulation of the parasubiculum

Neurons in the superficial layers of the entorhinal cortex provide the hippocampus with the majority of its cortical sensory input, and also receive the major output projection from the parasubiculum. This puts the parasubiculum in a position to modulate the activity of entorhinal neurons that project to the hippocampus. These brain areas receive cholinergic projections that are active during periods of theta- and gamma-frequency electroencephalographic (EEG) activity. The purpose of this study was to investigate how cholinergic receptor activation affects the strength of repetitive synaptic responses at these frequencies in the parasubiculo-entorhinal pathway and the cellular mechanisms involved. Whole-cell patch-clamp recordings of rat layer II medial entorhinal neurons were conducted using an acute slice preparation, and responses to 5-pulse trains of stimulation at theta- and gamma-frequency delivered to the parasubiculum were recorded. The cholinergic agonist carbachol (CCh) suppressed the amplitude of single synaptic responses, but also produced a relative facilitation of synaptic responses evoked during stimulation trains. The N-methyl-d-aspartate (NMDA) glutamate receptor blocker APV did not significantly reduce the relative facilitation effect. However, the hyperpolarization-activated cationic current (Ih) channel blocker ZD7288 mimicked the relative facilitation induced by CCh, suggesting that CCh-induced inhibition of Ih could produce the effect by increasing dendritic input resistance (Rin). Inward-rectifying and leak K+ currents are known to interact with Ih to affect synaptic excitability. Application of the K+ channel antagonist Ba2+ depolarized neurons and enhanced temporal summation, but did not block further facilitation of train-evoked responses by ZD7288. The Ih-dependent facilitation of synaptic responses can therefore occur during reductions in inward-rectifying potassium current (IKir) associated with dendritic depolarization. Thus, in addition to cholinergic reductions in transmitter release that are known to facilitate train-evoked responses, these findings emphasize the role of inhibition of Ih in the integration of synaptic inputs within the entorhinal cortex during cholinergically-induced oscillatory states, likely due to enhanced summation of excitatory postsynaptic potentials (EPSPs) induced by increases in dendritic Rin.

P853: Nerve excitability change by chronic administration of methylglyoxal: implication for diabetic neuropathy

Question: The methylglyoxal (MGO) pathway is an offshoot of glycolysis, which converts glucose into MGO. Frequently increased in diabetic patients, MGO is highly toxic, resulting in activation of apoptosis and mitochondrial damage. Recently, MGO was found to be a causative agent for neuropathic pain. Because abnormal nerve excitability is frequently present in peripheral neuropathy and neuropathic pain, MGO can alter overall nerve excitability. The objective of the present study was to observe the chronic effects of MGO on peripheral nerve excitability in vivo. Methods: Eight adult male mice were examined. Sensory nerve multiple excitability testing (QTRAC) was performed from a tail nerve under inhalational anesthesia. The parameters were compared between at baseline and 1 week after intraperitoneal injection of MGX (0.005 ml/10g body weight) nine times (3 times weekly for 3 weeks). Results: The tested animals were all survived. The excitability parameters were similar in strength-duration time constant and threshold changes by long depolarizing current (threshold electrotonus: TE). The threshold changes by long hyperpolarizing current (TE) became smaller by MGO; (1) TEh(peak) by -70% conditioning current: -203.8±7.5 at baseline to 162.3±9.1 post-injection, P=0.003; (2) TEh(90-100ms): -94.3±4.9 at baseline to -71.7±5.5 post-injection, P=0.006. The recovery cycle showed smaller refractoriness at 2ms by MGO: 23.5±3.0 at baseline to 7.9±4.8 post-injection, P=0.01. The overall nerve excitability assessed by strength-response curves was slightly reduced after injection. Conclusions: Chronic administration of MGO changed axonal excitability in vivo. It is likely that MGO facilitated hyperpolarization-activated cation current (Ih). The upregulation of Ih may be responsible for abnormal excitability and sensory symptoms of diabetic neuropathy.

Subthreshold delta‐frequency resonance in thalamic reticular neurons

The thalamic reticular nucleus (nRt) is an assembly of GABAergic projection neurons that participate in the generation of brain rhythms during synchronous sleep and absence epilepsy. NRt cells receive inhibitory and excitatory synaptic inputs, and are endowed with an intricate set of intrinsic conductances. However, little is known about how intrinsic and synaptic properties interact to generate rhythmic discharges in these neurons. In order to better understand this interaction, I studied the subthreshold responses of nRt cells to time-varying inputs. Patch-clamp recordings were performed in acute slices of rat thalamus (postnatal days 12-21). Sinusoidal current waveforms of linearly changing frequencies were injected into the soma, and the resulting voltage oscillations were recorded. At the resting membrane potential, the impedance profile showed a characteristic resonance at 1.7Hz. The relative strength of the resonance was 1.2, and increased with membrane hyperpolarization. Small suprathreshold current injections led to preferred spike generation at the resonance frequency. Bath application of ZD7288 or Cs(+) , inhibitors of the hyperpolarization-activated cation current (Ih ), transformed the resonance into low-pass behaviour, whereas the T-channel blockers mibefradil and Ni(2+) decreased the strength of the resonance. It is concluded that nRt cells have an Ih -mediated intrinsic frequency preference in the subthreshold voltage range that favours action potential generation in the delta-frequency band.

Cholinergic Neurons Excite Cortically Projecting Basal Forebrain GABAergic Neurons

The basal forebrain (BF) plays an important role in the control of cortical activation and attention. Understanding the modulation of BF neuronal activity is a prerequisite to treat disorders of cortical activation involving BF dysfunction, such as Alzheimer's disease. Here we reveal the interaction between cholinergic neurons and cortically projecting BF GABAergic neurons using immunohistochemistry and whole-cell recordings in vitro. In GAD67-GFP knock-in mice, BF cholinergic (choline acetyltransferase-positive) neurons were intermingled with GABAergic (GFP(+)) neurons. Immunohistochemistry for the vesicular acetylcholine transporter showed that cholinergic fibers apposed putative cortically projecting GABAergic neurons containing parvalbumin (PV). In coronal BF slices from GAD67-GFP knock-in or PV-tdTomato mice, pharmacological activation of cholinergic receptors with bath application of carbachol increased the firing rate of large (>20 μm diameter) BF GFP(+) and PV (tdTomato+) neurons, which exhibited the intrinsic membrane properties of cortically projecting neurons. The excitatory effect of carbachol was blocked by antagonists of M1 and M3 muscarinic receptors in two subpopulations of BF GABAergic neurons [large hyperpolarization-activated cation current (Ih) and small Ih, respectively]. Ion substitution experiments and reversal potential measurements suggested that the carbachol-induced inward current was mediated mainly by sodium-permeable cation channels. Carbachol also increased the frequency of spontaneous excitatory and inhibitory synaptic currents. Furthermore, optogenetic stimulation of cholinergic neurons/fibers caused a mecamylamine- and atropine-sensitive inward current in putative GABAergic neurons. Thus, cortically projecting, BF GABAergic/PV neurons are excited by neighboring BF and/or brainstem cholinergic neurons. Loss of cholinergic neurons in Alzheimer's disease may impair cortical activation, in part, through disfacilitation of BF cortically projecting GABAergic/PV neurons.

TRIP(8B)Ing up and Down HCN Channel Gating and Trafficking

The proper neuronal functioning of ion channels depends on their correct targeting to distinct polarized neuronal compartments, where the channels often mediate highly specific functions. HCN1 channels, which underlie the hyperpolarization-activated cation current (Ih) in many types of neurons, are targeted to the distal apical dendrites of hippocampal CA1 pyramidal neurons, where they regulate the integration of synaptic inputs and control excitability. Results from our laboratory and others indicate that the cytoplasmic protein TRIP8b is the major auxiliary subunit of HCN1 channels in the brain, where it plays an important role in regulating HCN1 function, expression and localization. TRIP8b undergoes extensive alternative splicing at its N-terminus, with at least 10 splice variants detected in brain. All splice variants interact strongly with the C-terminus of all four HCN channel isoforms (HCN1-4) at two different interaction surfaces. Whereas all TRIP8b isoforms inhibit channel gating by antagonizing the normal action of cAMP to facilitate opening, the various isoforms have distinct effects on channel trafficking. We identified two splice isoforms with opposing actions on HCN1 surface expression and distinct subcellular locales that are critical for HCN1 dendritic targeting. Our more recent results have identified the structural and functional bases for many of the regulatory actions of TRIP8b.

Binding of the auxiliary subunit TRIP8b to HCN channels shifts the mode of action of cAMP

Hyperpolarization-activated cyclic nucleotide–regulated cation (HCN) channels generate the hyperpolarization-activated cation current Ih present in many neurons. These channels are directly regulated by the binding of cAMP, which both shifts the voltage dependence of HCN channel opening to more positive potentials and increases maximal Ih at extreme negative voltages where voltage gating is complete. Here we report that the HCN channel brain-specific auxiliary subunit TRIP8b produces opposing actions on these two effects of cAMP. In the first action, TRIP8b inhibits the effect of cAMP to shift voltage gating, decreasing both the sensitivity of the channel to cAMP (K1/2) and the efficacy of cAMP (maximal voltage shift); conversely, cAMP binding inhibits these actions of TRIP8b. These mutually antagonistic actions are well described by a cyclic allosteric mechanism in which TRIP8b binding reduces the affinity of the channel for cAMP, with the affinity of the open state for cAMP being reduced to a greater extent than the cAMP affinity of the closed state. In a second apparently independent action, TRIP8b enhances the action of cAMP to increase maximal Ih. This latter effect cannot be explained by the cyclic allosteric model but results from a previously uncharacterized action of TRIP8b to reduce maximal current through the channel in the absence of cAMP. Because the binding of cAMP also antagonizes this second effect of TRIP8b, application of cAMP produces a larger increase in maximal Ih in the presence of TRIP8b than in its absence. These findings may provide a mechanistic explanation for the wide variability in the effects of modulatory transmitters on the voltage gating and maximal amplitude of Ih reported for different neurons in the brain.

Dopamine D4 receptor excitation of lateral habenula neurons via multiple cellular mechanisms.

Glutamatergic lateral habenula (LHb) output communicates negative motivational valence to ventral tegmental area (VTA) dopamine (DA) neurons via activation of the rostromedial tegmental nucleus (RMTg). However, the LHb also receives a poorly understood DA input from the VTA, which we hypothesized constitutes an important feedback loop regulating DA responses to stimuli. Using whole-cell electrophysiology in rat brain slices, we find that DA initiates a depolarizing inward current (I(DAi)) and increases spontaneous firing in 32% of LHb neurons. I(DAi) was also observed upon application of amphetamine or the DA uptake blockers cocaine or GBR12935, indicating involvement of endogenous DA. I(DAi) was blocked by D4 receptor (D4R) antagonists (L745,870 or L741,742), and mimicked by a selective D4R agonist (A412997). I(DAi) was associated with increased whole-cell conductance and was blocked by Cs+ or a selective blocker of hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel, ZD7288. I(DAi) was also associated with a depolarizing shift in half-activation voltage for the hyperpolarization-activated cation current (Ih) mediated by HCN channels. Recordings from LHb neurons containing fluorescent retrograde tracers revealed that I(DAi) was observed only in cells projecting to the RMTg and not the VTA. In parallel with direct depolarization, DA also strongly increased synaptic glutamate release and reduced synaptic GABA release onto LHb cells. These results demonstrate that DA can excite glutamatergic LHb output to RMTg via multiple cellular mechanisms. Since the RMTg strongly inhibits midbrain DA neurons, activation of LHb output to RMTg by DA represents a negative feedback loop that may dampen DA neuron output following activation.

Persistent Sodium Current Drives Conditional Pacemaking in CA1 Pyramidal Neurons under Muscarinic Stimulation

Hippocampal CA1 pyramidal neurons are normally quiescent but can fire spontaneously when stimulated by muscarinic agonists. In brain slice recordings from mouse CA1 pyramidal neurons, we examined the ionic basis of this activity using interleaved current-clamp and voltage-clamp experiments. Both in control and after muscarinic stimulation, the steady-state current-voltage curve was dominated by inward TTX-sensitive persistent sodium current (I(NaP)) that activated near -75 mV and increased steeply with depolarization. In control, total membrane current was net outward (hyperpolarizing) near -70 mV so that cells had a stable resting potential. Muscarinic stimulation activated a small nonselective cation current so that total membrane current near -70 mV shifted to become barely net inward (depolarizing). The small depolarization triggers regenerative activation of I(NaP), which then depolarizes the cell from -70 mV to spike threshold. We quantified the relative contributions of I(NaP), hyperpolarization-activated cation current (I(h)), and calcium current to pacemaking by using the cell's own firing as a voltage command along with specific blockers. TTX-sensitive sodium current was substantial throughout the entire interspike interval, increasing as the membrane potential approached threshold, while both Ih and calcium current were minimal. Thus, spontaneous activity is driven primarily by activation of I(NaP) in a positive feedback loop starting near -70 mV and providing increasing inward current to threshold. These results show that the pacemaking "engine" from I(NaP) is an inherent property of CA1 pyramidal neurons that can be engaged or disengaged by small shifts in net membrane current near -70 mV, as by muscarinic stimulation.

Interspike intervals under the constraint of linear synaptic integration and background synaptic activity

Previous work [1] using a one-compartment steady-state computer model of a neuron shows how combinations of two active dendritic conductances can produce a linear dendritic response over a voltage range of ca. 15 to 22 mV. Here we extend this linearization issue to a dynamic, non-steady state situation for a neuron model with a soma, dendrite and distal compartment. It is known that synaptically-induced voltage pulses that originate in distal compartments will be both attenuated and broader by the time they reach the soma due to the passive cable properties of neurons. Additionally, neurons in intact brains are not isolated but are under constant synaptic bombardment. While a single synaptic event will not significantly affect the cell properties, thousands of such events will significantly change the integrative properties of neurons [2]. Due to the reduction in membrane resistance caused by synaptic activity, attenuation of individual voltage pulses is even stronger in the presence of background synaptic activity. This study shows that the presence of active conductances distributed along the cell membrane can mitigate some of the effects of electrical filtering on voltage pulses propagating to the soma, even in the presence of background synaptic activity. We consider three uniformly distributed conductances found in pyramidal cells of the forebrain: persistent sodium current (INaP) [3,4], hyperpolarization-activated mixed cation current (Ih) [5], and A-type potassium current (IKA) [6]. Added to an otherwise passive cell, select combinations of these voltage-activated ion channels can generate linear synaptic integration over the entire interspike interval (ISI) membrane potential range, that is from rest (here -72 mV) to threshold (here roughly -45 mV). In the presence of various levels of background synaptic activity, the ratio of these active conductances changes and contributes to the inverse ISI effect. Under the constraint of linear synaptic integration, the length of the ISI is seen to be inversely related to the intensity of the background synaptic activity. This inverse relationship results from a decrease of the effective time constant of the cell by increased levels of membrane conductance induced by the combination of synaptic activity and active conductances.

Bifidobacterium longum NCC3001 inhibits AH neuron excitability

Bifidobacterium longum (B. longum) NCC3001 can affect behavior and brain biochemistry, but identification of the cellular targets needs further investigation. Our hypothesis was that the communication with the brain might start with action on enteric sensory neurons. Ileal segments from adult mice were used to create a longitudinal muscle-myenteric-plexus preparation to expose sensory after-hyperpolarizing (AH) neurons in the myenteric plexus to allow access with microelectrodes. The intrinsic excitability of AH neurons was tested in response to the perfusion of conditioned media (B.longum culture supernatant) or unconditioned media (growth medium, MRS). B. longum conditioned medium significantly reduced the excitability of AH neurons compared to perfusion with the unconditioned medium. Specifically, a reduction was seen in the number of action potentials fired per depolarizing test pulse, the instantaneous and time-dependent input resistances and the magnitude of the hyperpolarization-activated cationic current (Ih ). The probiotic B.longum reduces excitability of AH sensory neurons likely via opening of potassium channels and closing of hyperpolarization-activated cation channels.

Hyperpolarization-Activated Current (Ih) Is Reduced in Hippocampal Neurons from Gabra5−/− Mice

Changes in the expression of γ-aminobutyric acid type A (GABAA) receptors can either drive or mediate homeostatic alterations in neuronal excitability. A homeostatic relationship between α5 subunit-containing GABAA (α5GABAA) receptors that generate a tonic inhibitory conductance, and HCN channels that generate a hyperpolarization-activated cation current (Ih) was recently described for cortical neurons, where a reduction in Ih was accompanied by a reciprocal increase in the expression of α5GABAA receptors resulting in the preservation of dendritosomatic synaptic function. Here, we report that in mice that lack the α5 subunit gene (Gabra5−/−), cultured embryonic hippocampal pyramidal neurons and ex vivo CA1 hippocampal neurons unexpectedly exhibited a decrease in Ih current density (by 40% and 28%, respectively), compared with neurons from wild-type (WT) mice. The resting membrane potential and membrane hyperpolarization induced by blockade of Ih with ZD-7288 were similar in cultured WT and Gabra5−/− neurons. In contrast, membrane hyperpolarization measured after a train of action potentials was lower in Gabra5−/− neurons than in WT neurons. Also, membrane impedance measured in response to low frequency stimulation was greater in cultured Gabra5−/− neurons. Finally, the expression of HCN1 protein that generates Ih was reduced by 41% in the hippocampus of Gabra5−/− mice. These data indicate that loss of a tonic GABAergic inhibitory conductance was followed by a compensatory reduction in Ih. The results further suggest that the maintenance of resting membrane potential is preferentially maintained in mature and immature hippocampal neurons through the homeostatic co-regulation of structurally and biophysically distinct cation and anion channels.

The h-Current in the Substantia Nigra pars Compacta Neurons: A Re-examination

The properties of the hyperpolarization-activated cation current (Ih) were investigated in rat substantia nigra - pars compacta (SNc) principal neurons using patch-clamp recordings in thin slices. A reliable identification of single dopaminergic neurons was made possible by the use of a transgenic line of mice expressing eGFP under the tyrosine hydroxylase promoter. The effects of temperature and different protocols on the Ih kinetics showed that, at 37°C and minimizing the disturbance of the intracellular milieu with perforated patch, this current actually activates at potentials more positive than what is generally indicated, with a half-activation potential of −77.05 mV and with a significant level of opening already at rest, thereby substantially contributing to the control of membrane potential, and ultimately playing a relevant function in the regulation of the cell excitability. The implications of the known influence of intracellular cAMP levels on Ih amplitude and kinetics were examined. The direct application of neurotransmitters (DA, 5-HT and noradrenaline) physiologically released onto SNc neurons and known to act on metabotropic receptors coupled to the cAMP pathway modify the Ih amplitude. Here, we show that direct activation of dopaminergic and of 5-HT receptors results in Ih inhibition of SNc DA cells, whereas noradrenaline has the opposite effect. Together, these data suggest that the modulation of Ih by endogenously released neurotransmitters acting on metabotropic receptors –mainly but not exclusively linked to the cAMP pathway- could contribute significantly to the control of SNc neuron excitability.