The slow afterhyperpolarizing current (sIAHP) is a calcium-dependent potassium current that underlies the late phase of spike frequency adaptation in hippocampal and neocortical neurons. sIAHP is a well-known target of modulation by several neurotransmitters acting via the cyclic AMP (cAMP) and protein kinase A (PKA)-dependent pathway. The neuropeptide pituitary adenylate cyclase activating peptide (PACAP) and its receptors are present in the hippocampal formation. In this study we have investigated the effect of PACAP on the sIAHP and the signal transduction pathway used to modulate intrinsic excitability of hippocampal pyramidal neurons. We show that PACAP inhibits the sIAHP, resulting in a decrease of spike frequency adaptation, in rat CA1 pyramidal cells. The suppression of sIAHP by PACAP is mediated by PAC1 and VPAC1 receptors. Inhibition of PKA reduced the effect of PACAP on sIAHP, suggesting that PACAP exerts part of its inhibitory effect on sIAHP by increasing cAMP and activating PKA. The suppression of sIAHP by PACAP was also strongly hindered by the inhibition of p38 MAP kinase (p38 MAPK). Concomitant inhibition of PKA and p38 MAPK indicates that these two kinases act in a sequential manner in the same pathway leading to the suppression of sIAHP. Conversely, protein kinase C is not part of the signal transduction pathway used by PACAP to inhibit sIAHP in CA1 neurons. Our results show that PACAP enhances the excitability of CA1 pyramidal neurons by inhibiting the sIAHP through the activation of multiple signaling pathways, most prominently cAMP/PKA and p38 MAPK. Our findings disclose a novel modulatory action of p38 MAPK on intrinsic excitability and the sIAHP, underscoring the role of this current as a neuromodulatory hub regulated by multiple protein kinases in cortical neurons. © 2013 The Authors. Hippocampus Published by Wiley Periodicals, Inc.
hippocampus; slow afterhyperpolarization; neuropeptide; protein kinase A; p38 MAP kinase
We have shown previously that stimulation of heterologously expressed P2Y1 nucleotide receptors inhibits M-type K+ currents in sympathetic neurons. We now report that activation of endogenous P2Y1 receptors induces inhibition of the M-current in rat CA1/CA3 hippocampal pyramidal cells in primary neuron cultures. The P2Y1 agonist adenosine 5′-[β-thio]diphosphate trilithium salt (ADPβS) inhibited M-current by up to 52% with an IC50 of 84 nM. The hydrolyzable agonist ADP (10 μM) produced 32% inhibition, whereas the metabotropic glutamate receptor 1/5 agonist DHPG [(S)-3,5-dihydroxyphenylglycine] (10 μM) inhibited M-current by 44%. The M-channel blocker XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride] produced 73% inhibition at 3 μM; neither ADPβS nor ADP produced additional inhibition in the presence of XE991. The effect of ADPβS was prevented by a specific P2Y1 antagonist, MRS 2179 (2′-deoxy-N′-methyladenosine-3′,5′-bisphosphate tetra-ammonium salt) (30 μM). Inhibition of the M-current by ADPβS was accompanied by increased neuronal firing in response to injected current pulses. The neurons responding to ADPβS were judged to be pyramidal cells on the basis of (1) morphology, (2) firing characteristics, and (3) their distinctive staining for the pyramidal cell marker neurogranin. Strong immunostaining for P2Y1 receptors was shown in most cells in these cultures: 74% of the cells were positive for both P2Y1 and neurogranin, whereas 16% were only P2Y1 positive. These results show the presence of functional M-current-inhibitory P2Y1 receptors on hippocampal pyramidal neurons, as predicted from their effects when expressed in sympathetic neurons. However, the mechanism of inhibition in the two cell types seems to differ because, unlike nucleotide-mediated M-current inhibition in sympathetic neurons, that in hippocampal neurons did not appear to result from raised intracellular calcium
nucleotide receptors; P2Y receptors; hippocampus; pyramidal neurons; potassium channels; M-current
In this study we characterized the pharmacological selectivity and physiological actions of a new arylaspartate glutamate transporter blocker, L-threo-ß-benzylaspartate (L-TBA). At concentrations up to 100 µM, L-TBA did not act as an AMPA receptor (AMPAR) or NMDA receptor (NMDAR) agonist or antagonist when applied to outside-out patches from mouse hippocampal CA1 pyramidal neurons. L-TBA had no effect on the amplitude of field excitatory postsynaptic potentials (fEPSPs) recorded at the Schaffer collateral-CA1 pyramidal cell synapse. Excitatory postsynaptic currents (EPSCs) in CA1 pyramidal neurons were unaffected by L-TBA in the presence of physiological extracellular Mg2+ concentrations, but in Mg2+-free solution, EPSCs were significantly prolonged as a consequence of increased NMDAR activity. Although L-TBA exhibited approximately four-fold selectivity for neuronal EAAT3 over glial EAAT1/EAAT2 transporter subtypes expressed in Xenopus oocytes, the L-TBA concentration-dependence of the EPSC charge transfer increase in the absence of Mg2+ was the same in hippocampal slices from EAAT3 +/+ and EAAT3 −/− mice, suggesting that TBA effects were primarily due to block of glial transporters. Consistent with this, L-TBA blocked synaptically evoked transporter currents in CA1 astrocytes with a potency in accord with its block of heterologously expressed glial transporters. Extracellular recording in the presence of physiological Mg2+ revealed that L-TBA prolonged fEPSPs in a frequency-dependent manner by selectively increasing the NMDAR-mediated component of the fEPSP during short bursts of activity. The data indicate that glial glutamate transporters play a dominant role in limiting extrasynaptic transmitter diffusion and binding to NMDARs. Furthermore, NMDAR signaling is primarily limited by voltage-dependent Mg2+ block during low-frequency activity, while the relative contribution of transport increases during short bursts of higher frequency signaling.
Adenosine protects neurons during hypoxia by inhibiting excitatory synaptic transmission and preventing NMDA receptor activation. Using an adeno-associated viral (AAV) vector containing Cre recombinase, we have focally deleted adenosine A1 receptors in specific hippocampal regions of adult mice. Recently, we found that deletion of A1 receptors in the CA1 area blocks the postsynaptic responses to adenosine in CA1 pyramidal neurons, and deletion of A1 receptors in CA3 neurons abolishes the presynaptic effects of adenosine on the Schaffer collateral input [J Neurosci 23 (2003) 5762]. In the current study, we used this technique to delete A1 receptors focally from CA3 neurons to investigate whether presynaptic A1 receptors protect synaptic transmission from hypoxia. We studied the effects of prolonged (1 h) hypoxia on the evoked field excitatory postsynaptic potentials (fEPSPs) in the CA1 region using in vitro slices. Focal deletion of the presynaptic A1 receptors on the Schaffer collateral input slowed the depression of the fEPSPs in response to hypoxia and impaired the recovery of the fEPSPs after hypoxia. Delayed responses to hypoxia linearly correlated with impaired recovery. These findings provide direct evidence that the neuroprotective role of adenosine during hypoxia depends on the rapid inhibition of synaptic transmission by the activation of presynaptic A1 receptors.
CA1; Schaffer collaterals; electrophysiology; AAV; Cre recombinase; inducible knock-out mice
Many neurons of the central and peripheral nervous system express a slow afterhyperpolarization that is mediated by a slow calcium-activated potassium current. Previous work has shown that this aftercurrent regulates repetitive firing and is an important target for neuromodulators signaling through receptors coupled to G proteins of the Gαq-11 and Gαs subtypes. Yet, in spite of considerable effort, a molecular-level understanding of the potassium current underlying the slow afterhyperpolarization and its modulation has proven elusive. Here we use a combination of pharmacological and molecular biological approaches in cortical brain slices to show that the functional expression of the slow calcium-activated afterhyperpolarizing current in pyramidal cells is critically dependent on membrane PtdIns(4,5)P2 and that this dependence accounts for its inhibition by 5-HT2A receptors. Furthermore we show that PtdIns(4,5)P2 regulates the calcium sensitivity of IsAHP in a manner that suggests it acts downstream from the rise in intracellular calcium. These results clarify key functional aspects of the slow afterhyperpolarization current and its modulation by 5-HT2A receptors and point to a key role for PtdIns(4,5)P2 in the gating of this current.
In the early neonatal period activation of GABAB receptors attenuates calcium current through N-type calcium channels while enhancing current through L-type calcium channels in rat hippocampal neurons. The attenuation of N-type calcium current has been previously demonstrated to occur through direct interactions of the βγ subunits of Gi/o G-proteins, but the signal transduction pathway for the enhancement of L-type calcium channels in mammalian neurons remains unknown. In the present study, calcium currents were elicited in acute cultures from postnatal day 6–8 rat hippocampi in the presence of various modulators of protein kinase A (PKA) and protein kinase C (PKC) pathways. Overnight treatment with an inhibitor of Gi/o (pertussis toxin, 200 ng/ml) abolished the attenuation of calcium current by the GABAB agonist, baclofen (10 μM) with no effect on the enhancement of calcium current. These data indicate that while the attenuation of N-type calcium current is mediated by the Gi/o subtype of G-protein, the enhancement of L-type calcium current requires activation of a different G-protein. The enhancement of the sustained component of calcium current by baclofen was blocked by PKC inhibitors, GF-109203X (500 nM), chelerythrine chloride (5 μM), and PKC fragment 19–36 (2 μM) and mimicked by the PKC activator phorbol-12-myristate-13-acetate (1 μM). The enhancement of the sustained component of calcium current was blocked by PKA inhibitors H-89 (1 μM) and PKA fragment 6–22 (500 nM) but not Rp-cAMPS (30 μM) and it was not mimicked by the PKA activator, 8-Br-cAMP (500 μM – 1 mM). The data suggest that activation of PKC alone is sufficient to enhance L-type calcium current but that PKA may also be involved in the GABAB receptor mediated effect.
L-type calcium channel; hippocampus; GABAB receptor; protein kinase C; protein kinase A; G-protein
Stress facilitates the development of psychiatric disorders in vulnerable individuals. It affects physiological functions of hippocampal excitatory neurons, but little is known about the impact of stress on the GABAergic network. Here, we studied the effects of stress and a synthetic glucocorticoid on hippocampal GABAergic neurotransmission and network function focusing on two perisomatic interneurons, the parvalbumin (PV)- and the cholecystokinin (CCK)-positive neurons. In acute hippocampal slices of rat, application of the potent glucocorticoid receptor (GR) agonist dexamethasone (DEX) caused a rapid increase in spontaneous inhibitory postsynaptic currents (sIPSCs) in CA1 pyramidal neurons. This effect was mediated by a nongenomic GR that evoked nitric oxide (NO) release from pyramidal neurons. Retrograde NO signaling caused the augmentation of GABA release from the interneurons and increased CCK release, which in turn further enhanced the activity of the PV-positive cells. Interestingly, chronic restraint stress also resulted in increased sIPSCs in CA1 pyramidal neurons that were Ca2+-dependent and an additional DEX application elicited no further effect. Concomitantly, chronic stress reduced the number of PV-immunoreactive cells and impaired rhythmic sIPSCs originating from the PV-positive neurons. In contrast, the CCK-positive neurons remained unaffected. We therefore propose that, in addition to the immediate effect, the sustained activation of nongenomic GRs during chronic stress injures the PV neuron network and results in an imbalance in perisomatic inhibition mediated by the PV and CCK interneurons. This stress-induced dysfunctional inhibitory network may in turn impair rhythmic oscillations and thus lead to cognitive deficits that are common in stress-related psychiatric disorders.
GABA; parvalbumin; chronic stress; cholecystokinin; neuroplasticity; nitric oxide; GABA; Mood/Anxiety/Stress Disorders; Neurophysiology; Plasticity; glucocorticoid; Parvalbumin; nongenomic
Small conductance calcium-activated potassium (SK or KCa2) channels link intracellular calcium transients to membrane potential changes. SK channel subtypes present different pharmacology and distribution in the nervous system. The selective blocker apamin, SK enhancers and mice lacking specific SK channel subunits have revealed multifaceted functions of these channels in neurons, glia and cerebral blood vessels. SK channels regulate neuronal firing by contributing to the afterhyperpolarization following action potentials and mediating IAHP, and partake in a calcium-mediated feedback loop with NMDA receptors, controlling the threshold for induction of hippocampal long-term potentiation. The function of distinct SK channel subtypes in different neurons often results from their specific coupling to different calcium sources. The prominent role of SK channels in the modulation of excitability and synaptic function of limbic, dopaminergic and cerebellar neurons hints at their possible involvement in neuronal dysfunction, either as part of the causal mechanism or as potential therapeutic targets.
Calcium-activated potassium channel; afterhyperpolarization; IAHP; apamin; long-term potentiation; pacemaking neuron; glial cell; cerebral blood vessel endothelium
The excitability of hippocampal pyramidal neurons is regulated by activation of metabotropic glutamate receptors, an effect that is mediated by modulation of R-type calcium channels.
Activation of group I metabotropic glutamate receptors (subtypes mGluR1 and mGluR5) regulates neural activity in a variety of ways. In CA1 pyramidal neurons, activation of group I mGluRs eliminates the post-burst afterhyperpolarization (AHP) and produces an afterdepolarization (ADP) in its place. Here we show that upregulation of Cav2.3 R-type calcium channels is responsible for a component of the ADP lasting several hundred milliseconds. This medium-duration ADP is rapidly and reversibly induced by activation of mGluR5 and requires activation of phospholipase C (PLC) and release of calcium from internal stores. Effects of mGluR activation on subthreshold membrane potential changes are negligible but are large following action potential firing. Furthermore, the medium ADP exhibits a biphasic activity dependence consisting of short-term facilitation and longer-term inhibition. These findings suggest that mGluRs may dramatically alter the firing of CA1 pyramidal neurons via a complex, activity-dependent modulation of Cav2.3 R-type channels that are activated during spiking at physiologically relevant rates and patterns.
The hippocampus is an essential structure in the brain for the formation of new declarative memories. Understanding the cellular basis of memory formation, storage, and recall in the hippocampus requires a knowledge of the properties of the relevant neurons and how they are modulated by activity in the neural circuit. For many years, we have known that various chemical neurotransmitters can modulate the electrical excitability of neurons in the hippocampus. Here, we report new experiments to reveal how the chemical neurotransmitter glutamate increases neuronal excitability. The effect we study is the conversion of the afterhyperpolarization (a cellular consequence of firing an action potential) to an afterdepolarization. We identified the metabotropic glutamate receptors involved in this conversion (receptors called mGluR1 and mGluR5) as well as the final target of modulation (R-type calcium channels composed of Cav2.3 subunits), which cause the neurons to exhibit altered excitability in the presence of glutamate. We also determined some of the intermediate steps between activation of the glutamate receptors and modulation of the calcium channels responsible for the change in excitability, offering further mechanistic insight into how synaptic transmission can regulate cellular and network activity.
A fundamental property of small neuronal ensembles is their ability to be selectively activated by distinct stimuli. One cellular mechanism by which neurons achieve this input selectivity is by modulating the temporal dynamics of excitation and inhibition. We explored the interplay of excitation and inhibition in synapses between pyramidal neurons of cornu ammonis field 3 of the hippocampal formation (CA3) in cultured rat hippocampal slices, where activation of a single excitatory cell can readily recruit local interneurons. Simultaneous whole-cell recordings from pairs of CA3 pyramidal neurons revealed that the strength of connections was neither uniform nor balanced. Rather, stimulation of presynaptic neurons elicited distinct combinations of excitatory postsynaptic current–inhibitory postsynaptic current (EPSC–IPSC) amplitudes in the postsynaptic neurons. EPSC–IPSC sequences with small EPSCs had large IPSCs and sequences that contained large EPSCs had small IPSCs. In addition to differences in the amplitudes of the responses, the kinetics of the EPSCs were also different, creating distinct temporal dynamics of excitation and inhibition. Weaker EPSCs had significantly slower kinetics and were efficiently occluded by IPSCs, thereby further limiting their contribution to depolarizing the postsynaptic membrane. Our data suggest that hippocampal pyramidal cells may use an imbalance between excitation and inhibition as a filter to enhance selectivity toward preferential excitatory connections.
excitatory postsynaptic current; feed-forward inhibition; inhibitory postsynaptic current; network connectivity; rat; timing
Sigma (σ) receptors have been shown to regulate multiple ion channel types in intracardiac ganglion neurons, including voltage-gated calcium and potassium channels. However, the inhibition of these channels alone cannot fully account for σ receptor-induced changes in neuronal excitability previously reported. Whole-cell patch clamp experiments were conducted under current-clamp mode in isolated intracardiac neurons from neonatal rats to assess the effects of σ receptor activation on the active membrane properties of these cells. Bath application of the pan-selective σ receptor agonist, 1,3-Di-o-tolylguanidine (DTG), and the σ-1-selective agonist, (+)-pentazocine, significantly increased the action potential latency and decreased action potential overshoot in response to depolarizing current ramps, which suggests inhibition of voltage-gated sodium channels. Whole-cell voltage clamp experiments showed that these σ agonists reversibly decrease depolarization-activated Na+ currents in these cells at all potentials tested. The peak currents generated by membrane depolarizations were decreased in a dose dependent manner with IC50 values for DTG and (+)-pentazocine of 32 μM and 49 μM, respectively. The σ-1 receptor-selective antagonist, BD 1063 (100 nM), inhibited DTG (30 μM) block of Na+ currents by ∼ 50%, suggesting that the effects are mediated by activation of σ-1 receptors. DTG also shifted the steady-state inactivation curve of Na+ channels to more negative potentials, with the membrane potential of half-activation shifting from -49 mV to -63 mV in the absence and presence of 30 μM DTG, respectively. Taken together, these results suggest that σ-1 receptor activation decreases intracardiac ganglion neuron excitability by modulating voltage-gated Na+ channels.
Sigma-1 receptor; sodium channel; intracardiac neurons; action potential; parasympathetic
Although the potent anti-parkinsonian action of the atypical D1-like receptor agonist SKF83959 has been attributed to the selective activation of phosphoinositol(PI)-linked D1 receptor, whereas the mechanism underlying its potent neuroprotective effect is not fully understood. In the present study, the actions of SKF83959 on neuronal membrane potential and neuronal excitability were investigated in CA1 pyramidal neurons of rat hippocampal slices. SKF83959 (10–100 µM) caused a concentration-dependent depolarization, associated with a reduction of input resistance in CA1 pyramidal neurons. The depolarization was blocked neither by antagonists for D1, D2, 5-HT2A/2C receptors and α1-adrenoceptor, nor by intracellular dialysis of GDP-β-S. However, the specific HCN channel blocker ZD7288 (10 µM) antagonized both the depolarization and reduction of input resistance caused by SKF83959. In voltage-clamp experiments, SKF83959 (10–100 µM) caused a concentration-dependent increase of Ih current in CA1 pyramidal neurons, which was independent of D1 receptor activation. Moreover, SKF83959 (50 µM) caused a 6 mV positive shift in the activation curve of Ih and significantly accelerated the activation of Ih current. In addition, SKF83959 also reduced the neuronal excitability of CA1 pyramidal neurons, which was manifested by the decrease in the number and amplitude of action potentials evoked by depolarizing currents, and by the increase of firing threshold and rhoebase current. The above results suggest that SKF83959 increased Ih current through a D1 receptor-independent mechanism, which led to the depolarization of hippocampal CA1 pyramidal neurons. These findings provide a novel mechanism for the drug's neuroprotective effects, which may contributes to its therapeutic benefits in Parkinson's disease.
Caffeine is consumed worldwide to enhance wakefulness, but the cellular mechanisms are poorly understood. Caffeine blocks adenosine receptors suggesting that adenosine decreases cortical arousal. Given the widespread innervation of the cerebral cortex by thalamic fibers, adenosine receptors on thalamocortical terminals could provide an efficient method of limiting thalamic activation of the cortex. Using a thalamocortical slice preparation and whole-cell patch clamp recordings, we examined whether thalamocortical terminals are modulated by adenosine receptors. Bath application of adenosine decreased excitatory postsynaptic currents (EPSCs) elicited by stimulation of the ventrobasal thalamus. Thalamocortical synapses onto inhibitory and excitatory neurons were equally affected by adenosine. Adenosine also increased the paired pulse ratio and the coefficient of variation of the EPSCs, suggesting that adenosine decreased glutamate release. The inhibition produced by adenosine was reversed by a selective antagonist of adenosine A1 receptors (CPT) and mimicked by a selective A1 receptor agonist (CPA). Our results indicate that thalamocortical excitation is regulated by presynaptic adenosine A1 receptors and provide a mechanism by which increased adenosine levels can directly reduce cortical excitability.
somatosensory; glutamate; interneurons; spiny stellate cells
Metabotropic GABAB receptors play a fundamental role in modulating the excitability of neurons and circuits throughout the brain. These receptors influence synaptic transmission by inhibiting presynaptic release or activating postsynaptic potassium channels. However, their ability to directly influence different types of postsynaptic glutamate receptors remains unresolved. Here we examine GABAB receptor modulation in layer 2/3 pyramidal neurons from the mouse prefrontal cortex. We use two-photon laser-scanning microscopy to study synaptic modulation at individual dendritic spines. Using two-photon optical quantal analysis, we first demonstrate robust presynaptic modulation of multivesicular release at single synapses. Using two-photon glutamate uncaging, we then reveal that GABAB receptors strongly inhibit NMDA receptor calcium signals. This postsynaptic modulation occurs via the PKA pathway and does not affect synaptic currents mediated by AMPA or NMDA receptors. This novel form of GABAB receptor modulation has widespread implications for the control of calcium-dependent neuronal function.
GABAB receptor; NMDA receptor; PKA; prefrontal cortex; pyramidal neurons; dendrite; spine; two-photon microscopy; two-photon uncaging
Excess glutamate release and stimulation of post-synaptic glutamatergic receptors have been implicated in the pathophysiology of many neurological diseases. The hippocampus, and the pyramidal cell layer of the cornu ammonus 1 (CA1) region in particular, has been noted for its selective sensitivity to excitotoxic insults. The current studies examined the role of N-methyl-D-aspartate (NMDA) receptor subunit composition and sensitivity to stimulatory effects of the polyamine spermidine, an allosteric modulator of NMDA NR2 subunit activity, in hippocampal CA1 region sensitivity to excitotoxic insult. Organotypic hippocampal slice cultures of 8 day-old neonatal rat were obtained and maintained in vitro for 5 days. At this time, immunohistochemical analysis of mature neuron density (NeuN); microtubule associated protein-2(a,b) density (MAP-2); and NMDA receptor NR1 and NR2B subunit density in the primary cell layers of the dentate gyrus (DG), CA3, and CA1 regions, was conducted. Further, autoradiographic analysis of NMDA receptor distribution and density (i.e. [125I]MK-801 binding) and spermidine (100 μM)-potentiated [125I]MK-801 binding in the primary cell layers of these regions was examined. A final series of studies examined effects of prolonged exposure to NMDA (0.1–10 μM) on neurodegeneration in the primary cell layers of the DG, CA3, and CA1 regions, in the absence and presence of spermidine (100 μM) or ifenprodil (100 μM), an allosteric inhibitor of NR2B polypeptide subunit activity. The pyramidal cell layer of the CA1 region demonstrated significantly greater density of mature neurons, MAP-2, NR1 and NR2B subunits, and [125I]MK-801 binding than the CA3 region or DG. Twenty-four hour NMDA (10 μM) exposure produced marked neurodegeneration (~350% of control cultures) in the CA1 pyramidal cell region that was significantly reduced by co-exposure to ifenprodil or APV. The addition of spermidine significantly potentiated [125I]MK-801 binding and neurodegeneration induced by exposure to a non-toxic concentration of NMDA, exclusively in the CA1 region. This neurodegeneration was markedly reduced with co-exposure to ifenprodil. These data suggest that selective sensitivity of the CA1 region to excitotoxic stimuli may be attributable to the density of mature neurons expressing polyamine-sensitive NR2B polypeptide subunits.
glutamate; calcium; head injury; amino acid; spermidine
Corticotropin-releasing hormone (CRH) excites hippocampal neurons and induces death of selected CA3 pyramidal cells in immature rats. These actions of CRH require activation of specific receptors that are abundant in CA3 during early postnatal development. Given the dramatic effects of CRH on hippocampal neurons and the absence of CRH-containing afferents to this region, we hypothesized that a significant population of CRHergic neurons exists in developing rat hippocampus. This study defined and characterized hippocampal CRH-containing cells by using immunocytochemistry, ultrastructural examination, and colocalization with gamma-aminobutyric acid (GABA)-synthesizing enzyme and calcium-binding proteins. Numerous, large CRH-immunoreactive (ir) neurons were demonstrated in CA3 strata pyramidale and oriens, fewer were observed in the corresponding layers of CA1, and smaller CRH-ir cells were found in stratum lacunosum-moleculare of Ammon's horn. In the dentate gyrus, CRH-ir somata resided in the granule cell layer and hilus. Ultrastructurally, CRH-ir neurons had aspiny dendrites and were postsynaptic to both asymmetric and symmetric synapses. CRH-ir axon terminals formed axosomatic and axodendritic symmetric synapses with pyramidal and granule cells. Other CRH-ir terminals synapsed on axon initial segments of principal neurons. Most CRH-ir neurons were coimmunolabeled for glutamate decarboxylase (GAD)-65 and GAD-67 and the majority also contained parvalbumin, but none were labeled for calbindin. These results confirm the identity of hippocampal CRH-ir cells as GABAergic interneurons. Further, a subpopulation of neurons immunoreactive for both CRH and parvalbumin and located within and adjacent to the principal cell layers consists of basket and chandelier cells. Thus, axon terminals of CRH-ir interneurons are strategically positioned to influence the excitability of the principal hippocampal neurons via release of both CRH and GABA.
hippocampus; interneurons; neuropeptides; parvalbumin; development
SK2 potassium channels control excitability and contribute to plasticity by reducing excitatory postsynaptic potentials. Recent evidence suggests that SK2 channels form a calcium-dependent negative-feedback loop with synaptic NMDA receptors. Addiction to alcohol and other drugs of abuse induces plastic changes in glutamatergic synapses that include the targeting of NMDA receptors to synaptic sites; however, the role of SK2 channels in alcohol-associated homeostatic plasticity is unknown.
Electrophysiology, Western blot, and behavioral analyses were used to quantify changes in hippocampal SK channel function and expression using well-characterized in-vitro and in-vivo models of chronic alcohol exposure.
Chronic ethanol reduced apamin-sensitive SK currents in CA1 pyramidal neurons that were associated with a down-regulation of surface SK2 channels. Blocking SK channels with apamin potentiated excitatory post-synaptic potentials in control but not ethanol treated CA1 pyramidal neurons, suggesting that chronic ethanol disrupts the SK channel-NMDA receptor feedback loop. Alcohol reduced expression of SK2 channels and increased expression of NMDA receptors at synaptic sites in a mouse model. Positive modulation of SK function by 1-EBIO decreased alcohol withdrawal hyperexcitability and attenuated ethanol withdrawal neurotoxicity in hippocampus. 1-EBIO also reduced seizure activity in mice undergoing withdrawal.
These results provide evidence that SK2 channels contribute to alcohol-associated adaptive plasticity of glutamatergic synapses and that positive modulation of SK channels reduces the severity of withdrawal-related hyperexcitability. Therefore, SK2 channels appear to be critical regulators of alcohol-associated plasticity and may be novel therapeutic targets for the treatment of addiction.
SK2; adaptive plasticity; alcoholism; glutamatergic synapses; withdrawal hyperexcitability; 1-EBIO
Astrocytes display excitability in the form of intracellular calcium concentration ([Ca 2+]i) increases, but the signaling impact of these for neurons remains debated and controversial. A key unresolved issue is whether astrocyte [Ca2+]i elevations impact neurons or not. Here we report that in the CA1 region of the hippocampus, agonists of native P2Y1 and PAR-1 receptors, which are preferentially expressed in astrocytes, equally elevated [Ca2+]i levels without affecting the passive membrane properties of pyramidal neurons. However, under conditions chosen to isolate NMDA receptor responses, we found that activation of PAR-1 receptors led to the appearance of NMDA receptor-mediated slow inward currents (SICs) in pyramidal neurons. In stark contrast, activation of P2Y1 receptors was ineffective in this regard. The PAR-1 receptor-mediated increased SICs were abolished by several strategies that selectively impaired astrocyte [Ca2+]i excitability and function. Our studies therefore indicate that evoked astrocyte [Ca2+]i transients are not a binary signal for interactions with neurons, and that astrocytes result in neuronal NMDA receptor-mediated SICs only when appropriately excited. The data thus provide a basis to rationalize recent contradictory data on astrocyte–neuron interactions.
astrocyte; calcium; SIC; gliotransmitter; astrocytic glutamate release; glia
The hyperpolarization-activated cation current IH regulates the electrical activity of many excitable cells, but its precise function varies across cell types. The antiepileptic drug lamotrigine (LTG) recently was shown to enhance IH in hippocampal CA1 pyramidal neurons, revealing a potential anticonvulsant mechanism, as IH can dampen dendrito-somatic propagation of excitatory postsynaptic potentials in these cells. However, IH also is expressed in many hippocampal interneurons that provide synaptic inhibition to CA1 pyramidal neurons, and thus, IH modulation may indirectly regulate inhibitory control of principal cells via direct modulation of interneuron activity. Whether IH in hippocampal interneurons is sensitive to modulation by LTG, and how this may affect synaptic inhibition of pyramidal cells has not been investigated. In this study, we examined the effects of LTG on IH and spontaneous firing of area CA1 s.o. interneurons, and on spontaneous inhibitory postsynaptic currents (sIPSCs) in CA1 pyramidal neurons in immature rat brain slices. LTG (100 µM) significantly increased IH in the majority of interneurons, and depolarized interneurons from rest, promoting spontaneous firing. LTG also caused an increase in the frequency of spontaneous (but not miniature) IPSCs in pyramidal neurons without significantly altering amplitudes or rise and decay times. These data indicate that IH in CA1 interneurons can be increased by LTG, similarly to IH in pyramidal neurons, that IH enhancement increases interneuron excitability, and that these effects are associated with increased basal synaptic inhibition of CA1 pyramidal neurons.
Anticonvulsant; GABA; Interneuron; Patch-clamp; Epilepsy; H-current
The hyperpolarization-activated cation current (IH) regulates the electrical activity of many excitable cells, but its precise function varies across cell types. The antiepileptic drug lamotrigine (LTG) was recently shown to enhance IH in hippocampal CA1 pyramidal neurons, showing a potential anticonvulsant mechanism, as IH can dampen dendrito-somatic propagation of excitatory postsynaptic potentials in these cells. However, IH is also expressed in many hippocampal interneurons that provide synaptic inhibition to CA1 pyramidal neurons, and thus, IH modulation may indirectly regulate the inhibitory control of principal cells by direct modulation of interneuron activity. Whether IH in hippocampal interneurons is sensitive to modulation by LTG, and the manner by which this may affect the synaptic inhibition of pyramidal cells has not been investigated. In this study, we examined the effects of LTG on IH and spontaneous firing of area CA1 stratum oriens interneurons, as well as on spontaneous inhibitory postsynaptic currents in CA1 pyramidal neurons in immature rat brain slices. LTG (100 μM) significantly increased IH in the majority of interneurons, and depolarized interneurons from rest, promoting spontaneous firing. LTG also caused an increase in the frequency of spontaneous (but not miniature) IPSCs in pyramidal neurons without significantly altering amplitudes or rise and decay times. These data indicate that IH in CA1 interneurons can be increased by LTG, similarly to IH in pyramidal neurons, that IH enhancement increases interneuron excitability, and that these effects are associated with increased basal synaptic inhibition of CA1 pyramidal neurons.
anticonvulsant; GABA; interneuron; patch-clamp; epilepsy; H-current
Place fields of hippocampal pyramidal cells expand asymmetrically when adult rats repeatedly follow the same route. This behaviorally-induced expression of neuronal plasticity utilizes an NMDAR-dependent, LTP-like mechanism and could be used by hippocampal networks to store information. Aged spatial memory-impaired rats exhibit defective experience-dependent place field expansion plasticity. One possible explanation for this aged-associated deficit is alterations in glutamatergic function. In fact, both NMDAR- and AMPAR-mediated field excitatory postsynaptic potentials in CA1 decrease with aging. The current study investigated whether modulation of either AMPA or NDMA receptor activity could restore this experience-dependent plasticity by prolonging AMPAR activity with the ampakine CX516, and modulating the NMDAR with the noncompetitive antagonist memantine. The spatial firing characteristics of multiple CA1 pyramidal cells were monitored under both treatment conditions as aged rats repeatedly traversed a circular track. Compared to the saline baseline condition, acute administration of memantine but not CX516, reinstated experience-dependent place field expansion. Taken together, these data suggest that pharmacological manipulation of the NMDAR can improve the function of hippocampal networks critical to optimal cognition in aging.
aging; CA1; hippocampus; place cell; theta phase precession
The sodium-potassium ATPase (i.e., the “sodium pump”) plays a central role in maintaining ionic homeostasis in all cells. Although the sodium pump is intrinsically electrogenic and responsive to dynamic changes in intracellular sodium concentration, its role in regulating neuronal excitability remains unclear. Here we describe a physiological role for the sodium pump in regulating the excitability of mouse neocortical layer 5 and hippocampal CA1 pyramidal neurons. Trains of action potentials produced long-lasting (∼20 s) afterhyperpolarizations (AHPs) that were insensitive to blockade of voltage-gated calcium channels or chelation of intracellular calcium, but were blocked by tetrodotoxin, ouabain, or the removal of extracellular potassium. Correspondingly, the AHP time course was similar to the decay of activity-induced increases in intracellular sodium, whereas intracellular calcium decayed at much faster rates. To determine whether physiological patterns of activity engage the sodium pump, we replayed in vitro a place-specific burst of 15 action potentials recorded originally in vivo in a CA1 “place cell” as the animal traversed the associated place field. In both layer 5 and CA1 pyramidal neurons, this “place cell train” generated small, long-lasting AHPs capable of reducing neuronal excitability for many seconds. Place-cell-train-induced AHPs were blocked by ouabain or removal of extracellular potassium, but not by intracellular calcium chelation. Finally, we found calcium contributions to the AHP to be temperature dependent: prominent at room temperature, but largely absent at 35°C. Our results demonstrate a previously unappreciated role for the sodium-potassium ATPase in regulating the excitability of neocortical and hippocampal pyramidal neurons.
Corticosteroids regulate gene expression through the activation of mineralocorticoid and glucocorticoid receptors. The hippocampus contains the highest density of mineralocorticoid and glucocorticoid receptors in the central nervous system. The modulation of neuron excitability by corticosteroids in hippocampal subfield CA1 is well documented. However, it is not known whether corticosteroids produce different effects across the various hippocampal subfields. Therefore, we used intracellular recording techniques to examine the actions of chronic corticosteroid treatment (2 weeks) on the electrophysiological properties of rat hippocampal subfield CA3 pyramidal cells. The treatment groups used in this investigation were: adrenalectomy (ADX), selective mineralocorticoid receptor activation with aldosterone (ALD), mineralocorticoid and glucocorticoid receptor activation with high levels of corticosterone (HCT), and SHAM. Corticosteroid treatment altered the percentage of nonburst and burst firing neurons. The percentages of nonbursting cells were 74 and 62% in tissue from ADX and HCT animals compared to 42 and 41% in ALD and SHAM animals, respectively. The corticosteroid-induced effect on the ratio of nonbursting to bursting cells does not appear to be secondary to changes in the cell's membrane input resistance, resting potential, time constant, action potential, slow-or fast-afterhyperpolarizing potential properties. Based on these results we conclude that corticosteroids are important for maintaining the ratio of nonburst and burst firing pyramidal neurons in subfield CA3. These novel results are distinct from those previously reported for subfield CA1, suggesting that corticosteroids have different effects across hippocampal subfields.
Adrenal steroids; Hippocampus; Adrenal steroid receptors; Electrophysiology
In the hippocampus, the inhibitory neurotransmitter GABA shapes the activity of the output pyramidal neurons and plays important role in cognition. Most of its inhibitory effects are mediated by signaling from GABAB receptor to the G protein-gated Inwardly-rectifying K+ (GIRK) channels. Here, we show that RGS7, in cooperation with its binding partner R7BP, regulates GABABR-GIRK signaling in hippocampal pyramidal neurons. Deletion of RGS7 in mice dramatically sensitizes GIRK responses to GABAB receptor stimulation and markedly slows channel deactivation kinetics. Enhanced activity of this signaling pathway leads to decreased neuronal excitability and selective disruption of inhibitory forms of synaptic plasticity. As a result, mice lacking RGS7 exhibit deficits in learning and memory. We further report that RGS7 is selectively modulated by its membrane anchoring subunit R7BP, which sets the dynamic range of GIRK responses. Together, these results demonstrate a novel role of RGS7 in hippocampal synaptic plasticity and memory formation.
Neurons communicate with one another at junctions called synapses. The arrival of an electrical signal known as an action potential at the first cell causes molecules known as neurotransmitters to be released into the synapse. These molecules diffuse across the gap between the neurons and bind to receptors on the receiving cell. Some neurotransmitters, such as glutamate, activate cells when they bind to receptors, thus making it easier for the second neuron to ‘fire’ (i.e., to generate an action potential). By contrast, other neurotransmitters, such as GABA, usually make it harder for the second neuron to fire.
Many of the effects of GABA involve a type of receptor called GABAB. When GABA binds to one of these receptors, a molecule called a G-protein is recruited to the receptor. This activates the G-protein, triggering a cascade of events inside the cell that lead ultimately to the opening of potassium ion channels, which as known as GIRKs, in the cell membrane. Positively charged potassium ions then leave the cell through these channels, and this makes it more difficult for the cell to fire.
Now, Ostrovskaya et al. have revealed that a complex of three proteins regulates the interaction between GABAB receptors and GIRK channels. In neurons that lack either of these proteins, the receptors have less influence on GIRKs than in normal cells. Moreover, mice that lack one of the proteins (called RGS7) perform less well in various learning and memory tests: for example, they take longer than normal animals to learn the location of an escape platform in a water maze, or to retain a memory of a fearful event.
By identifying the proteins that regulate the interaction between GABAB receptors and GIRKs, Ostrovskaya et al. have helped to unravel a key signaling cascade relevant to cognition. Given that GIRK channels have recently been implicated in Down’s syndrome, these insights may also increase understanding of cognitive impairments in neuropsychiatric disorders.
GPCR signaling; RGS proteins; synaptic plasticity; learning and memory; hippocampus; GIRK channels; mouse
Salvinorin A is a nonopioid, selective κ opioid–receptor agonist. Despite its high potential for clinical application, its pharmacologic profile is not well known. In the current study, we hypothesized that salvinorin A dilates pial arteries via activation of nitric oxide synthase, adenosine triphosphate–sensitive potassium channels, and opioid receptors.
Cerebral artery diameters and cyclic guanosine monophosphate in cortical periarachnoid cerebrospinal fluid were monitored in piglets equipped with closed cranial windows. Observation took place before and after salvinorin A administration in the presence or absence of an opioid antagonist (naloxone), a κ opioid receptor–selective antagonist (norbinaltorphimine), nitric oxide synthase inhibitors (N(G)-nitro-L-arginine and 7-nitroindazole), a dopamine receptor D2 antagonist (sulpiride), and adenosine triphosphate–sensitive potassium and Ca2+-activated K channel antagonists (glibenclamide and iberiotoxin). The effects of salvinorin A on the constricted cerebral artery induced by hypocarbia and endothelin were investigated. Data were analyzed by repeated measures ANOVA (n = 5) with statistical significance set at a P value of less than 0.05.
Salvinorin A induced immediate but brief vasodilatation that was sustained for 30 min via continual administration every 2 min. Vasodilatation and the associated cyclic guanosine monophosphate elevation in cerebrospinal fluid were abolished by preadministration N(G)-nitro-L-arginine, but not 7-nitroindazole. Although naloxone, norbinaltorphimine, and glibenclamide abolished salvinorin A–induced cerebrovasodilation, this response was unchanged by iberiotoxin and sulpiride. Hypocarbia and endothelin-constricted pial arteries responded similarly to salvinorin A, to the extent observed under resting tone.
Salvinorin A dilates cerebral arteries via activation of nitric oxide synthase, adenosine triphosphate–sensitive potassium channel, and the κ opioid receptor.