Structure-function analysis of SAP97, a modular scaffolding protein that drives dendrite growth.

Activation of AMPA receptors assembled with the GluA1 subunit can promote dendrite growth in a manner that depends on its direct binding partner, SAP97. SAP97 is a modular scaffolding protein that has at least seven recognizable protein-protein interaction domains. Several complementary approaches were employed to show that the dendrite branching promoting action of full length SAP97 depends on ligand(s) that bind to the PDZ3 domain. Ligand(s) to PDZ1, PDZ2 and I3 domains also contribute to dendrite growth. The ability of PDZ3 ligand(s) to promote dendrite growth depends on localization at the plasma membrane along with GluA1 and SAP97. These results suggest that the assembly of a multi-protein complex at or near synapses is vital for the translation of AMPA-R activity into dendrite growth.

Selective changes in single cell GABA(A) receptor subunit expression and function in temporal lobe epilepsy.

Temporal lobe epilepsy is the most prevalent seizure disorder in adults. Compromised inhibitory neurotransmitter function in the hippocampus contributes to the hyperexcitability generating this condition, but the underlying molecular mechanisms are unknown. Combining patch-clamp recording and single-cell mRNA amplification (aRNA) techniques in single dentate granule cells, we demonstrate that expression of GABA(A) receptor subunit mRNAs is substantially altered in neurons from epileptic rats. These changes in gene expression precede epilepsy onset by weeks and correlate with profound alterations in receptor function, indicating that aberrant GABA(A) receptor expression and function has an essential role in the process of epileptogenesis.

Swope Supernova Survey 2017a (SSS17a), the optical counterpart to a gravitational wave source.

On 17 August 2017, the Laser Interferometer Gravitational-Wave Observatory (LIGO) and the Virgo interferometer detected gravitational waves (GWs) emanating from a binary neutron star merger, GW170817. Nearly simultaneously, the Fermi and INTEGRAL (INTErnational Gamma-Ray Astrophysics Laboratory) telescopes detected a gamma-ray transient, GRB 170817A. At 10.9 hours after the GW trigger, we discovered a transient and fading optical source, Swope Supernova Survey 2017a (SSS17a), coincident with GW170817. SSS17a is located in NGC 4993, an S0 galaxy at a distance of 40 megaparsecs. The precise location of GW170817 provides an opportunity to probe the nature of these cataclysmic events by combining electromagnetic and GW observations.

Electromagnetic evidence that SSS17a is the result of a binary neutron star merger.

Eleven hours after the detection of gravitational wave source GW170817 by the Laser Interferometer Gravitational-Wave Observatory and Virgo Interferometers, an associated optical transient, SSS17a, was identified in the galaxy NGC 4993. Although the gravitational wave data indicate that GW170817 is consistent with the merger of two compact objects, the electromagnetic observations provide independent constraints on the nature of that system. We synthesize the optical to near-infrared photometry and spectroscopy of SSS17a collected by the One-Meter Two-Hemisphere collaboration, finding that SSS17a is unlike other known transients. The source is best described by theoretical models of a kilonova consisting of radioactive elements produced by rapid neutron capture (the r-process). We conclude that SSS17a was the result of a binary neutron star merger, reinforcing the gravitational wave result.

Early spectra of the gravitational wave source GW170817: Evolution of a neutron star merger.

On 17 August 2017, Swope Supernova Survey 2017a (SSS17a) was discovered as the optical counterpart of the binary neutron star gravitational wave event GW170817. We report time-series spectroscopy of SSS17a from 11.75 hours until 8.5 days after the merger. Over the first hour of observations, the ejecta rapidly expanded and cooled. Applying blackbody fits to the spectra, we measured the photosphere cooling from [Formula: see text] to [Formula: see text] kelvin, and determined a photospheric velocity of roughly 30% of the speed of light. The spectra of SSS17a began displaying broad features after 1.46 days and evolved qualitatively over each subsequent day, with distinct blue (early-time) and red (late-time) components. The late-time component is consistent with theoretical models of r-process-enriched neutron star ejecta, whereas the blue component requires high-velocity, lanthanide-free material.

Light curves of the neutron star merger GW170817/SSS17a: Implications for r-process nucleosynthesis.

On 17 August 2017, gravitational waves (GWs) were detected from a binary neutron star merger, GW170817, along with a coincident short gamma-ray burst, GRB 170817A. An optical transient source, Swope Supernova Survey 17a (SSS17a), was subsequently identified as the counterpart of this event. We present ultraviolet, optical, and infrared light curves of SSS17a extending from 10.9 hours to 18 days postmerger. We constrain the radioactively powered transient resulting from the ejection of neutron-rich material. The fast rise of the light curves, subsequent decay, and rapid color evolution are consistent with multiple ejecta components of differing lanthanide abundance. The late-time light curve indicates that SSS17a produced at least ~0.05 solar masses of heavy elements, demonstrating that neutron star mergers play a role in rapid neutron capture (r-process) nucleosynthesis in the universe.

Normal and epilepsy-associated pathologic function of the dentate gyrus.

The dentate gyrus plays critical roles both in cognitive processing, and in regulation of the induction and propagation of pathological activity. The cellular and circuit mechanisms underlying these diverse functions overlap extensively. At the cellular level, the intrinsic properties of dentate granule cells combine to endow these neurons with a fundamental reluctance to activate, one of their hallmark traits. At the circuit level, the dentate gyrus constitutes one of the more heavily inhibited regions of the brain, with strong, fast feedforward and feedback GABAergic inhibition dominating responses to afferent activation. In pathologic states such as epilepsy, a number of alterations within the dentate gyrus combine to compromise the regulatory properties of this circuit, culminating in a collapse of its normal function. This epilepsy-associated transformation in the fundamental properties of this critical regulatory hippocampal circuit may contribute both to seizure propensity, and cognitive and emotional comorbidities characteristic of this disease state.

Human neuronal gamma-aminobutyric acid(A) receptors: coordinated subunit mRNA expression and functional correlates in individual dentate granule cells.

gamma-Aminobutyric acid(A) receptors (GABARs) are heteromeric proteins composed of multiple subunits. Numerous subunit subtypes are expressed in individual neurons, which assemble in specific preferred GABAR configurations. Little is known, however, about the coordination of subunit expression within individual neurons or the impact this may have on GABAR function. To investigate this, it is necessary to profile quantitatively the expression of multiple subunit mRNAs within individual cells. In this study, single-cell antisense RNA amplification was used to examine the expression of 14 different GABAR subunit mRNAs simultaneously in individual human dentate granule cells (DGCs) harvested during hippocampectomy for intractable epilepsy. alpha4, beta2, and delta-mRNA levels were tightly correlated within individual DGCs, indicating that these subunits are expressed coordinately. Levels of alpha3- and beta2-mRNAs, as well as epsilon- and beta1-mRNAs, also were strongly correlated. No other subunit correlations were identified. Coordinated expression could not be explained by the chromosomal clustering of GABAR genes and was observed in control and epileptic rats as well as in humans, suggesting that it was not species-specific or secondary to epileptogenesis. Benzodiazepine augmentation of GABA-evoked currents also was examined to determine whether levels of subunit mRNA expression correlated with receptor pharmacology. This analysis delineated two distinct cell populations that differed in clonazepam modulation and patterns of alpha-subunit expression. Clonazepam augmentation correlated positively with the relative expression of alpha1- and gamma2-mRNAs and negatively with alpha4- and delta-mRNAs. These data demonstrate that specific GABAR subunit mRNAs exhibit coordinated control of expression in individual DGCs, which has significant impact on inhibitory function.

Effects of status epilepticus on hippocampal GABAA receptors are age-dependent.

Long-term GABA(A) receptor alterations occur in hippocampal dentate granule neurons of rats that develop epilepsy after status epilepticus in adulthood. Hippocampal GABA(A) receptor expression undergoes marked reorganization during the postnatal period, however, and the effects of neonatal status epilepticus on subsequent GABA(A) receptor development are unknown. In the current study, we utilize single cell electrophysiology and antisense mRNA amplification to determine the effect of status-epilepticus induced by lithium-pilocarpine in postnatal day 10 rat pups on GABA(A) receptor subunit expression and function in hippocampal dentate granule neurons. We find that rats subjected to lithium-pilocarpine-induced status epilepticus at postnatal day 10 show long-term GABA(A) receptor changes including a two-fold increase in alpha1 subunit expression (compared with lithium-injected controls) and enhanced type I benzodiazepine augmentation that are opposite of those seen after status epilepticus in adulthood and may serve to enhance dentate gyrus inhibition. Further, unlike adult rats, postnatal day 10 rats subjected to status epilepticus do not become epileptic. These findings suggest age-dependent differences in the effects of status epilepticus on hippocampal GABA(A) receptors that could contribute to the selective resistance of the immature brain to epileptogenesis.

Differential effects of petit mal anticonvulsants and convulsants on thalamic neurones: GABA current blockade.

1. Currents evoked by applications of gamma-aminobutyric acid (GABA) to acutely dissociated thalamic neurones were analysed by voltage-clamp techniques, and the effects of the anticonvulsant succinimides ethosuximide (ES) and alpha-methyl-alpha-phenylsuccinimide (MPS) and the convulsants tetramethylsuccinimide (TMS), picrotoxin, pentylenetetrazol (PTZ), and bicuculline methiodide were assessed. 2. TMS (1 microM-10 microM) reduced responses to iontophoretically applied GABA, as did picrotoxin (0.1-100 microM), PTZ (1-100 mM) and bicuculline (1-100 microM). 3. ES, in high concentrations (1-10 mM), reduced GABA responses to a lesser extent, and also occluded the reductions in GABA-evoked currents produced by TMS, picrotoxin, and PTZ. ES did not occlude the effects of bicuculline on GABA responses. Therefore, we propose that ES acts as a partial agonist at the picrotoxin GABA-blocking receptor. 4. MPS had no effect on GABA responses (at a concentration of 1 mM), and, like ES, occluded the GABA-blocking actions of TMS, apparently acting as a full antagonist. 5. The anticonvulsant actions of ES and MPS against TMS and PTZ-induced seizures may thus involve two independent mechanisms: (1) the occlusion of TMS and PTZ GABA-blocking effects; and (2) the previously described specific effect of ES and MPS on low-threshold calcium current of thalamic neurones. The latter cellular mechanism may be more closely related to petit mal anticonvulsant activity.

Differential effects of petit mal anticonvulsants and convulsants on thalamic neurones: calcium current reduction.

1. Succinimide derivatives can be either convulsant (tetramethylsuccinimide (TMS)), or anticonvulsant (ethosuximide (ES); alpha-methyl-alpha-phenylsuccinimide (MPS)). ES, an anticonvulsant succinimide, has previously been shown to block calcium currents of thalamic neurones, while the convulsant succinimide TMS blocks gamma-aminobutyric acid (GABA) responses in a similar fashion to the convulsant pentylenetetrazol (PTZ). 2. Using voltage-clamp techniques, we analysed the effects of the anticonvulsant succinimides ES and MPS and the convulsants TMS and PTZ on calcium currents of acutely isolated thalamic relay neurones of the rat. 3. MPS and ES reduced low-threshold calcium current (LTCC) in a voltage-dependent manner, without affecting steady-state inactivation. MPS was less potent than ES (IC50 of 1100 vs 200 microM) but greater in efficacy (100% maximal reduction vs 40% for ES). 4. PTZ had no effect on calcium currents, and TMS only reduced LTCC at very high concentrations, and did not occlude MPS effects when applied concurrently. 5. These results, which demonstrate that anticonvulsant, but not convulsant, succinimides block LTCC, provide additional support for the hypothesis that LTCC reduction is a mechanism of action of the anticonvulsant succinimides related to their effects in petit mal epilepsy.

Thalamocortical rhythm generation in vitro: extra- and intracellular recordings in mouse thalamocortical slices perfused with low Mg2+ medium.

Mouse thalamocortical slices maintain synaptic connections between thalamus and cortex. When perfused with low Mg2+ medium, thalamocortical slices exhibited spontaneous thalamocortical 2-6 s long bursts of rhythmic activity every 15-30 s, which was potentiated by pentylenetetrazol and abolished by severing thalamocortical connections. These oscillations were similar in frequency and duration to normal and pathological thalamocortical rhythms in vivo. In vitro studies of these thalamocortical rhythms may prove valuable in understanding cellular factors important in rhythm generation.

Neurotoxic activation of glutamate receptors induces an extended neuronal depolarization in cultured hippocampal neurons.

Intracellular recording revealed that cytotoxic activation of excitatory amino acid receptors by glutamate or N-methyl-D-aspartate (NMDA) elicited an extended neuronal depolarization (END) of at least 5 h duration following washout of glutamate in hippocampal neurons in culture. During END, cells were still responsive to glutamate, and still able to fire sodium spikes. END induction could be blocked by concurrent application of D-2-amino-5-phosphonovalerate (APV) or MK-801, but not 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), during the glutamate exposure. The induction of END by excitotoxic glutamate receptor activation may play a role in the pathophysiology of glutamate toxicity.

AHP reductions in rabbit hippocampal neurons during conditioning correlate with acquisition of the learned response.

Young adult male albino rabbits were conditioned using a free field auditory conditioned stimulus (CS) and periorbital shock unconditioned stimulus (US) in a short delay eye blink paradigm. All rabbits received two 80-trial training sessions. Intracellular recordings were made from hippocampal CA1 pyramidal neurons within brain slices prepared 24 h following the second training session. All 46 CA1 neurons included in the analysis had stable penetration, at least 70 mV impulse amplitudes and at least 40 M omega input resistance. Recording and initial data analysis were done 'blind' regarding behavioral training performance of the rabbit from which the slices were prepared. The animals were separated into a High (86 +/- 6% CRs, n = 12), and Low (12 +/- 4% CRs, n = 10) Acquisition group based on the number of blink CRs shown on the second training day (P less than 0.001). CA1 pyramidal neurons from the High Acquisition group (n = 20) showed a significant reduction in the afterhypolarization (AHP) response following 4 impulses elicited by intracellular current injection as compared to neurons from the Low Acquisition group (n = 26). The mean maximal AHP amplitudes after 4 spikes were -2.9 +/- 0.34 mV and -4.0 +/- 0.31 mV, respectively, in the High and Low Acquisition groups (P less than 0.01). The size of the AHP examined at 100 ms intervals during the first 1.7 s after the current pulse proved to be reduced in the High group both when evaluated for all points (F = 5.88, df = 1.44, P less than 0.02) and for each of the individual time points (at least P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

GABAA alpha 2 mRNA levels are decreased following induction of spontaneous epileptiform discharges in hippocampal-entorhinal cortical slices.

Exposure of hippocampal slices to Mg2+ free media (0 Mg) has been shown to trigger full production of stimulus-induced seizure activity after restoration of physiological conditions [1]. In the present study employing hippocampal entorhinal cortical slices (HEC), spontaneous epileptiform discharges (SEDs) were induced using 0 Mg treatment following the return of the slices to physiological conditions. To evaluate the effect of sustained epileptiform activity on gene expression in this HEC slice preparation, changes in mRNA levels of the GABAA alpha 1 and alpha 2 and beta CaM Kinase II subunits were measured using in situ hybridization. HEC slices were incubated in oxygenated artificial cerebrospinal fluid (ACSF) in the presence or absence of Mg2+ for 3 h, then placed in oxygenated ACSF containing Mg2+ for up to 3 h. Control slices were maintained in Mg2+ containing ACSF for up to 6 h. Recurrent SEDs were observed in 0 Mg pre-treated slices while no epileptiform discharges were seen in control slices. Following induction of SEDs by 0 Mg pre-treatment, a significant decrease in mRNA encoding GABAA alpha 2 was found in the CA1, CA2, CA3 and dentate gyrus (DG) regions of the hippocampus for up to 3 h after treatment. Levels of mRNA for GABAA alpha 1 and beta CaM Kinase II were not affected. The results document a decrease in GABAA alpha 2 gene expression following the induction of SEDs in the HEC slice preparation and suggest that rapid changes in neuronal gene expression may contribute to long lasting excitability changes associated with the induction of epilepsy.

Specific petit mal anticonvulsants reduce calcium currents in thalamic neurons.

Low-threshold calcium current (LTCC) in thalamic neurons is important in generation of normal thalamocortical rhythms, and may be involved in the genesis of abnormal activities such as spike-wave discharges that characterize petit mal epilepsy. Ethosuximide and dimethadione, anticonvulsants effective in petit mal, reduced the LTCC when applied to thalamic neurons at clinically relevant concentrations. Therapeutic concentrations of phenytoin and carbamazepine, drugs ineffective in the control of petit mal, had minimal effects on calcium conductances. Reduction in LTCC may be an important mechanism of action by which specific petit mal anticonvulsants depress spike-wave activity.

Dextrorphan and dextromethorphan, common antitussives, are antiepileptic and antagonize N-methyl-D-aspartate in brain slices.

The antitussive, dextromethorphan (DM), and its metabolite, dextrorphan (DX), were evaluated for antiepileptic properties in vitro. Interictal bursts and prolonged ictal epileptiform afterdischarges, induced by perfusion of guinea pig neocortical brain slices with Mg2+-free solution, were blocked by DX (1-250 microM) or DM (100 microM). Intracellular records showed that these agents blocked N-methyl-D-aspartate (NMDA)-induced depolarizations without altering intrinsic membrane properties. DX blocked NMDA but not quisqualate-evoked multi-unit excitatory responses. DM is a widely available, orally effective drug with low toxicity in antitussive doses, which has antiepileptic and NMDA-antagonist properties in vitro. Its toxicity and effectiveness as an anticonvulsant should be expeditiously examined in clinical trials.

Anticonvulsant drug effects on spontaneous thalamocortical rhythms in vitro: phenytoin, carbamazepine, and phenobarbital.

When perfused with a medium containing no added Mg2+, rodent thalamocortical brain slices generate spontaneous generalized thalamocortical discharges of several types. Two of these discharges, termed simple and complex thalamocortical burst complexes (sTBCs and cTBCs), are physiologically and pharmacologically similar to the spike-wave discharges of generalized absence epilepsy and to the discharges underlying generalized tonic-clonic seizures, respectively. In a further characterization of the pharmacology of generalized thalamocortical discharges recorded in rodent thalamocortical slices, the actions of anticonvulsants effective in control of partial and generalized tonic-clonic seizures, but not generalized absence seizures, were studied on these rhythms. The effects of phenytoin, carbamazepine, and phenobarbital were tested against sTBCs and cTBCs recorded in vitro in rodent thalamocortical slices. When applied in clinically relevant concentrations, phenytoin and carbamazepine were very effective in reducing or blocking cTBCs. These drugs were much less effective in controlling sTBCs. Phenobarbital was effective in controlling both sTBCs and cTBCs, but the level of block was greater for cTBCs. Therefore, it appears that sTBCs and cTBCs are quite distinct in their relative sensitivity to anticonvulsant drugs, and this differential sensitivity parallels the relative effectiveness of these drugs in controlling generalized absence and generalized tonic-clonic seizures.

Anticonvulsant drug effects on spontaneous thalamocortical rhythms in vitro: ethosuximide, trimethadione, and dimethadione.

Spontaneous generalized epileptiform discharges were elicited in rodent thalamocortical slices by perfusion with a medium containing no added Mg2+. In multiple-channel extracellular field potential recordings in thalamus and cortex, several distinct types of discharges were recorded, with two principal variants bearing marked similarity to spike-wave and generalized tonic-clonic seizure discharges recorded in patients with generalized seizure disorders. These discharges were termed sTBCs and cTBCs, respectively, for simple and complex thalamocortical burst complexes. The sensitivity of these discharges to the generalized absence anticonvulsants ethosuximide, trimethadione and dimethadione (the active metabolite of trimethadione) was studied. sTBCs were reduced or blocked by ethosuximide and dimethadione, when these drugs were applied in clinically relevant concentrations. The order of effectiveness of these agents was dimethadione > or = ethosuximide >> trimethadione. This paralleled the relative efficacy of these drugs in blocking T current in thalamic neurons. cTBCs were unaffected or exacerbated by these drugs. Structural control drugs including succinimide, the behaviorally inactive ring base of ethosuximide, and alpha, alpha-dimethyl-beta-methylsuccinimide, a convulsant succinimide, were inactive or exacerbated either sTBCs or cTBCs, respectively. These spontaneous generalized thalamocortical discharges in rodent thalamocortical slices may represent a potentially valuable in vitro model of generalized seizure discharges, with marked pharmacological and physiological similarities to various forms of clinical epileptic seizure activity.

Anticonvulsant drug effects on spontaneous thalamocortical rhythms in vitro: valproic acid, clonazepam, and alpha-methyl-alpha-phenylsuccinimide.

Spontaneous thalamocortical epileptiform activity was elicited in rodent thalamocortical slices by a medium containing no added Mg2+. Multiple varieties of activity were generated in these slices, including simple thalamocortical burst complex (sTBC) activity that resembled the spike-wave discharges of generalized absence epilepsy, and complex thalamocortical burst complex (cTBC) activity that resembled generalized tonic-clonic seizure discharges. In a further pharmacological characterization of this activity, the effects of the broad-spectrum anticonvulsants valproic acid, alpha-methyl-alpha-phenylsuccinimide (the active metabolite of methsuximide) and clonazepam were studied. All three drugs were found to be effective in controlling both sTBC and cTBC activity when applied in clinically relevant concentration ranges. The effectiveness of valproic acid against spontaneous rhythms in vitro was not due to augmentation of GABAergic inhibition. No effect of valproic acid on GABA-activated chloride currents was evident in patch-clamp recordings of acutely isolated thalamic or cortical neurons. The equivalent general clinical and experimental spectrum of action of broadly effective anticonvulsants provided an additional correlation between the clinical efficacy of anticonvulsant drugs and their effects against epileptiform discharges in rodent thalamocortical slices. This further validates spontaneous generalized low-Mg2+ thalamocortical activity as a potentially valuable in vitro model of the primary generalized epilepsies, in which the cellular mechanisms underlying generation and control of these seizure discharges can be studied.