Downregulation of Ribosomal Protein Genes Is Revealed in a Model of Rat Hippocampal Neuronal Culture Activation with GABA(A)R/GlyRa2 Antagonist Picrotoxin.

Long-read transcriptome sequencing provides us with a convenient tool for the thorough study of biological processes such as neuronal plasticity. Here, we aimed to perform transcriptional profiling of rat hippocampal primary neuron cultures after stimulation with picrotoxin (PTX) to further understand molecular mechanisms of neuronal activation. To overcome the limitations of short-read RNA-Seq approaches, we performed an Oxford Nanopore Technologies MinION-based long-read sequencing and transcriptome assembly of rat primary hippocampal culture mRNA at three time points after the PTX activation. We used a specific approach to exclude uncapped mRNAs during sample preparation. Overall, we found 23,652 novel transcripts in comparison to reference annotations, out of which ~6000 were entirely novel and mostly transposon-derived loci. Analysis of differentially expressed genes (DEG) showed that 3046 genes were differentially expressed, of which 2037 were upregulated and 1009 were downregulated at 30 min after the PTX application, with only 446 and 13 genes differentially expressed at 1 h and 5 h time points, respectively. Most notably, multiple genes encoding ribosomal proteins, with a high basal expression level, were downregulated after 30 min incubation with PTX; we suggest that this indicates redistribution of transcriptional resources towards activity-induced genes. Novel loci and isoforms observed in this study may help us further understand the functional mRNA repertoire in neuronal plasticity processes. Together with other NGS techniques, differential gene expression analysis of sequencing data obtained using MinION platform might provide a simple method to optimize further study of neuronal plasticity.

Factors promoting the release of picrotoxin from the trap in the GABA(A) receptor pore.

Picrotoxin (PTX), a convulsant of plant origin, has been used in many studies as research tool. PTX is the open channel blocker of the GABAA receptor (GABAAR). Being in the pore, PTX initiates transfer of the channel to the closed state and thus it falls into the "trap". The consequence of this PTX trapping is so-called aftereffect, i.e. continuation of the blockade of the GABA-induced chloride current (IGABA) after removal of PTX from the external solution. The present work shows that the positive allosteric modulators (PAMs) of the GABAA receptor, allopregnanolone (Allo) and zolpidem (Zolp) as well as a high concentration of GABA shortened the PTX aftereffect. Experiments were carried out on isolated Purkinje neurons of the rat cerebellum using the whole-cell patch-clamp method. IGABA was induced by applications of 5 µM GABA (EC30) for 1 s with 30 s intervals. 50 µM PTX completely blocked IGABA, and recovery upon PTX washout occurred with a time constant (τrec) of 20.2 min. 1 µM Allo reduced the blocking effect of PTX by 30% and accelerated the recovery of IGABA by almost 10 times (τrec = 2.4 min). 0.5 µM Zolp did not change the IGABA block in the presence of PTX but accelerated the recovery of IGABA by more than 3 times (τrec = 5.6 min). Increasing the GABA concentration to 20 µM did not change the blocking effect of PTX, but accelerated recovery by 6 times (τrec = 3.3 min). The mechanism of the shortening of the PTX aftereffect is presumably the expansion of the GABAAR pore in the presence of PAMs and a high concentration of the agonist and, as a consequence, the escape of PTX from the "trap". The work describes new pharmacological properties of Allo and Zolp.

Functional Deficiency of Interneurons and Negative BOLD fMRI Response.

The functional deficiency of the inhibitory system typically appears during development and can progress to psychiatric disorders or epilepsy, depending on its severity, in later years. It is known that interneurons, the major source of GABAergic inhibition in the cerebral cortex, can make direct connections with arterioles and participate in the regulation of vasomotion. The goal of this study was to mimic the functional deficiency of interneurons through the use of localized microinjections of the GABA antagonist, picrotoxin, in such a concentration that it did not elicit epileptiform neuronal activity. First, we recorded the dynamics of resting-state neuronal activity in response to picrotoxin injections in the somatosensory cortex of an awake rabbit; second, we assessed the altered neuronal and hemodynamic responses to whisker stimulation using BOLD fMRI and electrophysiology recordings; third, we evaluated brain tissue oxygen levels before and after picrotoxin injection. Our results showed that neuronal activity typically increased after picrotoxin administration, the BOLD responses to stimulation became negative, and the oxygen response was nearly abolished. Vasoconstriction during the resting baseline was not observed. These results indicate that picrotoxin provoked imbalanced hemodynamics either due to increased neuronal activity, decreased vascular response, or a combination of both.

Chemical Kindling as an Experimental Model to Assess the Conventional Drugs in the Treatment of Post-traumatic Epilepsy.

BACKGROUND: Traumatic brain injury (TBI) is one of the leading causes of morbidity and mortality today, which will surpass many infectious diseases in the coming years/decades. Posttraumatic epilepsy (PTE) is one of the most common debilitating consequences of TBI. PTE is a secondary, acquired epilepsy that causes recurrent, spontaneous seizures more than a week after TBI. The extent of head injury in individuals who develop PTE is unknown; however, trauma is thought to account for 20% of symptomatic epilepsy worldwide. Understanding the mechanisms of epilepsy following TBI is crucial for the discovery of new anticonvulsant drugs for the treatment of PTE, as well as for improving the quality of life of patients with PTE. OBJECTIVE: This review article explains the rationale for the usage of a chemical model to access new treatments for post-traumatic epilepsy. RESULTS: There are multiple methods to control and manage PTE. The essential and available remedy for the management of epilepsy is the use of antiepileptic drugs. Antiepileptic drugs (AEDs) decrease the frequency of seizures without affecting the disease's causality. Antiepileptic drugs are administrated for the prevention and treatment of PTE; however, 30% of epilepsy patients are drug-resistant, and AED side effects are significant in PTE patients. There are different types of animal models, such as the liquid percussion model, intracortical ferric chloride injection, and cortical subincision model, to study PTE and neurophysiological mechanisms underlying the development of epilepsy after head injury. However, these animal models do not easily mimic the pathological events occurring in epilepsy. Therefore, animal models of PTE are an inappropriate tool for screening new and putatively effective AEDs. Chemical kindling is the most common animal model used to study epilepsy. There is a strong similarity between the kindling model and different types of human epilepsy. CONCLUSION: Today, researchers use experimental animal models to evaluate new anticonvulsant drugs. The chemical kindling models, such as pentylenetetrazol, bicuculline, and picrotoxin-induced seizures, are important experimental models to analyze the impact of putative antiepileptic drugs.

Aconitum Alkaloid Songorine Exerts Potent Gamma-Aminobutyric Acid-A Receptor Agonist Action In Vivo and Effectively Decreases Anxiety without Adverse Sedative or Psychomotor Effects in the Rat.

Songorine (SON) is a diterpenoid alkaloid from Aconitum plants. Preparations of Aconitum roots have been employed in traditional oriental herbal medicine, however, their mechanisms of action are still unclear. Since GABA-receptors are possible brain targets of SON, we investigated which subtypes of GABA-receptors contribute to the effects of SON, and how SON affects anxiety-like trait behavior and psychomotor cognitive performance of rats. First, we investigated the effects of microiontophoretically applied SON alone and combined with GABA-receptor agents picrotoxin and saclofen on neuronal firing activity in various brain areas. Next, putative anxiolytic effects of SON (1.0-3.0 mg/kg) were tested against the GABA-receptor positive allosteric modulator reference compound diazepam (1.0-5.0 mg/kg) in the elevated zero maze (EOM). Furthermore, basic cognitive effects were assessed in a rodent version of the psychomotor vigilance task (PVT). Local application of SON predominantly inhibited the firing activity of neurons. This inhibitory effect of SON was successfully blocked by GABA(A)-receptor antagonist picrotoxin but not by GABA(B)-receptor antagonist saclofen. Similar to GABA(A)-receptor positive allosteric modulator diazepam, SON increased the time spent by animals in the open quadrants of the EOM without any signs of adverse psychomotor and cognitive effects observed in the PVT. We showed that, under in vivo conditions, SON acts as a potent GABA(A)-receptor agonist and effectively decreases anxiety without observable side effects. The present findings facilitate the deeper understanding of the mechanism of action and the widespread pharmacological use of diterpene alkaloids in various CNS indications.

The CaV1.2 G406R mutation decreases synaptic inhibition and alters L-type Ca2+ channel-dependent LTP at hippocampal synapses in a mouse model of Timothy Syndrome.

Genetic alterations in autism spectrum disorders (ASD) frequently disrupt balance between synaptic excitation and inhibition and alter plasticity in the hippocampal CA1 region. Individuals with Timothy Syndrome (TS), a genetic disorder caused by CaV1.2 L-type Ca2+ channel (LTCC) gain-of function mutations, such as G406R, exhibit social deficits, repetitive behaviors, and cognitive impairments characteristic of ASD that are phenocopied in TS2-neo mice expressing G406R. Here, we characterized hippocampal CA1 synaptic function in male TS2-neo mice and found basal excitatory transmission was slightly increased and inhibitory transmission strongly decreased. We also found distinct impacts on two LTCC-dependent forms of long-term potentiation (LTP) synaptic plasticity that were not readily consistent with LTCC gain-of-function. LTP induced by high-frequency stimulation (HFS) was strongly impaired in TS2-neo mice, suggesting decreased LTCC function. Yet, CaV1.2 expression, basal phosphorylation, and current density were similar for WT and TS2-neo. However, this HFS-LTP also required GABAA receptor activity, and thus may be impaired in TS2-neo due to decreased inhibitory transmission. In contrast, LTP induced in WT mice by prolonged theta-train (PTT) stimulation in the presence of a ß-adrenergic receptor agonist to increase CaV1.2 phosphorylation was partially induced in TS2-neo mice by PTT stimulation alone, consistent with increased LTCC function. Overall, our findings provide insights regarding how altered CaV1.2 channel function disrupts basal transmission and plasticity that could be relevant for neurobehavioral alterations in ASD.

Picrotoxin Delineates Different Transport Configurations for Malate and γ Aminobutyric Acid through TaALMT1.

Plant-derived pharmacological agents have been used extensively to dissect the structure-function relationships of mammalian GABA receptors and ion channels. Picrotoxin is a non-competitive antagonist of mammalian GABAA receptors. Here, we report that picrotoxin inhibits the anion (malate) efflux mediated by wheat (Triticum aestivum) ALMT1 but has no effect on GABA transport. The EC50 for inhibition was 0.14 nM and 0.18 nM when the ALMTs were expressed in tobacco BY2 cells and in Xenopus oocytes, respectively. Patch clamping of the oocyte plasma membrane expressing wheat ALMT1 showed that picrotoxin inhibited malate currents from both sides of the membrane. These results demonstrate that picrotoxin inhibits anion efflux effectively and can be used as a new inhibitor to study the ion fluxes mediated by ALMT proteins that allow either GABA or anion transport.

Retinal ganglion cell desensitization is mitigated by varying parameter constant excitation pulse trains.

Retinal prostheses partially restore vision in patients blinded by retinitis pigmentosa (RP) and age-related macular degeneration (AMD). One issue that limits the effectiveness of retinal stimulation is the desensitization of the retina response to repeated pulses. Rapid fading of percepts is reported in clinical studies. We studied the retinal output evoked by fixed pulse trains vs. pulse trains that have variable parameters pulse-to-pulse. We used the current clamp to record RGC spiking in the isolated mouse retina. Trains of biphasic current pulses at different frequencies and amplitudes were applied. The main results we report are: (1) RGC desensitization was induced by increasing stimulus frequency, but was unrelated to stimulus amplitude. Desensitization persisted when the 20 Hz stimulation pulses were applied to the retinal ganglion cells at 65 µA, 85 µA, and 105 µA. Subsequent pulses in the train evoked fewer spikes. There was no obvious desensitization when 2 Hz stimulation pulse trains were applied. (2) Blocking inhibitory GABAA receptor increased spontaneous activity but did not reduce desensitization. (3) Pulse trains with constant charge or excitation (based on strength-duration curves) but varying pulse width, amplitude, and shape increased the number of evoked spikes/pulse throughout the pulse train. This suggests that retinal desensitization can be partially overcome by introducing variability into each pulse.

Exposure to GABAA Receptor Antagonist Picrotoxin in Pregnant Mice Causes Autism-Like Behaviors and Aberrant Gene Expression in Offspring.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is characterized by impairments in social interaction and restricted/repetitive behaviors. The neurotransmitter γ-aminobutyric acid (GABA) through GABAA receptor signaling in the immature brain plays a key role in the development of neuronal circuits. Excitatory/inhibitory imbalance in the mature brain has been investigated as a pathophysiological mechanism of ASD. However, whether and how disturbances of GABA signaling in embryos that are caused by GABAA receptor inhibitors cause ASD-like pathophysiology are poorly understood. The present study examined whether exposure to the GABAA receptor antagonist picrotoxin causes ASD-like pathophysiology in offspring by conducting behavioral tests from the juvenile period to adulthood and performing gene expression analyses in mature mouse brains. Here, we found that male mice that were prenatally exposed to picrotoxin exhibited a reduction of active interaction time in the social interaction test in both adolescence and adulthood. The gene expression analyses showed that picrotoxin-exposed male mice exhibited a significant increase in the gene expression of odorant receptors. Weighted gene co-expression network analysis showed a strong correlation between social interaction and enrichment of the "odorant binding" pathway gene module. Our findings suggest that exposure to a GABAA receptor inhibitor during the embryonic period induces ASD-like behavior, and impairments in odorant function may contribute to social deficits in offspring.

GABA Neurotransmission of the Suprachiasmatic Nucleus Is Modified During Rat Postnatal Development.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the brain structure that controls circadian rhythms in mammals. The SCN is formed by two neuroanatomical regions: the ventral and dorsal. Gamma-aminobutyric acid (GABA) neurotransmission is important for the regulation of circadian rhythms. Excitatory GABA effects have been described in both SCN regions displaying a circadian variation. Moreover, the GABAergic system transfers photic information from the ventral to the dorsal SCN. However, there is almost no knowledge about GABA neurotransmission during the prenatal or postnatal development of the SCN. Here, we used whole-cell patch-clamp recordings to study spontaneous inhibitory postsynaptic currents (IPSCs) in the two SCN regions, at two zeitgeber times (day or night), and at four postnatal (P) ages: P3-5, P7-9, P12-15, and P20-25. The results herein show that the three analyzed parameters of the IPSCs, frequency, amplitude, and decay time, were significantly affected by the postnatal age: mostly, the IPSC frequency increased with age, principally in the ventral SCN in both day and night recordings; similarly, the amplitude of IPSCs augmented with age, especially at night, whereas the IPSC decay time was reduced (it was faster) with postnatal age, mainly during the day. Our findings first reveal that parameters of GABA neurotransmission are modified by postnatal development, implying that synaptic adjustments are required for an appropriate maturation of the GABAergic system in the SCN.

Revision of the Unstable Picrotoxinin Hydrolysis Product.

The plant metabolite picrotoxinin (PXN) is a widely used tool in neuroscience for the identification of GABAergic signaling. Its hydrolysis in weakly alkaline media has been observed for over a century and the structure of the unstable hydrolysis intermediate was assigned by analogy to the degradation product picrotoxic acid. Here we show this assignment to be in error and we revise the structure of the hydrolysis product by spectroscopic characterization in situ. Counterintuitively, hydrolysis occurs at a lactone that remains closed in the major isolable degradation product, which accounts for the longstanding mistake in the literature.

The role of ligand-gated chloride channels in behavioural alterations at elevated CO2 in a cephalopod.

Projected future carbon dioxide (CO2) levels in the ocean can alter marine animal behaviours. Disrupted functioning of γ-aminobutyric acid type A (GABAA) receptors (ligand-gated chloride channels) is suggested to underlie CO2-induced behavioural changes in fish. However, the mechanisms underlying behavioural changes in marine invertebrates are poorly understood. We pharmacologically tested the role of GABA-, glutamate-, acetylcholine- and dopamine-gated chloride channels in CO2-induced behavioural changes in a cephalopod, the two-toned pygmy squid (Idiosepius pygmaeus). We exposed squid to ambient (∼450 µatm) or elevated (∼1000 µatm) CO2 for 7 days. Squid were treated with sham, the GABAA receptor antagonist gabazine or the non-specific GABAA receptor antagonist picrotoxin, before measurement of conspecific-directed behaviours and activity levels upon mirror exposure. Elevated CO2 increased conspecific-directed attraction and aggression, as well as activity levels. For some CO2-affected behaviours, both gabazine and picrotoxin had a different effect at elevated compared with ambient CO2, providing robust support for the GABA hypothesis within cephalopods. In another behavioural trait, picrotoxin but not gabazine had a different effect in elevated compared with ambient CO2, providing the first pharmacological evidence, in fish and marine invertebrates, for altered functioning of ligand-gated chloride channels, other than the GABAAR, underlying CO2-induced behavioural changes. For some other behaviours, both gabazine and picrotoxin had a similar effect in elevated and ambient CO2, suggesting altered function of ligand-gated chloride channels was not responsible for these CO2-induced changes. Multiple mechanisms may be involved, which could explain the variability in the CO2 and drug treatment effects across behaviours.

Antagonistic surround responses in different cones are mediated by feedback synapses from different horizontal cells.

Cone photoreceptors are the first neurons along the visual pathway that exhibit center-surround antagonistic receptive fields, the basic building blocks for spatial information processing in the visual system. The surround responses in cones are mediated by the horizontal cells (HCs) via multiple feedback synaptic mechanisms. It has been controversial on which mechanisms are responsible for the surround-elicited depolarizing responses in cones (ΔVCone(s)), and whether the surround responses of various types of cones are mediated by the same HC feedback mechanisms. In this report, we studied ΔVCone(s)) of four types of cones in the salamander retina, and found that they are mediated by feedback synapses from A-type, B-type or A- and B-type HCs. ΔVCone(s) are observable in the presence of concomitant center light spots, and surround + center light stimuli of various intensity, size and wavelength differentially activate the feedback synapses from A- and B-type HCs to cones. We found that ΔVCone(s) of the L-cones are mediated by both A- and B-type HCs, those of the P- and S-cones by B-type HCs, and those of the A-cones by the A-type HCs. Moreover, our results suggest that B-type HCs mediate ΔVCone(s) through both GABAergic and GluT-ClC feedback synaptic mechanisms, and A-type HCs mediate ΔVCone(s) via the GluT-ClC feedback mechanism. Feedback synaptic mechanisms that increase calcium influx in cone synaptic terminals play important roles in mediating the antagonistic surround responses in the postsynaptic bipolar cells, but they may not generate enough current to depolarize the cones and significantly contribute to ΔVCone(s).

Functions of the GABAergic system on serum LH concentrations in pre-pubertal Nellore heifers.

This study was conducted to evaluate the luteinizing hormone (LH) secretion pattern after gamma-aminobutyric acid (GABAA) antagonist to determine the effects of the GABAergic system on LH secretion during reproductive maturation in pre-pubertal Nellore heifers. Nellore heifers (n = 10) were administered a picrotoxin injection of 0.18 mg/kg, i.v. Blood samples were collected every 15 min for 3 h at different developmental stages (8, 10, 14 and 17 mo of age). Plasma concentrations of LH were quantified using an RIA (sensitivity of 0.04 ng/mL and CV of 15 %). There was an interaction between treatment and age (P = 0.034). Picrotoxin-treated heifers had lesser (P ≤  0.05) LH mean concentrations during a 3 h period at 10 and 17 mo of age compared to control heifers (P ≤  0.05). Comparing the period before and after Picrotoxin injection in the same animals, there was a 33 % decrease in LH concentration at 8 mo of age (P = 0.0165). These results indicate the GABAergic system has a stimulatory function in inducing LH secretion in pre-pubertal Nellore heifers. These findings corroborate previous results that GABA increases GnRH/LH secretion in other species during the pre-pubertal period.

Effects of Tempol on Epileptic Activity in Picrotoxin-Induced Epilepsy in Rats.

INTRODUCTION: Epilepsy is a common neurological disease, although its etiology and pathophysiology are not yet fully understood. Oxidative stress plays a key role in the pathogenesis of many neurological diseases, including epilepsy, and there have been many studies reporting that antiepileptic medicines with neuroprotective and antioxidant activity inhibit free oxygen radicals. This study evaluates the effects of tempol on epileptic activity through behavioral parameters in acute picrotoxin (Ptx) models. MATERIALS AND METHODS: This experimental study was conducted on 42 adult male Wistar Albino rats weighing 450-500 g. Ptx (2.5 mg/kg) was injected i.p. as a single dose and observed for one hour to establish the acute Ptx model. Following injection, the animals were observed for 30 min in glass observation cages measuring 35 cm x 35 cm x 35 cm. RESULTS: In picrotoxin-induced epilepsy, the total number of seizures and the total duration of seizures were decreased significantly with Ptx + tempol 100 mg/kg and Ptx + Tempol 150 mg/kg. The seizure phases were reduced significantly by Ptx + tempol 150 mg/kg (P < 0.05). CONCLUSION: Tempol 100 mg/kg and tempol 150 mg/kg are found to be effective in epilepsy models caused by Ptx, with tempol 150 mg/kg found especially to be more effective.

Direct Structural Insights into GABAA Receptor Pharmacology.

GABAA receptors are pentameric ligand-gated ion channels that mediate most fast neuronal inhibition in the brain. In addition to their important physiological roles, they are noteworthy in their rich pharmacology; prominent drugs used for anxiety, insomnia, and general anesthesia act through positive modulation of GABAA receptors. Direct structural information for how these drugs work was absent until recently. Efforts in structural biology over the past few years have revealed how important drug classes and natural products interact with the GABAA receptor, providing a foundation for studies in dynamics and structure-guided drug design. Here, we review recent developments in GABAA receptor structural pharmacology, focusing on subunit assemblies of the receptor found at synapses.

Chrysin reduces anxiety-like behavior through actions on GABAA receptors during metestrus-diestrus in the rat.

Low concentrations of ovarian hormones, among other factors, are associated with greater vulnerability to negative effects of environmental stressors and may trigger anxiety symptoms in females. The flavonoid chrysin (5,7-dihydroxyflavone) exerts anxiolytic-like effects in male and ovariectomized female rats, but it is unknown if chrysin could reduce anxiety-like behavior that naturally occurs through the ovarian cycle phases. The present study evaluated the effect of chrysin on anxiety-like behavior associated with the ovarian cycle phases in rats and the participation of γ-aminobutyric acid-A (GABAA) receptors in these actions. The acute effects of chrysin (2 mg/kg) were investigated in female cycling Wistar rats in the elevated plus maze, locomotor activity test, and light/dark test. Diazepam (2 mg/kg) was used as reference anxiolytic drug. The participation of GABAA receptor in the anxiolytic actions of chrysin was explored by pretreating the rats with the noncompetitive GABAA chloride ion channel antagonist picrotoxin (1 mg/kg). Chrysin and diazepam prevented anxiety-like behavior that was associated with the metestrus-diestrus phase in both the elevated plus maze and light/dark test, and these effects were reversed by picrotoxin, with no significant changes in spontaneous locomotor activity. No significant motor effects of chrysin were detected in either behavioral test during proestrus-estrus or metestrus-diestrus phases, whereas diazepam produced motor hypoactivity in the locomotor activity test during proestrus-estrus phase. These results indicate that the flavonoid chrysin prevents anxiety-like behavior that naturally occurs during metestrus-diestrus in two unconditioned models that are used to evaluate anxiety-like behavior, and these effects were mediated by actions on GABAA receptors.

Frequency- and state-dependent effects of hippocampal neural disinhibition on hippocampal local field potential oscillations in anesthetized rats.

Reduced inhibitory GABA function, so-called neural disinhibition, has been implicated in cognitive disorders, including schizophrenia and age-related cognitive decline. We previously showed in rats that hippocampal disinhibition by local microinfusion of the GABA-A receptor antagonist picrotoxin disrupted memory and attention and enhanced hippocampal multi-unit burst firing recorded around the infusion site under isoflurane anesthesia. Here, we analyzed the hippocampal local field potential (LFP) recorded alongside the multi-unit data. We predicted frequency-specific LFP changes, based on previous studies implicating GABA in hippocampal oscillations, with the weight of evidence suggesting that disinhibition would facilitate theta and disrupt gamma oscillations. Using a new semi-automated method based on the kurtosis of the LFP peak-amplitude distribution as well as on amplitude envelope thresholding, we separated three distinct hippocampal LFP states under isoflurane anesthesia: "burst" and "suppression" states-high-amplitude LFP spike bursts and the interspersed low-amplitudeperiods-and a medium-amplitude "continuous" state. The burst state showed greater overall power than suppression and continuous states and higher relative delta/theta power, but lower relative beta/gamma power. The burst state also showed reduced functional connectivity across the hippocampal recording area, especially around theta and beta frequencies. Overall neuronal firing was higher in the burst than the other two states, whereas the proportion of burst firing was higher in burst and continuous states than the suppression state. Disinhibition caused state- and frequency-dependent LFP changes, tending to increase power at lower frequencies (<20 Hz), but to decrease power and connectivity at higher frequencies (>20 Hz) in burst and suppression states. The disinhibition-induced enhancement of multi-unit bursting was also state-dependent, tending to be more pronounced in burst and suppression states than the continuous state. Overall, we characterized three distinct hippocampal LFP states in isoflurane-anesthetized rats. Disinhibition changed hippocampal LFP oscillations in a state- and frequency-dependent way. Moreover, the disinhibition-induced enhancement of multi-unit bursting was also LFP state-dependent.

Spontaneous and evoked neurotransmission are partially segregated at inhibitory synapses.

Synaptic transmission is initiated via spontaneous or action-potential evoked fusion of synaptic vesicles. At excitatory synapses, glutamatergic receptors activated by spontaneous and evoked neurotransmission are segregated. Although inhibitory synapses also transmit signals spontaneously or in response to action potentials, they differ from excitatory synapses in both structure and function. Therefore, we hypothesized that inhibitory synapses may have different organizing principles. We report picrotoxin, a GABAAR antagonist, blocks neurotransmission in a use-dependent manner at rat hippocampal synapses and therefore can be used to interrogate synaptic properties. Using this tool, we uncovered partial segregation of inhibitory spontaneous and evoked neurotransmission. We found up to 40% of the evoked response is mediated through GABAARs which are only activated by evoked neurotransmission. These data indicate GABAergic spontaneous and evoked neurotransmission processes are partially non-overlapping, suggesting they may serve divergent roles in neuronal signaling.

Glycinergic and GABAergic interneurons shift the location and differentially alter the size of ganglion cell receptive field centers in the mammalian retina.

By using the multi-electrode array (MEA) recording technique in conjunction with white-noise checkerboard stimuli and reverse correlation methods, we studied modulatory actions of glycinergic and GABAergic interneurons on spatiotemporal profiles of ganglion cells (GCs) in dark-adapted mouse retinas. We found that application of 2 µM strychnine decreased receptive field center radii of GCs by a mean value of 11%, and shifted the GC receptive field (RF) centers by a mean distance of 28.3 µm. On the other hand, 200 µM picrotoxin + 100 µM bicuculline + 50 µM TPMPA increased GC receptive field center radii by a mean value of 19%, and shifted the GC RF centers by a mean distance of 53.7 µm. Glycinergic neurons in the mouse retina are narrow-field amacrine cells that have been shown to mediate ON-OFF crossover inhibitory synapses within the RGs' RF center, therefore they may increase the size and shift the location of GC RF center by synergistic addition to bipolar cell inputs to GCs. GABAergic neurons are wide-field amacrine cells and horizontal cells that are known to mediate antagonistic surround responses of GCs, and thus they decrease the GCs' RF center size. Our results suggest that a major global function of glycinergic and GABAergic interneurons in the mammalian retina is to provide the flexibility for adjusting the size and location of GCs' RF centers. The apparent shifts of GC RF centers suggest that the synergistic addition by GlyACs and the surround inhibition by GABAergic interneurons are not spatially symmetrical within GC RFs.