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High levels of IL1β can result in chronic inflammation, which in turn can promote tumor growth and metastasis. Inhibition of IL1β could therefore be a promising therapeutic option in the treatment of cancer. Here, the effects of IL1β blockade induced by the mAbs canakinumab and gevokizumab were evaluated alone or in combination with docetaxel, anti–programmed cell death protein 1 (anti–PD-1), anti-VEGFα, and anti-TGFβ treatment in syngeneic and humanized mouse models of cancers of different origin. Canakinumab and gevokizumab did not show notable efficacy as single-agent therapies; however, IL1β blockade enhanced the effectiveness of docetaxel and anti–PD-1. Accompanying these effects, blockade of IL1β alone or in combination induced significant remodeling of the tumor microenvironment (TME), with decreased numbers of immune suppressive cells and increased tumor infiltration by dendritic cells (DC) and effector T cells. Further investigation revealed that cancer-associated fibroblasts (CAF) were the cell type most affected by treatment with canakinumab or gevokizumab in terms of change in gene expression. IL1β inhibition drove phenotypic changes in CAF populations, particularly those with the ability to influence immune cell recruitment. These results suggest that the observed remodeling of the TME following IL1β blockade may stem from changes in CAF populations. Overall, the results presented here support the potential use of IL1β inhibition in cancer treatment. Further exploration in ongoing clinical studies will help identify the best combination partners for different cancer types, cancer stages, and lines of treatment.

Supplementary tables S1-S5 and figures S1-S7

High levels of IL1β can result in chronic inflammation, which in turn can promote tumor growth and metastasis. Inhibition of IL1β could therefore be a promising therapeutic option in the treatment of cancer. Here, the effects of IL1β blockade induced by the mAbs canakinumab and gevokizumab were evaluated alone or in combination with docetaxel, anti–programmed cell death protein 1 (anti–PD-1), anti-VEGFα, and anti-TGFβ treatment in syngeneic and humanized mouse models of cancers of different origin. Canakinumab and gevokizumab did not show notable efficacy as single-agent therapies; however, IL1β blockade enhanced the effectiveness of docetaxel and anti–PD-1. Accompanying these effects, blockade of IL1β alone or in combination induced significant remodeling of the tumor microenvironment (TME), with decreased numbers of immune suppressive cells and increased tumor infiltration by dendritic cells (DC) and effector T cells. Further investigation revealed that cancer-associated fibroblasts (CAF) were the cell type most affected by treatment with canakinumab or gevokizumab in terms of change in gene expression. IL1β inhibition drove phenotypic changes in CAF populations, particularly those with the ability to influence immune cell recruitment. These results suggest that the observed remodeling of the TME following IL1β blockade may stem from changes in CAF populations. Overall, the results presented here support the potential use of IL1β inhibition in cancer treatment. Further exploration in ongoing clinical studies will help identify the best combination partners for different cancer types, cancer stages, and lines of treatment.

Immune-stimulator antibody conjugates (ISAC) combining tumor-targeting monoclonal antibodies with immunostimulatory agents allow targeted delivery of immune activators into tumors. NJH395 is a novel, first-in-class ISAC comprising a Toll‐like receptor 7 (TLR7) agonist conjugated to an anti-HER2 antibody via a noncleavable linker payload. Preclinical characterization showed ISAC-mediated activation of myeloid cells in the presence of antigen-expressing cancer cells, with antigen targeting and TLR7 agonism contributing to antitumor activity. Safety, efficacy, immunogenicity, pharmacokinetics, and pharmacodynamics were investigated in a phase I, multicenter, open-label study in patients with HER2+ non-breast advanced malignancies (NCT03696771). Data from 18 patients enrolled in single ascending dose escalation demonstrated delivery of the TLR7-agonist payload in HER2+ tumor cells and induction of type I IFN responses, which correlated with immune modulation in the tumor microenvironment. Cytokine release syndrome was a common, but manageable, drug-related adverse event. Antidrug antibodies and neuroinflammation at high doses represented significant clinical challenges. Data provide proof-of-mechanism and critical insights for novel immunotherapies.

Supplementary Figure from Preclinical Characterization and Phase I Study of an Anti–HER2-TLR7 Immune-Stimulator Antibody Conjugate in Patients with HER2+ Malignancies

Supplementary Data from Preclinical Characterization and Phase I Study of an Anti–HER2-TLR7 Immune-Stimulator Antibody Conjugate in Patients with HER2+ Malignancies

Supplementary Figures - PDF file 1385K, File contains Supplementary figures of resistant cell lines, proliferation assay of a panel of cell lines, TdF binding assay transcriptional out and kinome selectivity

Immune-stimulator antibody conjugates (ISAC) combining tumor-targeting monoclonal antibodies with immunostimulatory agents allow targeted delivery of immune activators into tumors. NJH395 is a novel, first-in-class ISAC comprising a Toll-like receptor 7 (TLR7) agonist conjugated to an anti-HER2 antibody via a noncleavable linker payload. Preclinical characterization showed ISAC-mediated activation of myeloid cells in the presence of antigen-expressing cancer cells, with antigen targeting and TLR7 agonism contributing to antitumor activity. Safety, efficacy, immunogenicity, pharmacokinetics, and pharmacodynamics were investigated in a phase I, multicenter, open-label study in patients with HER2+ non-breast advanced malignancies (NCT03696771). Data from 18 patients enrolled in single ascending dose escalation demonstrated delivery of the TLR7-agonist payload in HER2+ tumor cells and induction of type I IFN responses, which correlated with immune modulation in the tumor microenvironment. Cytokine release syndrome was a common, but manageable, drug-related adverse event. Antidrug antibodies and neuroinflammation at high doses represented significant clinical challenges. Data provide proof-of-mechanism and critical insights for novel immunotherapies.

Chronic (18 h) exposure of cultured hippocampal slices to the type-A GABA receptor blocker, bicuculline methiodide (BMI) 10 micro m increased the levels of connexin 43 (Cx43) and connexin 32 (Cx32) mRNAs, but not connexin 26 and connexin 36, as demonstrated by RNase protection assays. The levels of Cx43 and Cx32 proteins in membrane fractions detected by western blotting were also significantly increased. Immunoblotting indicated that BMI also promoted a significant expression of the transcription protein c-fos. The rate of fluorescence recovery after photobleaching, an index of gap junctional coupling, was also significantly increased, whereas it was blocked by the gap junctional blocker, carbenoxolone (100 micro m). Extracellular recordings in CA1 stratum pyramidale, performed in BMI-free solution, demonstrated that BMI-exposed cultures possessed synaptic responses characteristic of epileptiform discharges: (i) significantly greater frequency of spontaneous epileptiform discharges, (ii) post-synaptic potentials with multiple population spikes, and (iii) significantly longer duration of primary afterdischarges. Carbenoxolone (100 micro m), but not its inactive analog, oleanolic acid (100 micro m), reversibly inhibited spontaneous and evoked epileptiform discharges. The findings of BMI-induced parallel increases in levels of gap junction expression and function, and the increase in epileptiform discharges, which were sensitive to gap junctional blockers, are consistent with the hypothesis that increased gap junctional communication plays an intrinsic role in the epileptogenic process.

Seizure-induced cell death produced by repeated tetanic stimulation in vitro: possible role of endoplasmic reticulum calcium stores. Seizures may cause brain damage due to mechanisms initiated by excessive excitatory synaptic transmission. One such mechanism is the activation of death-promoting intracellular cascades by the influx and the perturbed homeostasis of Ca2+. The neuroprotective effects of preventing the entry of Ca2+from voltage-dependent Ca2+channels, NMDA receptors, and non-NMDA receptors, is well known. Less clear is the contribution to excitotoxicity of Ca2+released from endoplasmic reticulum (ER) stores. We produced epileptiform discharges in combined entorhinal cortex/hippocampus slices using repeated tetanic stimulation of the Schaffer collaterals and assessed cell death after 1, 3, or 12–14 h with gel electrophoresis of genomic DNA and immunohistologically using terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine 5′-triphosphate (dUTP) nick end labeling (TUNEL) staining. We manipulated ER Ca2+stores using two conventional drugs, dantrolene, which blocks the Ca2+release channel, and thapsigargin, which blocks sarco-endoplasmic reticulum Ca2+-ATPases resulting in depletion of ER Ca2+stores. To monitor epileptogenesis, and to assess effects attributable to dantrolene and thapsigargin on normal synaptic transmission, extracellular potentials were recorded in stratum pyramidale of the CA1 region. Repeated tetanic stimulation reliably produced primary afterdischarge and spontaneous epileptiform discharges, which persisted for 14 h, the longest time recorded. We did not observe indications of cell death attributable to seizures with either method when assessed after 1 or 3 h; however, qualitatively more degraded DNA always was observed in tetanized slices from the 12- to 14-h group compared with time-matched controls. Consistent with these data was a significant, fourfold, increase in the percentage of TUNEL-positive cells in CA3, CA1, and entorhinal cortex in tetanized slices from the 12- to 14-h group (16.5 ± 4.4, 33.7 ± 7.1, 11.6 ± 2.1, respectively; means ± SE; n = 7) compared with the appropriate time-matched control (4.1 ± 2.2, 7.3 ± 2.0, 2.8 ± 0.9, respectively; n = 6). Dantrolene (30 μM; n = 5) and thapsigargin (1 μM; n = 4) did not affect significantly normal synaptic transmission, assessed by the amplitude of the population spike after 30 min of exposure. Dantrolene and thapsigargin also were without effect on the induction or the persistence of epileptiform discharges, but both drugs prevented seizure-induced cell death when assessed with gel electrophoresis. We suggest that Ca2+entering a cell from the outside, in addition to the Ca2+contributed from ryanodine-sensitive stores (i.e., Ca2+-induced Ca2+release), may be necessary for seizure-induced cell death.

The expression of immunoglobulin (Ig) genes depends on tissue-specific elements in the promoter and enhancer regions of light chain and heavy chain genes. In contrast to the complex modular character of Ig enhancers, the promoters appear to be simple, depending primarily on a conserved TATA box and octamer elements. We have analyzed the role of proximal sequences for Igkappa promoter function. Igkappa promoter transcription critically depends on initiator-like sequences and on a downstream element located at +24 to +39 relative to the start site. Replacement of these sequences resulted in strong reduction of promoter activity. In vitro, these elements were found to be more effective in extracts of lymphoid than of non-lymphoid origin. Deletion of the downstream and initiation site regions had a comparable effect on promoter activity to obliteration of the TATA box or octamer element. The downstream sequence was bound by two nuclear proteins, identical to the previously identified Ig-specific C5 and C6 complexes. Whereas C5 is found in HeLa cells and in lymphoid cells, C6 is lymphoid specific. Thus, further specific sequences in addition to the previously characterized elements, the octamer and the TATA box, are required for efficient kappa promoter expression in B lymphocytes.

The operation of a motor vehicle requires the integrity of sensory, motor, and intellectual faculties. Impairment of these faculties following the consumption of alcohol has been studied extensively through laboratory, closed-course and on-road driving, and epidemiological studies. The scientific literature was reviewed critically, with a focus on low-to-moderate blood alcohol concentrations (BAC ≤ 0.100%), to identify the most reliable determinants of alcohol-impaired driving. Variables such as age, gender, driving skill, and tolerance were shown to have limited impact on impairment. It was concluded the most relevant variables are BAC and complexity of the driving task. The scientific literature provides a high degree of confidence to support the conclusion that a BAC of 0.050% impairs faculties required in the operation of a motor vehicle. Whether impairment is apparent depends upon the complexity of the driving task, which applies to both study design and actual driving.

Twenty-seven pregnant rats from three genetic lines, Roman High Avoidance (RHA), Roman Low Avoidance (RLA), and Satinder's Heterogeneous Stock (SHS), received daily SC injections of either 0.075 mg/kg CdCl2 (low dose), 0.225 mg/kg CdCl2 (high dose), or an equivalent volume of saline vehicle (control) throughout gestation. Cd-exposed progeny from the RHA genetic line weighed significantly less than RHA control progeny (pd 35-44); however, SHS progeny from the low-dose group weighed significantly more than progeny from any other group (pd 14-44). Unconditioned escape response (UER) level was determined on pd 39. Progeny from the high-dose group required a significantly lower UER level as compared to the low-dose group. Acquisition of conditioned avoidance responses was tested from pd 41 to 44. There were significant differences due to dose for one-way avoidance responses. Cd-related differences in one-way avoidance were restricted to progeny from the SHS genetic line. SHS progeny from the high-dose group demonstrated significantly more one-way responses when compared to the control group. Differences in avoidance responses are discussed in relation to Cd-induced hypernociception. The concentration of Cd in the liver and kidney of dams and progeny was determined by Inductively Coupled Plasma spectroscopy (ICP). There were significant dose-related differences in Cd concentration in maternal tissues.

Chronic (18 h) exposure of cultured hippocampal slices to the type-A GABA receptor blocker, bicuculline methiodide (BMI) 10 micro m increased the levels of connexin 43 (Cx43) and connexin 32 (Cx32) mRNAs, but not connexin 26 and connexin 36, as demonstrated by RNase protection assays. The levels of Cx43 and Cx32 proteins in membrane fractions detected by western blotting were also significantly increased. Immunoblotting indicated that BMI also promoted a significant expression of the transcription protein c-fos. The rate of fluorescence recovery after photobleaching, an index of gap junctional coupling, was also significantly increased, whereas it was blocked by the gap junctional blocker, carbenoxolone (100 micro m). Extracellular recordings in CA1 stratum pyramidale, performed in BMI-free solution, demonstrated that BMI-exposed cultures possessed synaptic responses characteristic of epileptiform discharges: (i) significantly greater frequency of spontaneous epileptiform discharges, (ii) post-synaptic potentials with multiple population spikes, and (iii) significantly longer duration of primary afterdischarges. Carbenoxolone (100 micro m), but not its inactive analog, oleanolic acid (100 micro m), reversibly inhibited spontaneous and evoked epileptiform discharges. The findings of BMI-induced parallel increases in levels of gap junction expression and function, and the increase in epileptiform discharges, which were sensitive to gap junctional blockers, are consistent with the hypothesis that increased gap junctional communication plays an intrinsic role in the epileptogenic process.

Dr. Carlen reviews the evidence that seizures may cause cell death and discusses possible strategies for preventing seizure-induced brain damage.

Lack of expression of the fragile X mental retardation protein (FMRP), due to silencing of the FMR1 gene, causes the Fragile X syndrome. Although FMRP was characterized previously to be an RNA binding protein, little is known about its function or the mechanisms underlying the Fragile X syndrome. Here we report that the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit, GluR1, was decreased in the cortical synapses, but not in the hippocampus or cerebellum, of FMR1 gene knockout mice. Reduced long-term potentiation (LTP) was also found in the cortex but not in the hippocampus. Another RNA binding protein, FXR; the N-methyl-D-aspartate receptor subunit, NR2; and other learning-related proteins including c-fos, synapsin, myelin proteolipid protein, and cAMP response element binding protein were not different between FMR1 gene knockout and wild-type mice. These findings suggest that the depressed cortical GluR1 expression and LTP associated with FMRP deficiency could contribute to the Fragile X phenotype.

ATP-sensitive potassium (KATP) channels are heteromultimer complexes of subunits from members of the inwardly rectifying K+channel and the ATP-binding cassette protein superfamilies. KATPchannels couple metabolic state to membrane excitability, are distributed widely, and participate in a variety of physiological functions. Understood best in pancreatic β cells, where their activation inhibits insulin release, KATPchannels have been implicated also in postischemia cardio- and neuroprotection. The dentate gyrus (DG) is a brain region with a high density of KATPchannels and is relatively resistant to ischemia/reperfusion-induced cell death. Therefore we were interested in describing the characteristics of single KATPchannels in DG granule cells. We recorded single KATPchannels in 59/105 cell-attached patches from DG granule cells in acutely prepared hippocampal slices. Single-channel openings had an EKclose to 0 mV (symmetrical K+) and were organized in bursts with a duration of 19.3 ± 1.6 (SE) ms and a frequency of 3.5 ± 0.8 Hz, a unitary slope conductance of 27 pS, and a low, voltage-independent, probability of opening ( Popen, 0.04 ± 0.01). Open and closed dwell-time histograms were fitted best with one (τopen= 1.3 ± 0.2 ms) and the sum of two (τclosed,fast= 2.6 ± 0.9 ms, τclosed,slow= 302.7 ± 67.7 ms) exponentials, respectively, consistent with a kinetic model having at least a single open and two closed states. The Popenwas reduced ostensibly to zero by the sulfonylureas, glybenclamide (500 nM, 2/6; 10 μM,11/14 patches) and tolbutamide (20 μM, 4/6; 100 μM, 4/4 patches). The blocking dynamics for glybenclamide included transition to a subconductance state (43.3 ± 2.6% of control Iopen channel). Unlike glybenclamide, the blockade produced by tolbutamide was reversible. In 5/5 patches, application of diazoxide (100 μM) increased significantly Popen(0.12 ± 0.02), which was attributable to a twofold increase in the frequency of bursts (8.3 ± 2.0 Hz). Diazoxide was without effect on τopenand τclosed,fastbut decreased significantly τclosed,slow(24.4 ± 2.6 ms). We observed similar effects in 6/7 patches after exposure to hypoxia/hypoglycemia, which increased significantly Popen(0.09 ± 0.03) and the frequency of bursts (7.1 ± 1.7 Hz) and decreased significantly τclosed,slow(29.5 ± 1.8 ms). We have presented convergent evidence consistent with single KATPchannel activity in DG granule cells. The subunit composition of KATPchannels native to DG granule cells is not known; however, the characteristics of the channel activity we recorded are representative of Kir6.1/SUR1, SUR2B-based channels.

It is well known that brain damage can cause epilepsy. Post-traumatic epilepsy (PTE), for instance, is a consequence of traumatic brain injury. The risk of PTE ranges from less than 5 percent of patients following blunt head trauma (e.g., simple skull fracture) to 50 percent in patients following penetrating wounds or those involving intracranial hematoma.11 One of the accepted causes of temporal-lobe epilepsy is perinatal brain injury, which can also lead to other epileptic syndromes, such as West’s syndrome and Lennox-Gastaut syndrome. Epilepsy can also be a presenting symptom of both brain tumors and strokes.

1. Focal cortical epilepsy was investigated by applying tetanic stimulation repeatedly (100 Hz. 2 s in duration, once every 10 min, 10 episodes) to layer III association fibers in rat piriform cortex slices and recording both extracellular and intracellular responses from the endopiriform nucleus. To promote excitability, piriform slices were incubated in artificial cerebrospinal fluid (ACSF) containing 0.9 mM Mg2+ and 5 mM K+, at an initial temperature of 10–12 degrees C, which was allowed to warm passively to room temperature. 2. Responses recorded extracellularly in the endopiriform nucleus consisted of two types: weak stimulation evoked an early-occurring, small-amplitude, negatively deflecting potential; strong stimulation evoked a more complex response comprising both an early potential of maximal amplitude and a later-occurring epileptiform potential of greater amplitude and longer duration. Late-occurring epileptiform potentials were not observed in slices incubated in ACSF at room temperature. 3. Both the early potential and the late-occurring epileptiform responses were abolished by the non-N-methyl-D-aspartic acid (non-NMDA) subtype of glutamate receptor blocker, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM). Application of D(-)-2-amino-5-phosphonopentanoic acid (APV; 50 microM) to block NMDA receptors was without effect on the early potential but diminished the late-occurring epileptiform potential. The late-occurring potential was unable to follow stimulation delivered at a frequency of 1 Hz. These results suggest that the early potential was generated monosynaptically and dependent solely on the activation of non-NMDA receptors, whereas the late-occurring epileptiform potential was polysynaptic in origin and possessed both a CNQX- and an APV-sensitive component. 4. Responses increased progressively in both amplitude and duration after tetanic stimulation. The threshold intensity required to evoke the complex dual-component potential was reduced by tetanic stimulation. An increase in multiunit spiking activity, indicating an increase in synchronous discharges, was also observed. A residual potential could be evoked in the presence of CNQX (10 microM) after the tetanic stimulation procedure. 5. Spontaneous discharges occurred as early as after the first episode of tetanic stimulation and persisted for the duration of the experiment. Spontaneous discharges were abolished by either CNQX or by a fourfold increase in extracellular Mg2+ concentration, the latter reversibly. APV reduced the frequency of spontaneous discharges by 38.6 +/- 9.3% (mean +/- SE). The conventional anticonvulsant drug 5,5-diphenylhydantoin, the benzodiazepine receptor agonist midazolam, and the benzodiazepine receptor antagonist flumazenil were without effect on the frequency of spontaneous discharges. Evoked responses were also unaffected by either 5,5-diphenylhydantoin or midazolam. Slices not exposed to cold ACSF, although demonstrating potentiation of evoked responses after tetanization did not produce spontaneous epileptiform discharges. 6. Intracellular recordings from endopiriform neurons revealed the cellular correlates of the extracellular responses. Weak stimulation evoked a small-amplitude depolarizing potential. Increasing the intensity of stimulation increased the amplitude of this response and also evoked a second depolarizing potential of greater amplitude occurring at variable latencies. Maximal stimulation evoked an action potential. After tetanic stimuli, responses resembling a paroxysmal depolarizing shift consisting of a depolarizing potential with superimposed multiple action potentials were evoked reliably. Passive membrane properties after repeated tetanic stimulation were not different when compared with control. 7. This novel model of in vitro focal cortical epilepsy has many features characteristic of conventional kindling including 1) progressive nature; 2) reduced threshold to evoke discharges; and 3) persist

Recordings were obtained from neurons in layer II/III of slices of rat frontal cortex maintained in vitro. We investigated whether brief application of the potassium channel blocker tetraethylammonium (TEA), which induces a novel form of synaptic plasticity in the CA1 region of the hippocampus referred to as LTPK, evokes similar responses in neocortex. Consistent with previous findings, TEA produced a persistent enhancement of excitatory transmission, which was independent of NMDA receptor activation but required the activation of nifedipine-sensitive voltage-dependent Ca2+ channels (VDCC), presumably the L-type. We also observed a persistent enhancement of presumptive CI(-)-dependent GABAA receptor-mediated transmission. Enhancement of excitatory and inhibitory synaptic transmission did not require activation of synapses with electrical stimulation during TEA application. The enhancement of excitatory, but not inhibitory synaptic transmission, was blocked when the Ca2+ chelator 1,2-bis(2-aminophenoxy)-ethane N,N,N',N'-tetraacetic acid (BAPTA) was included in the recording electrode. Under voltage clamp conditions that minimized the activation of L-type channels robust enhancement of both excitatory and inhibitory transmission was still observed. No enhancement of excitatory synaptic transmission was observed in the presence of NiCl2, a putative T-type channel blocker. The possible involvement of kinase activation was studied by including the non-specific and competitive kinase inhibitor (+/-)-1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) in the patch pipette. H-7 retarded the time course and reduced the magnitude of the enhancement of excitatory transmission. These results suggest that TEA-induced enhancement of excitatory transmission in the neocortex requires entry of Ca2+ into the postsynaptic neuron via VDCCs and possibly the activation of a kinase.

Summary: Purpose: The antiepileptic effects of benzodiaze-pine-receptor (BZR) agonists have been well documented. Surprisingly, an antiepileptic effect for the BZR antagonist, fluma-zenil, has also been described, the mechanism of which is unknown. We investigated the effects of nanomolar concentrations of flumazenil and a structurally dissimilar BZR antagonist, propyl-β-carboline-3-carboxylate (β-CCP), on normal synaptic responses and epileptiform discharges induced by a variety of methods in the CA1 region of rat hippocampal slices. Methods: Extracellular field potentials were recorded from stratum pyramidale of the CA1 region. Orthodromic stimulation was delivered by a bipolar electrode placed in the stratum radiatum at the border of the CA2/KA3 regions. Drugs were bath applied, and epileptiform discharges were quantified by using the Coastline Bursting Index, which calculates the total length of the discharge waveform of evoked multiple population spikes. For statistical comparisons, we calculated the Coastline Bursting Index for the average of five traces at the end of the control period (20 min), drug application (20 min), and washout (20–40 min). Results: Flumazenil was without effect on normal synaptic responses; however, flumazenil reduced epileptiform discharges evoked in the presence of high [K+]0, leu-enkephalin, the BZR inverse agonist, methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), or after a cold-shock procedure. β-CCP exhibited an action similar to that observed for flumazenil, suggesting that the antiepileptic effect is due to properties common to BZR antagonists. Conclusions: We suggest that the antiepileptic effect we observed for flumazenil and (β-CCP is mediated at the BZR and might be due to competition with endogenous BZR inverse agonists released preferentially during epileptiform activity.

Previous experiments have described highly specific effects of noradrenergic agonists on synaptic transmission in the dentate gyrus (DG). For example, perfusion of hippocampal slices with the beta-noradrenergic agonist isoproterenol induces a long-lasting potentiation (LLP) of extracellularly recorded responses following stimulation of the medial perforant path (PP), and long-lasting depression (LLD) of responses evoked by stimulation of the lateral PP (Dahl D, Sarvey JM, 1989, Proc Natl Acad Sci USA 86:4776–4780). To examine the possible interactions of LLP, LLD, and long-term potentiation induced by tetanic stimulation (LTP), the authors recorded extracellular field potentials evoked in the DG by stimulation of the lateral or medial perforant path following LTP and LLP or LLD, invoked in different orders. After establishment of LLP or LLD by path application of isoproternol, subsequent tetanization of the respective afferents resulted in additional potentiation of the medial PP-evoked response and return of the lateral PP-evoked response to baseline levels. In other slices application of isoproterenol after establishment of LTP resulted in further potentiation of medial PP-evoked responses but no change in the potentiated response evoked by lateral PP stimulation. Thus the pathway specificity was maintained irrespective of the history of previous potentiation or depression. Experiments using the specific beta1 antagonist metoprolol further confirmed pathway specificity. Perfusion with 20 μM of metoprolol appeared to reduce LTP evoked by stimulation of the medial but not lateral PP. In a subsequent experiment, metoprolol in the absence of tetanization produced LLD of the medial PP-evoked response and LLP of the lateral PP-evoked response, opposite to the effects of ISO. These results confirm the impressive extent of pathway specificity in the DG and reveal the persistent capacity for synaptic modification as exemplified by the processes of LLP, LLD, and LTP. © 1994 Wiley-Liss, Inc.

We reported previously that activation of alpha-2 adrenoceptors with infusions of clonidine into the amygdala/pyriform region is sufficient to retard kindling. To characterize further the involvement in kindling of alpha-2 receptors in the amygdala/pyriform, we exposed rats to unilateral intraamygdaloid infusions of a variety of noradrenergic drugs followed by either low-frequency stimulation of the amygdala, to induce rapid kindling, or conventional high-frequency stimulation. Infusions and electrical stimulation were administered once every 48 h. The prophylactic effects of clonidine were blocked by simultaneous infusion of idazoxan, an alpha-2 adrenergic antagonist, which suggests strongly that these effects were produced at an alpha-2 receptor. Intraamygdaloid infusions of xylazine, another alpha-2 agonist, also significantly retarded low-frequency kindling. Unexpectedly, intraamygdaloid infusions of the alpha-2 antagonists idazoxan, yohimbine, and SKF 104856 failed to accelerate kindling. Infusion of the alpha-1 antagonist corynanthine also failed to affect kindling. We propose that the alpha-2 adrenoceptors in the amygdala/pyriform region contribute to the prophylactic effects of systemically administered clonidine and that the facilitation of kindling observed after systemic administration of alpha-2 antagonists may be due to blockade of alpha-2 adrenoceptors outside of the amygdala/pyriform region.

It has been reported previously that systemic administration of clonidine, an agonist of alpha-2 noradrenergic receptors, significantly retards amygdaloid kindling by delaying the emergence from partial seizure. We examined the effect of either systemic administration (i.p.) or intra-amygdaloid infusions of clonidine on the kindling of seizures with electrical stimulation of the amygdala. Rats received either low-frequency stimulation of the amygdala, to induce rapid kindling, or conventional high-frequency stimulation. Clonidine and electrical stimulation were administered once every 48 h. We observed a significant retardation of kindling in rats receiving i.p. injections of clonidine (0.1 mg/kg) or infusions of clonidine in concentrations of 10(-7)-10(-4) M, regardless of the stimulation frequency. The prophylactic effect was due to a delay in the progression out of partial seizure. The effect was specific to the amygdala/pyriform region, because infusions of clonidine dorsal to the amygdala were without effect. Intra-amygdaloid clonidine had little effect on established generalized seizures, suggesting that it was producing a genuine prophylactic effect against kindling. We conclude that the subpopulation of alpha-2 adrenoceptors in the amygdala/pyriform region contributes to the antiepileptogenic effect observed after systemic administration of clonidine.

We examined the effects of the benzodiazepine antagonist, flumazenil, on epileptiform discharges evoked in the hippocampal CA1 region in vitro. Application of 100 nM flumazenil did not affect normal synaptic responses ; however, flumazenil did depress epileptiform discharges induced by 8 mM (K + ] o . Epileptiform discharges induced by the GABA A channel antagonist picrotoxin or by the K + channel blocker 4-aminopyridine were unaffected. Application of the high-affinity, low-efficacy benzodiazepine partial inverse agonist, Ro 19-4603, blocked the anticonvulsant effect of flumazenil, indicating that this action of flumazenil is mediated at a benzodiazepine binding site located on the GABA A receptor. A likely explanation of the present results is that flumazenil antagonizes the action of an endogenous benzodiazepine inverse agonist, which is released during epileptiform discharges evoked in high K + ACSF.

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