Mouse Neocortical Slices Research Articles

Progressive Circuit Hyperexcitability in Mouse Neocortical Slice Cultures with Increasing Duration of Activity Silencing.

Forebrain neurons deprived of activity become hyperactive when activity is restored. Rebound activity has been linked to spontaneous seizures in vivo following prolonged activity blockade. Here, we measured the time course of rebound activity and the contributing circuit mechanisms using calcium imaging, synaptic staining, and whole-cell patch clamp in organotypic slice cultures of mouse neocortex. Calcium imaging revealed hypersynchronous activity increasing in intensity with longer periods of deprivation. While activity partially recovered 3 d after slices were released from 5 d of deprivation, they were less able to recover after 10 d of deprivation. However, even after the longer period of deprivation, activity patterns eventually returned to baseline levels. The degree of deprivation-induced rebound was age-dependent, with the greatest effects occurring when silencing began in the second week. Pharmacological blockade of NMDA receptors indicated that hypersynchronous rebound activity did not require activation of Hebbian plasticity. In single-neuron recordings, input resistance roughly doubled with a concomitant increase in intrinsic excitability. Synaptic imaging of pre- and postsynaptic proteins revealed dramatic reductions in the number of presumptive synapses with a larger effect on inhibitory than excitatory synapses. Putative excitatory synapses colocalizing PSD-95 and Bassoon declined by 39 and 56% following 5 and 10 d of deprivation, but presumptive inhibitory synapses colocalizing gephyrin and VGAT declined by 55 and 73%, respectively. The results suggest that with prolonged deprivation, a progressive reduction in synapse number is accompanied by a shift in the balance between excitation and inhibition and increased cellular excitability.

The general anaesthetic propofol prevents cerebrocortical potentiation in neocortical mouse brain slices

Propofol is well known to cause amnesia independent of its sedative effect. Memory consolidation processes in the hippocampus have been proposed as a target — however the neural substrates for propofol’s amnesic actions remain understudied and poorly described. In particular, the potential role of the cerebral cortex has not been investigated.As an in vitro experimental model of cortical memory consolidation, potentiated cerebral cortex evoked responses were generated in mouse neocortical slices using high frequency (20 Hz) stimulation to layer IV cortical grey matter or subcortical white matter. In separate experiments, slices were pretreated with propofol at two concentrations, 2 µg/mL and 4 µg/mL, to determine the effect of clinically relevant propofol levels on the potentiation response.Only grey matter stimulation induced a significant and lasting increase in cortical evoked potential amplitude in the drug-free condition. Propofol at 2 µg/mL completely inhibited cortical evoked response potentiation, while the 4 µg/mL concentration caused a small but significant depressant effect consequent to the high frequency stimulation.These findings support the hypothesis that propofol disrupts memory consolidation and actively facilitates memory decay in the cerebral cortex. The results further highlight the importance of the cerebral cortex in the early phase of long term memory consolidation.

RedquorinXS Mutants with Enhanced Calcium Sensitivity and Bioluminescence Output Efficiently Report Cellular and Neuronal Network Activities.

Considerable efforts have been focused on shifting the wavelength of aequorin Ca2+-dependent blue bioluminescence through fusion with fluorescent proteins. This approach has notably yielded the widely used GFP-aequorin (GA) Ca2+ sensor emitting green light, and tdTomato-aequorin (Redquorin), whose bioluminescence is completely shifted to red, but whose Ca2+ sensitivity is low. In the present study, the screening of aequorin mutants generated at twenty-four amino acid positions in and around EF-hand Ca2+-binding domains resulted in the isolation of six aequorin single or double mutants (AequorinXS) in EF2, EF3, and C-terminal tail, which exhibited markedly higher Ca2+ sensitivity than wild-type aequorin in vitro. The corresponding Redquorin mutants all showed higher Ca2+ sensitivity than wild-type Redquorin, and four of them (RedquorinXS) matched the Ca2+ sensitivity of GA in vitro. RedquorinXS mutants exhibited unaltered thermostability and peak emission wavelengths. Upon stable expression in mammalian cell line, all RedquorinXS mutants reported the activation of the P2Y2 receptor by ATP with higher sensitivity and assay robustness than wt-Redquorin, and one, RedquorinXS-Q159T, outperformed GA. Finally, wide-field bioluminescence imaging in mouse neocortical slices showed that RedquorinXS-Q159T and GA similarly reported neuronal network activities elicited by the removal of extracellular Mg2+. Our results indicate that RedquorinXS-Q159T is a red light-emitting Ca2+ sensor suitable for the monitoring of intracellular signaling in a variety of applications in cells and tissues, and is a promising candidate for the transcranial monitoring of brain activities in living mice.

Transitions between neocortical seizure and non-seizure-like states and their association with presynaptic glutamate release

The transition between seizure and non-seizure states in neocortical epileptic networks is governed by distinct underlying dynamical processes. Based on the gamma distribution of seizure and inter-seizure durations, over time, seizures are highly likely to self-terminate; whereas, inter-seizure durations have a low chance of transitioning back into a seizure state. Yet, the chance of a state transition could be formed by multiple overlapping, unknown synaptic mechanisms. To identify the relationship between the underlying synaptic mechanisms and the chance of seizure-state transitions, we analyzed the skewed histograms of seizure durations in human intracranial EEG and seizure-like events (SLEs) in local field potential activity from mouse neocortical slices, using an objective method for seizure state classification. While seizures and SLE durations were demonstrated to have a unimodal distribution (gamma distribution shape parameter >1), suggesting a high likelihood of terminating, inter-SLE intervals were shown to have an asymptotic exponential distribution (gamma distribution shape parameter <1), suggesting lower probability of cessation. Then, to test cellular mechanisms for these distributions, we studied the modulation of synaptic neurotransmission during, and between, the in vitro SLEs. Using simultaneous local field potential and whole-cell voltage clamp recordings, we found a suppression of presynaptic glutamate release at SLE termination, as demonstrated by electrically- and optogenetically-evoked excitatory postsynaptic currents (EPSCs), and focal hypertonic sucrose application. Adenosine A1 receptor blockade interfered with the suppression of this release, changing the inter-SLE shape parameter from asymptotic exponential to unimodal, altering the chance of state transition occurrence with time. These findings reveal a critical role for presynaptic glutamate release in determining the chance of neocortical seizure state transitions.

Spreading depolarization and neuronal damage or survival in mouse neocortical brain slices immediately and 12 hours following middle cerebral artery occlusion.

Whereas many studies have examined the properties of the compromised neocortex in the first several days following ischemia, there is less information regarding the initial 12 h poststroke. In this study we examined live mouse neocortical slices harvested immediately and 12 h after a 30-min middle cerebral artery occlusion (MCAo). We compared nonischemic and ischemic hemispheres with regard to the propensity for tissue swelling and for generating spreading depolarization (SD), as well as evoked synaptic responses and single pyramidal neuron electrophysiological properties. We observed spontaneous SD in 7% of slices on the nonstroked side and 25% in the stroked side following the 30-min MCAo. Spontaneous SD was rare in 12-h recovery slices. The region of the ischemic core and surround in slices was not susceptible to SD induced by oxygen and glucose deprivation. At the neuronal level, neocortical gray matter is surprisingly unaltered in brain slices harvested immediately poststroke. However, by 12 h, the fields of pyramidal and striatal neurons that comprise the infarcted core are electrophysiologically silent because the majority are morphologically devastated. Yet, there remains a subset of diffusely distributed "healthy" pyramidal neurons in the core at 12 h post-MCAo that persist for days poststroke. Their intact electrophysiology and dendritic morphology indicate a surprisingly selective resilience to stroke at the neuronal level. NEW & NOTEWORTHY It is generally accepted that the injured core region of the brain resulting from a focal stroke contains no functioning neurons. Our study shows that some neurons, although surrounded by devastated neighbors, can maintain their structure and electrical activity. This surprising finding raises the possibility of discovering how these neurons are protected to pinpoint new strategies for reducing stroke injury.

Two-Photon Optogenetic Mapping of Excitatory Synaptic Connectivity and Strength

SummaryThe development of optical methods to activate neurons with single-cell resolution has enabled systematic mapping of inhibitory connections. In contrast, optical mapping of excitatory connections between pyramidal neurons (PCs) has been a major challenge due to their high densities in cortical tissue and their weak and stochastic connectivity. Here we present an optogenetic two-photon mapping method in mouse neocortical slices by activating PCs with the red-shifted opsin C1V1 while recording postsynaptic responses in whole-cell configuration. Comparison of delays from triggered action potentials (APs) with those from synaptic inputs allowed us to predict connected PCs in three dimensions. We confirmed these predictions with paired recordings, and used this method to map strong connections among large populations of layer 2/3 PCs. Our method can be used for fast, systematic mapping of synaptic connectivity and weights.

Firing Frequency Maxima of Fast-Spiking Neurons in Human, Monkey, and Mouse Neocortex.

Cortical fast-spiking (FS) neurons generate high-frequency action potentials (APs) without apparent frequency accommodation, thus providing fast and precise inhibition. However, the maximal firing frequency that they can reach, particularly in primate neocortex, remains unclear. Here, by recording in human, monkey, and mouse neocortical slices, we revealed that FS neurons in human association cortices (mostly temporal) could generate APs at a maximal mean frequency (Fmean) of 338 Hz and a maximal instantaneous frequency (Finst) of 453 Hz, and they increase with age. The maximal firing frequency of FS neurons in the association cortices (frontal and temporal) of monkey was even higher (Fmean 450 Hz, Finst 611 Hz), whereas in the association cortex (entorhinal) of mouse it was much lower (Fmean 215 Hz, Finst 342 Hz). Moreover, FS neurons in mouse primary visual cortex (V1) could fire at higher frequencies (Fmean 415 Hz, Finst 582 Hz) than those in association cortex. We further validated our in vitro data by examining spikes of putative FS neurons in behaving monkey and mouse. Together, our results demonstrate that the maximal firing frequency of FS neurons varies between species and cortical areas.

Clusters of synaptic inputs on dendrites of layer 5 pyramidal cells in mouse visual cortex.

The spatial organization of synaptic inputs on the dendritic tree of cortical neurons plays a major role for dendritic integration and neural computations, yet, remarkably little is known about it. We mapped the spatial organization of glutamatergic synapses between layer 5 pyramidal cells by combining optogenetics and 2-photon calcium imaging in mouse neocortical slices. To mathematically characterize the organization of inputs we developed an approach based on combinatorial analysis of the likelihoods of specific synapse arrangements. We found that the synapses of intralaminar inputs form clusters on the basal dendrites of layer 5 pyramidal cells. These clusters contain 4 to 14 synapses within ≤30 µm of dendrite. According to the spatiotemporal characteristics of synaptic summation, these numbers suggest that there will be non-linear dendritic integration of synaptic inputs during synchronous activation.

Inhibition of astrocyte metabolism is not the primary mechanism for anaesthetic hypnosis.

Astrocytes have been promoted as a possible mechanistic target for anaesthetic hypnosis. The aim of this study was to explore this using the neocortical brain slice preparation. The methods were in two parts. Firstly, multiple general anaesthetic compounds demonstrating varying in vivo hypnotic potency were analysed for their effect on “zero-magnesium” seizure-like event (SLE) activity in mouse neocortical slices. Subsequently, the effect of astrocyte metabolic inhibition was investigated in neocortical slices, and compared with that of the anaesthetic drugs. The rationale was that, if suppression of astrocytes was both necessary and sufficient to cause hypnosis in vivo, then inhibition of astrocytic metabolism in slices should mimic the anaesthetic effect. In vivo anaesthetic potency correlated strongly with the magnitude of reduction in SLE frequency in neocortical slices (R2 37.7 %, p = 0.002). Conversely, SLE frequency and length were significantly enhanced during exposure to both fluoroacetate (23 and 20 % increase, respectively, p < 0.01) and aminoadipate (12 and 38 % increase, respectively, p < 0.01 and p < 0.05). The capacity of an anaesthetic agent to reduce SLE frequency in the neocortical slice is a good indicator of its in vivo hypnotic potency. The results do not support the hypothesis that astrocytic metabolic inhibition is a mechanism of anaesthetic hypnosis.

Direct current stimulation induces mGluR5-dependent neocortical plasticity.

To obtain insights into mechanisms mediating changes in cortical excitability induced by cathodal transcranial direct current stimulation (tDCS). Neocortical slices were exposed to direct current stimulation (DCS) delivered through Ag/AgCl electrodes over a range of current orientations, magnitudes, and durations. DCS-induced cortical plasticity and its receptor dependency were measured as the change in layer II/III field excitatory postsynaptic potentials by a multielectrode array, both with and without neurotransmitter receptor blockers or allosteric modulators. In vivo, tDCS was delivered to intact mice scalp via surface electrodes. Molecular consequences of DCS in vitro or tDCS in vivo were tested by immunoblot of protein extracted from stimulated slices or the neocortex harvested from stimulated intact mice. Cathodal DCS in vitro induces a long-term depression (DCS-LTD) of excitatory synaptic strength in both human and mouse neocortical slices. DCS-LTD is abolished with an mGluR5 negative allosteric modulator, mechanistic target of rapamycin (mTOR) inhibitor, and inhibitor of protein synthesis. However, DCS-LTD persists despite either γ-aminobutyric acid type A receptor or N-methyl-D-aspartate receptor inhibition. An mGluR5-positive allosteric modulator, in contrast, transformed transient synaptic depression resultant from brief DCS application into durable DCS-LTD. We identify a novel molecular pathway by which tDCS modulates cortical excitability, and indicate a capacity for synergistic interaction between tDCS and pharmacologic mGluR5 facilitation. The findings support exploration of cathodal tDCS as a treatment of neurologic conditions characterized by aberrant regional cortical excitability referable to mGluR5-mTOR signaling. Ann Neurol 2016;80:233-246.

A distinct class of slow (~0.2-2 Hz) intrinsically bursting layer 5 pyramidal neurons determines UP/DOWN state dynamics in the neocortex.

During sleep and anesthesia, neocortical neurons exhibit rhythmic UP/DOWN membrane potential states. Although UP states are maintained by synaptic activity, the mechanisms that underlie the initiation and robust rhythmicity of UP states are unknown. Using a physiologically validated model of UP/DOWN state generation in mouse neocortical slices whereby the cholinergic tone present in vivo is reinstated, we show that the regular initiation of UP states is driven by an electrophysiologically distinct subset of morphologically identified layer 5 neurons, which exhibit intrinsic rhythmic low-frequency burst firing at ~0.2-2 Hz. This low-frequency bursting is resistant to block of glutamatergic and GABAergic transmission but is absent when slices are maintained in a low Ca(2+) medium (an alternative, widely used model of cortical UP/DOWN states), thus explaining the lack of rhythmic UP states and abnormally prolonged DOWN states in this condition. We also characterized the activity of various other pyramidal and nonpyramidal neurons during UP/DOWN states and found that an electrophysiologically distinct subset of layer 5 regular spiking pyramidal neurons fires earlier during the onset of network oscillations compared with all other types of neurons recorded. This study, therefore, identifies an important role for cell-type-specific neuronal activity in driving neocortical UP states.

Endogenous cortical oscillations constrain neuromodulation by weak electric fields.

Transcranial alternating current stimulation (tACS) is a non-invasive brain stimulation modality that may modulate cognition by enhancing endogenous neocortical oscillations by application of sine-wave electric fields. Yet, the role of endogenous network activity in enabling and shaping the effects of tACS has remained unclear. We combined optogenetic stimulation and multichannel slice electrophysiology to elucidate how the effect of a weak sine-wave electric field depends on the ongoing cortical oscillatory activity. We hypothesized that endogenous cortical oscillations constrain neuromodulation by tACS. We studied the effect of weak sine-wave electric fields on oscillatory activity in mouse neocortical slices. Optogenetic control of the network activity enabled the generation of in vivo-like cortical oscillations for studying the temporal relationship between network activity and sine-wave electric field stimulation. Weak electric fields enhanced endogenous oscillations but failed to induce a frequency shift of the ongoing oscillation for stimulation frequencies that were not matched to the endogenous oscillation. This constraint on the effect of electric field stimulation imposed by endogenous network dynamics was limited to the case of weak electric fields targeting in vivo-like network dynamics. Together, these results suggest that the key mechanism of tACS may be enhancing, but not overriding, intrinsic network dynamics. Our results contribute to understanding the inconsistent tACS results from human studies and propose that stimulation precisely adjusted in frequency to the endogenous oscillations is key to rational design of non-invasive brain stimulation paradigms.

Investigation into the role of gap junction modulation of intracortical connectivity in mouse neocortical brain slices

General anesthetics are hypothesized to cause unconsciousness by interrupting communication pathways within the cerebral cortex. A correlate of this has been demonstrated in mouse neocortical slices, where anesthetics disrupt the spread of population field potential activity—resulting in a “decoupling” of activity recorded across spatial locations within the slice. In this study we investigated whether this decoupling can be explained by gap junction blockade, with a particular focus on the connexin36 (Cx36) subtype. Baseline, coupled seizure-like event (SLE) activity was recorded from two extracellular electrodes in slices perfused with no-magnesium artificial cerebrospinal fluid (aCSF). The connexin36 gap junction blocker mefloquine (25µM) failed to decouple SLE activity in wild-type mice (median(range) decoupling rate of 0.70(0.03–3.00)%, not significantly different from controls). Slices from Cx36 knock-out mice exhibited coupled SLE activity under baseline conditions and readily decoupled when exposed to the general anesthetic etomidate. The general gap junction blocker carbenoxolone (CBX, 100µM) strongly decoupled SLE activity compared to controls in wild-type mice (2.7(0.1–42.5) % compared to 0.03(0.0–0.5)%, p=0.0001). Taken together, the results show that Cx36 gap junction blockade does not cause decoupling of intracortical population activity, but the involvement of other gap junction subtypes cannot be ruled out.

Investigation into the effect of the general anaesthetic etomidate on local neuronal synchrony in the mouse neocortical slice

How general anaesthetic drugs cause unconsciousness is a topic of ongoing clinical and scientific interest. It is becoming increasingly apparent that they disrupt cortical information processing, but the effects appear to depend on the spatial scale under investigation. In this study we investigated whether the intravenous anaesthetic etomidate synchronises neuronal activity on a sub-millimetre scale in mouse neocortical slices. In slices generating no-magnesium seizure-like event (SLE) field activity, we analysed the morphology of field potential activity recorded with 50µm extracellular electrodes. The analysis was based on the understanding that the amplitude and sheerness of field potential oscillations correlates with the synchrony of the underlying neural activity. When recorded from the region of the slice initiating SLE activity, etomidate consistently increased both population event amplitude (median(range) 85(24–350) to 101(30–427) µV) and slope 16.6(1.5–106.2) to 20.2(1.7–111.1) µV/ms (p=0.016 and p=0.0013, respectively). The results are consistent with an increase in neuronal synchrony within the receptive field of the recording electrode, estimated to be a circle diameter of 300µm. In conclusion, the neocortical slice preparation supports in vivo data showing that general anaesthetics increase neuronal synchrony on a local scale and provides an ideal model for investigating underlying mechanisms.

The role of voltage dependence of the NMDA receptor in cellular and network oscillation

Unraveling the mechanisms underlying oscillatory behavior is critical for understanding normal and pathological brain processes. Here we used electrophysiology in mouse neocortical slices and principles of nonlinear dynamics to demonstrate how an increase in the N-methyl-d-aspartic acid receptor (NMDAR) conductance can create a nonlinear whole-cell current-voltage (I-V) relationship which leads to changes in cellular stability. We discovered two behaviorally and morphologically distinct pyramidal cell populations. Under control conditions, both cell types responded to depolarizing current injection with regular spiking patterns. However, upon NMDAR activation, an intrinsic oscillatory (IO) cell type (n = 44) showed a nonlinear whole-cell I-V relationship, intrinsic voltage-dependent oscillations plus amplification of alternating input current, and these properties persisted after disabling action potential generation with tetrodotoxin (TTX). The other non-oscillatory (NO) neuronal population (n = 24) demonstrated none of these behaviors. Simultaneous intra- and extracellular recordings demonstrated the NMDAR's capacity to promote low-frequency seizure-like network oscillations via its effects on intrinsic neuronal properties. The two pyramidal cell types demonstrated different relationships with network oscillation--the IO cells were leaders that were activated early in the population activity cycle while the activation of the NO cell type was distributed across network bursts. The properties of IO neurons disappeared in a low-magnesium environment where the voltage dependence of the receptor is abolished; concurrently, the cellular contribution to network oscillation switched to synchronous firing. Thus, depending upon the efficacy of NMDAR in altering the linearity of the whole-cell I-V relationship, the two cell populations played different roles in sustaining network oscillation.

Activation of cortical 5-HT3 receptor-expressing interneurons induces NO mediated vasodilatations and NPY mediated vasoconstrictions

GABAergic interneurons are local integrators of cortical activity that have been reported to be involved in the control of cerebral blood flow (CBF) through their ability to produce vasoactive molecules and their rich innervation of neighboring blood vessels. They form a highly diverse population among which the serotonin 5-hydroxytryptamine 3A receptor (5-HT3A)-expressing interneurons share a common developmental origin, in addition to the responsiveness to serotonergic ascending pathway. We have recently shown that these neurons regroup two distinct subpopulations within the somatosensory cortex: Neuropeptide Y (NPY)-expressing interneurons, displaying morphological properties similar to those of neurogliaform cells and Vasoactive Intestinal Peptide (VIP)-expressing bipolar/bitufted interneurons. The aim of the present study was to determine the role of these neuronal populations in the control of vascular tone by monitoring blood vessels diameter changes, using infrared videomicroscopy in mouse neocortical slices. Bath applications of 1-(3-Chlorophenyl)biguanide hydrochloride (mCPBG), a 5-HT3R agonist, induced both constrictions (30%) and dilations (70%) of penetrating arterioles within supragranular layers. All vasoconstrictions were abolished in the presence of the NPY receptor antagonist (BIBP 3226), suggesting that they were elicited by NPY release. Vasodilations persisted in the presence of the VIP receptor antagonist VPAC1 (PG-97-269), whereas they were blocked in the presence of the neuronal Nitric Oxide (NO) Synthase (nNOS) inhibitor, L-NNA. Altogether, these results strongly suggest that activation of neocortical 5-HT3A-expressing interneurons by serotoninergic input could induces NO mediated vasodilatations and NPY mediated vasoconstrictions.

Investigating paradoxical hysteresis effects in the mouse neocortical slice model

Clinically, anesthetic drugs show hysteresis in the plasma drug concentrations at induction versus emergence from anesthesia induced unconsciousness. This is assumed to be the result of pharmacokinetic lag between the plasma and brain effect-site and vice versa. However, recent mathematical and experimental studies demonstrate that anesthetic hysteresis might be due in part to lag in the brain physiology, independent of drug transport delay — so-called “neural inertia”. The aim of this study was to investigate neural inertia in the reduced neocortical mouse slice model. Seizure-like event (SLE) activity was generated by exposing cortical slices to no-magnesium artificial cerebrospinal fluid (aCSF). Concentration–effect loops were generated by manipulating SLE frequency, using the general anesthetic drug etomidate and by altering the aCSF magnesium concentration. The etomidate (24 μM) concentration–effect relationship showed a clear hysteresis, consistent with the slow diffusion of etomidate into slice tissue. Manipulation of tissue excitability, using either carbachol (50 μM) or elevated potassium (5 mM vs 2.5 mM) did not significantly alter the size of etomidate hysteresis loops. Hysteresis in the magnesium concentration–effect relationship was evident, but only when the starting condition was magnesium-containing “normal” aCSF. The in vitro cortical slice manifests pathway-dependent “neural inertia” and may be a valuable model for future investigations into the mechanisms of neural inertia in the cerebral cortex.

Connexin36 Gap Junction Blockade Is Ineffective at Reducing Seizure-Like Event Activity in Neocortical Mouse Slices

Despite much research, there remains controversy over the role of gap junctions in seizure processes. Many studies report anticonvulsant effects of gap junction blockade, but contradictory results have also been reported. The aim of this study was to clarify the role of connexin36 (Cx36) gap junctions in neocortical seizures. We used the mouse neocortical slice preparation to investigate the effect of pharmacological (mefloquine) and genetic (Cx36 knockout mice (Cx36KO)) manipulation of Cx36 gap junctions on two seizure models: low-magnesium artificial cerebrospinal fluid (ACSF) and aconitine perfusion in low-magnesium ACSF. Low-magnesium- (nominally zero) and aconitine- (230 nM) induced seizure-like event (SLE) population activity was recorded extracellularly. The results were consistent in showing that neither mefloquine (25 μM) nor genetic knockdown of Cx36 expression had anticonvulsant effects on SLE activity generated by either method. These findings call into question the widely held idea that open Cx36 gap junctions promote seizure activity.

The HIV-1 Viral Protein Tat Increases Glutamate and Decreases GABA Exocytosis from Human and Mouse Neocortical Nerve Endings

Human immunodeficiency virus-1 (HIV-1)-encoded transactivator of transcription (Tat) potentiated the depolarization-evoked exocytosis of [(3)H]D-aspartate ([(3)H]D-ASP) from human neocortical terminals. The metabotropic glutamate (mGlu) 1 receptor antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) prevented this effect, whereas the mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP) was ineffective. Western blot analysis showed that human neocortex synaptosomes possess mGlu1 and mGlu5 receptors. Tat potentiated the K(+)-evoked release of [(3)H]D-ASP or of endogenous glutamate from mouse neocortical synaptosomes in a CPCCOEt-sensitive and MPEP-insensitive manner. Deletion of mGlu1 receptors (crv4/crv4 mice) or mGlu5 receptors (mGlu5(-/-)mouse) silenced Tat effects. Tat enhanced inositol 1,4,5-trisphosphate production in human and mouse neocortical synaptosomes, consistent with the involvement of group I mGlu receptors. Tat inhibited the K(+)-evoked release of [(3)H]gamma-aminobutyric acid ([(3)H]GABA) from human synaptosomes and that of endogenous GABA or [(3)H]GABA from mouse nerve terminals; the inhibition was insensitive to CPCCOEt or MPEP. Tat-induced effects were retained by Tat(37-72) but not by Tat(48-85). In mouse neocortical slices, Tat facilitated the K(+)- and the veratridine-induced release of [(3)H]D-ASP in a CPCCOEt-sensitive manner and was ineffective in crv4/crv4 mouse slices. These observations are relevant to the comprehension of the pathophysiological effects of Tat in central nervous system and may suggest new potential therapeutic approaches to the cure of HIV-1-associated dementia.

D1-Like Dopamine Receptor Activation Modulates GABAergic Inhibition But Not Electrical Coupling between Neocortical Fast-Spiking Interneurons

Dopamine, acting through D(1) receptors, is thought to play an important role in cognitive functions of the frontal cortex such as working memory. D(1) receptors are widely expressed in fast-spiking (FS) interneurons, a prominent class of inhibitory cells that exert a powerful control of neuronal firing through proximal synapses on their postsynaptic targets. FS cells are extensively mutually interconnected by both GABA(A) receptor-mediated synapses and gap junction-mediated electrical synapses, and networks of FS cells play a crucial role in the generation of rhythmic synchronous activity. Although recent studies have documented the effects of dopamine modulation of neocortical synaptic connections among excitatory cells and between excitatory and various inhibitory cells, the effects of dopamine receptor activation on GABAergic and electrical interactions among FS cells is not known. To resolve this, we recorded from pairs of FS cells in the infragranular layers of mouse neocortical slices and tested the effects of D(1)-like (D(1)/D(5)) receptor activation on these connections. We found that D(1)-like receptor activation modulated GABAergic but not electrical connections between them. A D(1)-like receptor agonist preserved the strength of electrical coupling but reduced the amplitude of IPSPs and IPSCs between FS cells. Our results suggest that D(1)-like receptor activation has synapse-specific effects within networks of FS cells, with potential implications for the generation of rhythmic activity in the neocortex.