The level of expression of functional α7-containing nicotinic acetylcholine receptors (nAChRs) in hippocampal CA1 pyramidal neurons is believed to be very low compared to hippocampal CA1 interneurons, and for many years this expression was largely overlooked. However, high densities of expression of functional α7-containing nAChRs in CA1 pyramidal neurons may not be necessary for triggering important cellular and network functions, especially if activation of α7-containing nAChRs occurs in the presence of positive allosteric modulators such as PNU-120596.
An approach previously developed for α7-containing nAChRs expressed in tuberomammillary neurons was applied to investigate functional CA1 pyramidal α7-containing nAChRs using rat coronal hippocampal slices and patch-clamp electrophysiology. The majority (∼71%) of tested CA1 pyramidal neurons expressed low densities of functional α7-containing nAChRs as evidenced by small whole-cell responses to choline, a selective endogenous agonist of α7 nAChRs. These responses were potentiated by PNU-120596, a novel positive allosteric modulator of α7 nAChRs. The density of functional α7-containing nAChRs expressed in CA1 pyramidal neurons (and thus, the normalized net effect of activation, i.e., response net charge per unit of membrane capacitance per unit of time) was estimated to be ∼5% of the density observed in CA1 interneurons. The results of this study demonstrate that despite low levels of expression of functional pyramidal α7-containing nAChRs, physiological levels of choline (∼10 µM) are sufficient to activate these receptors and transiently depolarize and even excite CA1 pyramidal neurons in the presence of PNU-120596. The observed effects are possible because in the presence of 10 µM choline and 1–5 µM PNU-120596, a single opening of an individual pyramidal α7-containing nAChR ion channel appears to transiently depolarize (∼4 mV) the entire pyramidal neuron and occasionally trigger action potentials.
1) The majority of hippocampal CA1 pyramidal neurons express functional α7-containing nAChRs. In the absence of PNU-120596, a positive allosteric modulator of α7 nAChRs, a lack of responsiveness of some hippocampal CA1 pyramidal neurons to focal application of 0.5–1 mM choline does not imply a lack of expression of functional α7-containing nAChRs in these neurons. Rather, it may indicate a lack of detection of α7-containing nAChR-mediated currents by patch-clamp electrophysiology. 2) PNU-120596 can serve as a powerful tool for detection and enhancement of responsiveness of low densities of functional α7-containing nAChRs such as those present in hippocampal CA1 pyramidal neurons. 3) In the presence of PNU-120596, physiological concentrations of choline activate functional CA1 pyramidal α7-containing nAChRs and produce step-like currents that cause repetitive step-like depolarizations, occasionally triggering bursts of action potentials in CA1 pyramidal neurons. Therefore, the results of this study suggest that in the presence of PNU-120596 and possibly other positive allosteric modulators, endogenous choline may persistently activate CA1 pyramidal α7-containing nAChRs, enhance the excitability of CA1 pyramidal neurons and thus act as a potent therapeutic agent with potential neuroprotective and cognition-enhancing properties.
The basolateral amygdala plays a critical role in the etiology of anxiety disorders and addiction. Pyramidal neurons, the primary output cells of this region, display increased firing following exposure to stressors, and it is thought that this increase in excitability contributes to stress responsivity and the expression of anxiety-like behaviors. However, much remains unknown about the underlying mechanisms that regulate the intrinsic excitability of basolateral amygdala pyramidal neurons.
Ex vivo gramicidin perforated patch recordings were conducted in current clamp mode where hyper- and depolarizing current steps were applied to basolateral amygdala pyramidal neurons to assess the effects of adenosine A2A receptor modulation on intrinsic excitability.
Activation of adenosine A2A receptors with the selective A2A receptor agonist CGS-21680 significantly increased the firing rate of basolateral amygdala pyramidal neurons in rat amygdala brain slices, likely via inhibition of the slow afterhyperpolarization potential. Both of these A2A receptor-mediated effects were blocked by preapplication of a selective A2A receptor antagonist (ZM-241385) or by intra-pipette infusion of a protein kinase A inhibitor, suggesting a postsynaptic locus of A2A receptors on basolateral amygdala pyramidal neurons. Interestingly, bath application of the A2A receptor antagonist alone significantly attenuated basolateral amygdala pyramidal cell firing, consistent with a role for tonic adenosine in the regulation of the intrinsic excitability of these neurons.
Collectively, these data suggest that adenosine, via activation of A2A receptors, may directly facilitate basolateral amygdala pyramidal cell output, providing a possible balance for the recently described inhibitory effects of adenosine A1 receptor activation on glutamatergic excitation of basolateral amygdala pyramidal cells.
basolateral amygdala; intrinsic excitability; adenosine; A2A; AHP
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
Corticotropin-releasing hormone (CRH) plays an important role in a substantial number of patients with stress-related mental disorders, such as anxiety disorders and depression. CRH has been shown to increase neuronal excitability in the hippocampus, but the underlying mechanisms are poorly understood. The effects of CRH on neuronal excitability were investigated in acute hippocampal brain slices. Population spikes (PS) and field excitatory postsynaptic potentials (fEPSP) were evoked by stimulating Schaffer-collaterals and recorded simultaneously from the somatic and dendritic region of CA1 pyramidal neurons. CRH was found to increase PS amplitudes (mean ± Standard error of the mean; 231.8 ± 31.2% of control; n = 10) while neither affecting fEPSPs (104.3 ± 4.2%; n = 10) nor long-term potentiation (LTP). However, when Schaffer-collaterals were excited via action potentials (APs) generated by stimulation of CA3 pyramidal neurons, CRH increased fEPSP amplitudes (119.8 ± 3.6%; n = 8) and the magnitude of LTP in the CA1 region. Experiments in slices from transgenic mice revealed that the effect on PS amplitude is mediated exclusively by CRH receptor 1 (CRHR1) expressed on glutamatergic neurons. The effects of CRH on PS were dependent on phosphatase-2B, L- and T-type calcium channels and voltage-gated potassium channels but independent on intracellular Ca2+-elevation. In patch-clamp experiments, CRH increased the frequency and decay times of APs and decreased currents through A-type and delayed-rectifier potassium channels. These results suggest that CRH does not affect synaptic transmission per se, but modulates voltage-gated ion currents important for the generation of APs and hence elevates by this route overall neuronal activity.
CRH; CRH receptor; neuronal excitability; potassium channels; protein kinases
Background and purpose:
α7 Nicotinic receptors have been suggested to play an important role in hippocampal learning and memory. However, the direct action of this receptor subtype on hippocampal pyramidal neurones in vivo has not yet been fully investigated. The availability of selective agonists for α7 receptors [AR-R17779 and (R)-(-)-5′-phenylspiro[1-azabicyclo[2.2.2] octane-3,2′-(3′H)furo[2,3-b]pyridine (PSAB-OFP)] has now allowed this role to be investigated.
Single-cell extracellular recordings were made from hippocampal CA3 pyramidal neurones in anaesthetized rats. The effects of nicotine, AR-R17779 and PSAB-OFP, applied either systemically or iontophoretically, were studied on the activity of these neurones.
Intravenous injection of cumulative doses of nicotine and PSAB-OFP induced dose-related, significant increases in neuronal firing in the majority of neurones tested. This excitation could be inhibited by intravenous administration of methyllycaconitine (MLA), a selective α7 nicotinic receptor antagonist. Furthermore, iontophoretic application of nicotine, AR-R17779 and PSAB-OFP each evoked current-dependent excitation of most CA3 pyramidal neurones studied, and this excitation was antagonized by co-iontophoretic application of MLA. In addition, the excitation induced by iontophoretic application of nicotine, AR-R17779 or PSAB-OFP was also blocked by co-iontophoretic application of either 6,7-dinitroquinoxaline-2,3-dione (DNQX) or D(2)-2-amino-5-phosphonopentanoate (D-AP5), selective N-methyl-D-aspartic acid (NMDA) and non-NMDA receptor antagonists respectively.
Conclusions and implications:
CA3 pyramidal neurones are modulated by activation of presynaptic α7 nicotinic receptors, which, at least in part, enhances glutamate release onto post-synaptic (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid and NMDA receptors on these CA3 neurones. This mechanism probably contributes to the effects of nicotine on hippocampal learning and memory.
presynaptic α7 nAChR; nicotine; AR-R17779; PSAB-OFP; hippocampus; CA3; iontophoresis; rat
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
1. The effects of the mixed A1 and A2 adenosine receptor agonist N6-L-phenyl-isopropyladenosine (L-PIA) were tested on ischaemia-induced hippocampal neuronal injury in gerbils subjected to 5-min bilateral carotid occlusion. For comparison, the effects of the selective A2 adenosine receptor agonist, CGS 21680 were tested. 2. Five-min bilateral carotid occlusion produced within 1 week an irreversible suppression of the CA1, but not of the dentate extracellular electrical somatic responses, in 30% of gerbil hippocampal slices with respect to controls. In addition, a significant reduction occurred in the density of CA1 hippocampal pyramidal neurones but not of dentate granule cells with respect to controls. 3. Injection 1 h before or after bilateral carotid occlusion of L-PIA (0.8-1.5 mg kg-1, i.p.) but not of CGS 21680 (5 mg kg-1, i.p.), significantly prevented the irreversible disappearance of the CA1 extracellular electrical somatic responses with respect to controls. In addition, the CA1 pyramidal neuronal loss was also prevented. 4. The results show that activation of A1 adenosine receptors is able to prevent or block the electrophysiological and morphological correlates of hippocampal neuronal injury after global ischaemia in the gerbil, suggesting that adenosine receptor agonists might have a useful role in the treatment of neuronal functional and anatomical injury due to ischaemia.
Extracellular adenosine, a key regulator of neuronal excitability, is metabolized by astrocyte-based enzyme adenosine kinase (ADK). We hypothesized that ADK might be an upstream regulator of adenosine-based homeostatic brain functions by simultaneously affecting several downstream pathways. We therefore studied the relationship between ADK expression, levels of extracellular adenosine, synaptic transmission, intrinsic excitability, and brain-derived neurotrophic factor (BDNF)-dependent synaptic actions in transgenic mice underexpressing or overexpressing ADK. We demonstrate that ADK: 1) Critically influences the basal tone of adenosine, evaluated by microelectrode adenosine biosensors, and its release following stimulation; 2) determines the degree of tonic adenosine-dependent synaptic inhibition, which correlates with differential plasticity at hippocampal synapses with low release probability; 3) modulates the age-dependent effects of BDNF on hippocampal synaptic transmission, an action dependent upon co-activation of adenosine A2A receptors; and 4) influences GABAA receptor-mediated currents in CA3 pyramidal neurons. We conclude that ADK provides important upstream regulation of adenosine-based homeostatic function of the brain and that this mechanism is necessary and permissive to synaptic actions of adenosine acting on multiple pathways. These mechanistic studies support previous therapeutic studies and implicate ADK as a promising therapeutic target for upstream control of multiple neuronal signaling pathways crucial for a variety of neurological disorders.
adenosine; brain-derived neurotrophic factor; GABA; homeostasis; transgenic mice
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.
The pharmacological effects of opioid- and adenosine-receptor agonists on neural signalling were investigated by measuring drug actions on barium current flowing through calcium channels in acutely-dissociated neurons of the rat nucleus accumbens (NAc).Under whole-cell voltage clamp, opioids acted via μ, but not δ or κ, receptors to partially inhibit barium current. Mean inhibition was 35±2% (±s.e.mean, n=33) for methionine-enkephalin and 37±1% (n=65) for the selective μ receptor agonist DAMGO, both measured at saturating agonist concentrations in neurons with diameter ⩾20 μm. EC50 for DAMGO was 100 nM. Perfusion of naloxone reversed the current inhibition by DAMGO.Adenosine also partially inhibited barium current in these neurons. Mean inhibition was 28±2% (n=29) for adenosine and 33±3% (n=27) for the selective A1 receptor agonist N6CPA, both at saturating concentrations in neurons with diameter ⩾20 μm. EC50 for N6CPA was 34 nM. Adenosine inhibition was reversed by perfusion of an A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine, while the selective A2A receptor agonist, CGS 21680, had no effect.Inhibition by opioids and adenosine was mutually occlusive, suggesting a converging pathway onto calcium channels.These actions involved a G-protein-coupled mechanism, as demonstrated by the partial relief of inhibition by strong depolarization and by the application of N-ethylmaleimide or GTP-γ-S.Inhibition of barium current by opioids had their greatest effect in large neurons, that is, in presumed interneurons. In contrast, opioid inhibition in neurons with diameter ⩽15 μm was 11±2% (n=26) for methionine-enkephalin and 11±4% (n=17) for DAMGO, both measured at saturating agonist concentrations. Adenosine inhibition in neurons with diameter ⩽15 μm was 22±5% (n=9).These results implicate the interneurons as a locus for the modulation of the excitability of projection neurons in the NAc during the processes of addiction and withdrawal.
Opioid; DAMGO; nucleus accumbens; calcium channel; opioid withdrawal; interneuron; adenosine
SUMMARY AND CONCLUSIONS
The role of synaptic activation of NMDA (N-methyl-d-aspartate) receptor-mediated conductances on CA1 hippocampal pyramidal cells in short-term excitability changes was studied with the use of a computational model. Model parameters were based on experimental recordings from dendrites and somata and previous hippocampal simulations. Representation of CA1 neurons included NMDA and non-NMDA excitatory dendritic synapses, dendritic and somatic inhibition, five intrinsic membrane conductances, and provision for activity-dependent intracellular and extracellular ion concentration changes.The model simulated somatic and dendritic potentials recorded experimentally. The characteristic CA1 spike afterdepolarization was a consequence of the longitudinal spread of dendritic charge, reactivation of slow Ca2+-dependent K+ conductances, slow synaptic processes (NMDA-dependent depolarizing and γ-aminobutyric acid–mediated hyperpolarizing currents) and was sensitive to extracellular potassium accumulation. Calcium currents were found to be less important in generating the spike afterdepolarization.Repetitive activity was influenced by the cumulative activation of the NMDA-mediated synaptic conductances, the frequency-dependent depression of inhibitory synaptic responses, and a shift in the potassium reversal potential. NMDA receptor activation produced a transient potentiation of the excitatory postsynaptic potential (EPSP). The frequency dependence of EPSP potentiation was similar to the experimental data, reaching a maximal value near 10 Hz.Although the present model did not have compartments for dendritic spines, Ca2+ accumulation was simulated in a restricted space near the intracellular surface of the dendritic membrane. The simulations demonstrated that the Ca2+ component of the NMDA-operated synaptic current can be a significant factor in increasing the Ca2+ concentration at submembrane regions, even in the absence of Ca2+ spikes.Elevation of the extracellular K+ concentration enhanced the dendritic synaptic response during repetitive activity and led to an increase in intracellular Ca2+ levels. This increase in dendritic excitability was partly mediated by NMDA receptor-mediated conductances.Blockade of Ca2+-sensitive K+ conductances in the dendrites increased the size of EPSPs leading to a facilitation of dendritic and somatic spike activity and increased [Ca2+]i. NMDA receptor-mediated conductances appeared as an amplifying component in this mechanism, activated by the relatively depolarized membrane potential.The results suggest that dendritic NMDA receptors, by virtue of their voltage-dependency, can interact with a number of voltage-sensitive conductances to increase the dendritic excitatory response during periods of repetitive synaptic activation. These findings support experimental results that implicate NMDA receptor-mediated conductances in the short-term response plasticity of the CA1 hippocampal pyramidal neuron.
The efficacy, potency, and selectivity of the compound 2-Chloro-5-hydroxyphenylglycine (CHPG), a nominally selective agonist for metabotropic glutamate receptor 5 (mGluR5), were examined with select mGluRs by examining their ability to induce modulation of the native voltage dependent ion channels in isolated sympathetic neurons from the rat superior cervical ganglion (SCG). SCG neurons offer a null mGluR-background in which specific mGluR subtypes can be made to express via intranuclear cDNA injection.
Consistent with previous reports, CHPG strongly activated mGluR5b expressed in SCG neurons with an apparent EC50 around 60 μM. Surprisingly, CHPG also activated two mGluR1 splice variants with a similar potency as at mGluR5 when calcium current inhibition was used as an assay for receptor function. No effect of 1 mM CHPG was seen in cells expressing mGluR2 or mGluR4, suggesting that CHPG only activates group I mGluRs (mGluR1 and 5). CHPG was also able to induce modulation of M-type potassium current through mGluR1, but not as consistently as glutamate. Since this channel is modulated through a Gq-dependent pathway, these data indicate that CHPG may exhibit some biased agonist properties on mGluR1. Closer examination of the voltage-independent, Gq-mediated component of mGluR-induced calcium current modulation data confirmed that some biased agonism was evident, but the effect was weak and inconsistent.
These data contrast with the established literature which suggests that CHPG is a selective mGluR5 agonist. Instead, CHPG appears to act equally well as an agonist at mGluR1. While some weak biased agonism was observed with CHPG acting on mGluR1, but not mGluR5, favoring Gi/o signaling over Gq/11, this effect does not appear sufficient to fully explain the discrepancies in the literature.
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
The medial prefrontal cortex (mPFC) is a region of neocortex that plays an integral role in several cognitive processes which are abnormal in schizophrenic patients. As with other cortical regions, large‐bodied layer 5 pyramidal neurons serve as the principle subcortical output of microcircuits of the mPFC. The coexpression of both inhibitory serotonin 5‐HT1A receptors on the axon initial segments, and excitatory 5‐HT2A receptors throughout the somatodendritic compartments, by layer 5 pyramidal neurons allows serotonin to provide potent top–down regulation of input–output relationships within cortical microcircuits. Application of 5‐HT2A agonists has previously been shown to enhance synaptic input to layer 5 pyramidal neurons, as well as increase the gain in neuronal firing rate in response to increasing depolarizing current steps. Using whole‐cell patch‐clamp recordings obtained from layer 5 pyramidal neurons of the mPFC of C57/bl6 mice, the aim of our present study was to investigate the modulation of long‐term spike trains by the selective 5‐HT2A agonist TCB‐2. We found that in the presence of synaptic blockers, TCB‐2 induced recurrent oscillatory bursting (ROB) after 15–20 sec of tonic spiking in 7 of the 14 cells. In those seven cells, ROB discharge was accurately predicted by the presence of a voltage sag in response to a hyperpolarizing current injection. This effect was reversed by 5–10 min of drug washout and ROB discharge was inhibited by both synaptic activity and coapplication of the 5‐HT2A/2C antagonist ketanserin. While the full implications of this work are not yet understood, it may provide important insight into serotonergic modulation of cortical networks.
Using whole‐cell patch‐clamp recordings obtained from layer 5 pyramidal neurons of the mouse mPFC, we investigated the modulation of long‐term spike trains by the selective 5‐HT2A agonist TCB‐2. In the presence of synaptic blockers, TCB‐2 induced recurrent oscillatory bursting (ROB) after 15–20 sec of tonic spiking in 7 of the 14 cells; ROB discharge was accurately predicted by the presence of a voltage sag in response to a hyperpolarizing current injection. We have identified a novel modulation of pyramidal neuron excitability by a 5HT receptor known to contribute to the pathophysiology of schizophrenia.
5‐HT2A; layer 5 pyramidal neuron; mPFC; serotonin; TCB‐2
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
Hippocampal CA1 pyramidal neurons receive two excitatory glutamatergic synaptic inputs: their most distal dendritic regions in the stratum lacunosum-moleculare (SLM) are innervated by the perforant path (PP), originating from layer III of the entorhinal cortex, while their more proximal regions of the apical dendrites in the stratum radiatum (SR) are innervated by the Schaffer-collaterals (SC), originating from hippocampal CA3 neurons. Endocannabinoids (eCBs) are naturally occurring mediators capable of modulating both GABAergic and glutamatergic synaptic transmission and plasticity via the CB1 receptor. Previous work on eCB modulation of excitatory synapses in the CA1 region largely focuses on the SC pathway. However, little information is available on whether and how eCBs modulate glutamatergic synaptic transmission and plasticity at PP synapses.
By employing somatic and dendritic patch-clamp recordings, Ca2+ uncaging, and immunostaining, we demonstrate that there are significant differences in low-frequency stimulation (LFS)- or DHPG-, an agonist of group I metabotropic glutamate receptors (mGluRs), induced long-term depression (LTD) of excitatory synaptic transmission between SC and PP synapses in the same pyramidal neurons. These differences are eliminated by pharmacological inhibition with selective CB1 receptor antagonists or genetic deletion of the CB1 receptor, indicating that these differences likely result from differential modulation via a CB1 receptor-dependent mechanism. We also revealed that depolarization-induced suppression of excitation (DSE), a form of short-term synaptic plasticity, and photolysis of caged Ca2+-induced suppression of Excitatory postsynaptic currents (EPSCs) were less at the PP than that at the SC. In addition, application of WIN55212 (WIN) induced a more pronounced inhibition of EPSCs at the SC when compared to that at the PP.
Our results suggest that CB1 dependent LTD and DSE are differentially expressed at the PP versus SC synapses in the same neurons, which may have an impact on synaptic scaling, integration and plasticity of hippocampal CA1 pyramidal neurons.
1. The potential neuroprotective actions of the A3 adenosine receptor (A3AR) were investigated using mice with functional deletions of the A3AR (A3AR−/−) in behavioral assessments of analgesia, locomotion, tests predictive of depression and anxiety, and the effects of mild hypoxia on cognition and neuronal survival.
2. Untreated A3AR−/− mice were tested in standard behavioral paradigms, including activity in the open field, performance in the hot-plate, tail-flick, tail-suspension, and swim tests, and in the elevated plus maze. In addition, mice were exposed repeatedly to a hypoxic environment containing carbon monoxide (CO). The cognitive effects of this treatment were assessed using the contextual fear conditioning test. After testing, the density of pyramidal neurons in the CA1, 2, and 3 subfields of the hippocampus was determined using standard histological and morphometric techniques.
3. A3AR−/− mice showed increased locomotion in the open field test, elevated plus maze (number of arm entries) and light/dark box (number of transitions). However, they spent more time immobile in two different tests of antidepressant activity (Swim and tail suspension tests). A3AR−/− mice also showed evidence of decreased nociception in the hot-plate, but not tail-flick tests. Further, A3AR−/− mice were more vulnerable to hippocampal pyramidal neuron damage following episodes of carbon monoxide (CO)-induced hypoxia. One week after exposure to CO a moderate loss of pyramidal neurons was observed in all hippocampal subfields of both wild-type (A3AR+/+) and A3AR−/− mice. However, the extent of neuronal death in the CA2–3 subfields was less pronounced in A3AR+/+ than A3AR−/− mice. This neuronal loss was accompanied by a decline in cognitive function as determined using contextual fear conditioning. These histological and cognitive changes were reproduced in wild-type mice by repeatedly administering the A3AR-selective antagonist MRS 1523 (5-propyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate 1 mg/kg i.p.).
4. These results indicate that pharmacologic or genetic suppression of A3AR function enhances some aspects of motor function and suppresses pain processing at supraspinal levels, while acting as a depressant in tests predictive of antidepressant action. Consistent with previous reports of the neuroprotective actions of A3AR agonists, A3AR−/− mice show an increase in neurodegeneration in response to repeated episodes of hypoxia.
Adenosine receptor; neurodegeneration; knockout mice; analgesia; depression
Disturbances of GABAergic inhibition are a major cause of epileptic seizures. GABA exerts its actions via ionotropic GABAA receptors and metabotropic G protein‐coupled GABAB receptors. Malfunction of GABAA inhibition has long been recognized in seizure genesis but the role of GABAB receptors in controlling seizure activity is still not well understood. Here, we examined the anticonvulsive, or inhibitory effects, of GABAB receptors in a mouse model of hippocampal kindling as well as mouse hippocampal slices through the use of GS 39783, a positive allosteric GABAB receptor modulator, and CGP 55845, a selective GABAB receptor antagonist. When administered via intraperitoneal injections in kindled mice, GS 39783 (5 mg/kg) did not attenuate hippocampal EEG discharges, but did reduce aberrant hippocampal spikes, whereas CGP 55845 (10 mg/kg) prolonged hippocampal discharges and increased spike incidences. When examined in hippocampal slices, neither GS 39783 at 5 μmol/L nor the GABAB receptor agonist baclofen at 0.1 μmol/L alone significantly altered repetitive excitatory field potentials, but GS 39783 and baclofen together reversibly abolished these field potentials. In contrast, CGP 55845 at 1 μmol/L facilitated induction and incidence of these field potentials. In addition, CGP 55845 attenuated the paired pulse depression of CA3 population spikes and increased the frequency of EPSCs in individual CA3 pyramidal neurons. Collectively, these data suggest that GABABB receptors regulate hippocampal hyperexcitability by inhibiting CA3 glutamatergic synapses. We postulate that positive allosteric modulation of GABAB receptors may be effective in reducing seizure‐related hyperexcitability.
GABAB positive modulator GS 39783 attenuated, whereas GABAB antagonist CGP55845 facilitated hippocampal EEG spikes in kindled mice and excitatory field potentials in hippocampal slices. We postulate that GABAB receptors may inhibit CA3 glutamate synapses and hence regulate hippocampal hyperexcitability.
Allosteric; EEG; epilepsy; hippocampus; kindling; mice; seizure; slices
The vasopressin 1b receptor (Avpr1b) is critical for social memory and social aggression in rodents, yet little is known about its specific roles in these behaviors. Some clues to Avpr1b function can be gained from its profile of expression in the brain, which is largely limited to the pyramidal neurons of the CA2 region of the hippocampus, and from experiments showing that inactivation of the gene or antagonism of the receptor leads to a reduction in social aggression. Here we show that partial replacement of the Avpr1b through lentiviral delivery into the dorsal CA2 region restored the probability of socially motivated attack behavior in total Avpr1b knockout mice, without altering anxiety-like behaviors. To further explore the role of the Avpr1b in this hippocampal region, we examined the effects of Avpr1b agonists on pyramidal neurons in mouse and rat hippocampal slices. We found that selective Avpr1b agonists induced significant potentiation of excitatory synaptic responses in CA2, but not in CA1 or in slices from Avpr1b knockout mice. In a way that is mechanistically very similar to synaptic potentiation induced by oxytocin, Avpr1b agonist-induced potentiation of CA2 synapses relies on NMDA receptor activation, calcium and calcium/calmodulin-dependent protein kinase II activity, but not on cAMP-dependent protein kinase activity or presynaptic mechanisms. Our data indicate that the hippocampal CA2 is important for attacking in response to a male intruder and that the Avpr1b, likely through its role in regulating CA2 synaptic plasticity, is a necessary mediator.
1. Activation of metabotropic glutamate receptors (mGluRs) in hippocampal CA1 pyramidal neurones leads to a depolarization, an increase in input resistance and a reduction in spike frequency adaptation (or accommodation). At least eight subtypes of mGluR have been identified which have been divided into three groups based on their biochemical, structural and pharmacological properties. It is unclear to which group the mGluRs which mediate these excitatory effects in hippocampal CA1 pyramidal neurones belong. We have attempted to address this question by using intracellular recording to test the effects of a range of mGluR agonists and antagonists, that exhibit different profiles of subtype specificity, on the excitability of CA1 pyramidal neurones in rat hippocampal slices. 2. (2S, 1'S,2'S)-2-(2'-carboxycyclopropyl)glycine (L-CCG1) caused a reduction in spike frequency adaptation and a depolarization (1-10 mV) associated with an increase in input resistance (10-30%) at concentrations (> or = 50 microM) that have been shown to activate mGluRs in groups I, II and III. Similar effects were observed with concentrations (50-100 microM) of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD) and (1S,3S)-ACPD that exhibit little or no activity at group III mGluRs but which activate groups I and II mGluRs. 3. Inhibition of the release of endogenous neurotransmitters through activation of GABAB receptors, by use of 200 microM (+/-)-baclofen, did not alter the effects of (1S,3R)-ACPD (50-100 microM), (1S,3S)-ACPD (100 microM) or L-CCG1 (100 microM). This suggests that mGluR agonists directly activate CA1 pyramidal neurones. 4. Like these broad spectrum mGluR agonists, the racemic mixture ((SR)-) or resolved (S)-isomer of the selective group I mGluR agonist 3,5-dihydroxyphenylglycine ((SR)-DHPG (50-100 microM) or (S)-DHPG (20-50 microM)) caused a reduction in spike frequency adaptation concomitant with postsynaptic depolarization and an increase in input resistance. In contrast, 2S,1'R,2'R,3'R-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV; 100 microM) and (S)-2-amino-4-phosphonobutanoic acid (L-AP4; 100-500 microM), which selectively activate group II mGluRs and group III mGluRs, respectively, had no effect on the passive membrane properties or spike frequency adaptation of CA1 pyramidal neurones. 5. The mGluR antagonists (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG; 1000 microM) and (S)-4-carboxyphenylglycine ((S)-4CPG; 1000 microM), which block groups I and II mGluRs and group I mGluRs, respectively, had no effect on membrane potential, input resistance or spike frequency adaptation per se.(ABSTRACT TRUNCATED AT 400 WORDS)
1. The effects of a range of structurally-dissimilar compounds which possess affinity for sigma binding sites were examined on the responses of cultured hippocampal pyramidal neurones to the excitatory amino acid analogues N-methyl-D-aspartate (NMDA), kainate and (RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). 2. In mouse hippocampal neurones under whole-cell voltage-clamp, the compounds tested reversibly attenuated NMDA-, but not kainate- or AMPA-, evoked currents with a rank order potency (IC50 values in microM): ifenprodil (0.8) > (+)-N-allylnormetazocine (1.1) > dextromethorphan (1.8) = haloperidol (1.9) > (+)-pentazocine (7.2) > 1S,2R-(-)-cis-N-methyl-N-[2-(3, 4-dichlorophenyl) ethyl]-2-(1-pyrrolidinyl)cyclohexylamine (17) = rimcazole (18) > 1,3-di(2-tolyl)guanidine (37) > opipramol (96) > caramiphen (110) = carbetapentane (112) > > (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (485). 3. The attenuation of NMDA-evoked responses was not mediated through interactions with the agonist, glycine (except haloperidol) or polyamine (except ifenprodil) binding sites on the NMDA receptor-channel complex but, in the light of the voltage- and, in some cases, use-dependent nature of their antagonism, an interaction with the ion channel appears to be a likely mechanism of action for many of the compounds. 4. Micromolar concentrations of selected sigma site ligands also reduced NMDA-evoked rises in intracellular free calcium concentration in Fura-2-loaded cultured hippocampal neurones of the rat with the same rank order potency as observed in the electrophysiological studies. 5. The data indicate that, at micromolar concentrations, the sigma site ligands tested act as NMDA receptor antagonists, an action which does not appear to be mediated by high-affinity sigma binding site(s). The functional effects of micromolar concentrations of sigma site ligands cannot, therefore, be attributed exclusively to interactions with high-affinity sigma binding sites.
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
Glutathione (GSH) plays an important role in neuronal oxidant defence. Depletion of cellular GSH is observed in neurodegenerative diseases and thereby contributes to the associated oxidative stress and Ca2+ dysregulation. Whether depletion of cellular GSH, associated with neuronal senescence, directly influences Ca2+ permeation pathways is not known. Transient receptor potential melastatin type 2 (TRPM2) is a Ca2+ permeable non-selective cation channel expressed in several cell types including hippocampal pyramidal neurons. Moreover, activation of TRPM2 during oxidative stress has been linked to cell death. Importantly, GSH has been reported to inhibit TRPM2 channels, suggesting they may directly contribute to Ca2+ dysregulation associated with neuronal senescence. Herein, we explore the relation between cellular GSH and TRPM2 channel activity in long-term cultures of hippocampal neurons.
In whole-cell voltage-clamp recordings, we observe that TRPM2 current density increases in cultured pyramidal neurons over time in vitro. The observed increase in current density was prevented by treatment with NAC, a precursor to GSH synthesis. Conversely, treatment of cultures maintained for 2 weeks in vitro with L-BSO, which depletes GSH by inhibiting its synthesis, augments TRPM2 currents. Additionally, we demonstrate that GSH inhibits TRPM2 currents through a thiol-independent mechanism, and produces a 3.5-fold shift in the dose-response curve generated by ADPR, the intracellular agonist for TRPM2.
These results indicate that GSH plays a physiologically relevant role in the regulation of TRPM2 currents in hippocampal pyramidal neurons. This interaction may play an important role in aging and neurological diseases associated with depletion of GSH.
TRPM2; Aging; Glutathione; Oxidative stress; Pyramidal neuron; Primary hippocampal culture
The Schaffer collaterals are among the major glutamatergic inputs to CA1 pyramidal neurons, the primary output of the hippocampus, which also receive sparse recurrent inputs from pyramidal neurons in the CA1 field. Although tonically active α7 nicotinic acetylcholine receptors (nAChRs) have been shown to sustain spontaneous glutamate transmission to CA1 pyramidal neurons in hippocampal slices under resting conditions, it remains to be determined whether these receptors are those expressed by CA3 or CA1 pyramidal neurons. This study was designed to test the hypothesis that the CA3 field of the hippocampus is a significant source of α7 nAChR-sustained glutamatergic transmission to CA1 pyramidal neurons. To this end, spontaneous excitatory postsynaptic currents (EPSCs) were recorded from CA1 and CA3 pyramidal neurons in intact rat hippocampal slices as well as from CA1 pyramidal neurons in CA3-ablated slices under various experimental conditions. Surgical removal of the CA3 region from the slices reduced by 20% the frequency of spontaneous EPSCs recorded from CA1 pyramidal neurons. This finding is in agreement with the concept that the CA3 field contributes significantly to the maintenance of spontaneous glutamatergic synaptic activity in CA1 pyramidal neurons. In addition, the α7 nAChR antagonist methyllycaconitine (MLA, 10 nM) reduced the frequency of spontaneous EPSCs recorded from CA1 pyramidal neurons by 30% in intact slices and 12% in CA3-ablated slices. Taken together, these results demonstrate that tonically active α7 nAChRs in CA3 pyramidal neurons and/or in the Mossy fibers that innervate the CA3 pyramidal neurons do in fact contribute to the maintenance of glutamatergic synaptic activity in CA1 pyramidal neurons of hippocampal slices under resting conditions.
hippocampus; EPSC; methyllycaconitine; tetrodotoxin; α7 nAChR; pyramidal neuron
Pretreatment with 17β-estradiol (E2) is profoundly neuroprotective in young animals subjected to focal and global ischemia. However, whether E2 retains its neuroprotective efficacy in aging animals, especially when administered after brain insult, is largely unknown.
We examined the neuroprotective effects of E2 and two agonists that bind to non-classical estrogen receptors, G1 and STX, when administered after ischemia in middle-aged rats after prolonged ovarian hormone withdrawal. Eight weeks after ovariectomy, middle-aged female rats underwent 10 minutes of global ischemia by four vessel occlusion. Immediately after reperfusion, animals received a single infusion of either E2 (2.25 µg), G1 (50 µg) or STX (50 µg) into the lateral ventricle (ICV) or a single systemic injection of E2 (100 µg/kg). Surviving pyramidal neurons in the hippocampal CA1 were quantified 1 week later. E2 and both agonists that target non-classical estrogen receptors (G1 and STX) administered ICV at the time of reperfusion provided significant levels of neuroprotection, with 55–60% of CA1 neurons surviving vs 15% survival in controls. A single systemic injection of a pharmacological dose of E2 also rescued approximately 50% of CA1 pyramidal neurons destined to die. To determine if E2 and G1 have similar mechanisms of action in hippocampal neurons, we compared the ability of E2 and G1 to modify CA1 pyramidal neuron responses to excitatory inputs from the Schaffer collaterals recorded in hippocampal slices derived from female rats not subjected to global ischemia. E2 and G1 (10 nM) significantly potentiated pyramidal neuron responses to excitatory inputs when applied to hippocampal slices.
These findings suggest (1) that middle-aged female rats retain their responsiveness to E2 even after a long period of hormone withdrawal, (2) that non-classical estrogen receptors may mediate the neuroprotective actions of E2 when given after ischemia, and (3) that the neuroprotective efficacy of estrogens may be related to their modulation of synaptic activity in hippocampal slices.