Identification of a hemagglutinin-specific idiotype associated with reovirus recognition shared by lymphoid and neural cells.

A xenogeneic antiserum raised to antireovirus immunoglobulin was used to define an idiotypic determinant present on antibodies to reovirus type 3 hemagglutinin. The same idiotype was identified on nonimmune lymphoid cells and on neuronal cells that specifically bind the hemagglutinin of type 3 reovirus. This idiotypic determinant, called Id3, is shared by (a) a monoclonal antibody to the neutralization site of hemagglutinin from type 3 reovirus; (b) BALB/c serum antibodies to the hemagglutinin of reovirus type 3; (c) R1.1, a murine thymoma cell line that binds reovirus type 3; (d) primary cultures of murine neuronal cells. The presence of an idiotype shared by antihemagglutinin antibodies and by structures on nonlymphoid cells suggests a general relationship between disparate receptors that recognize a common determinant. Furthermore, this suggests a novel approach for the study of viral receptor interactions and for analysis of mechanisms of autoimmune responses.

Cellular mechanisms of epilepsy: a status report.

The cellular phenomena underlying focal epilepsy are currently understood in the context of contemporary concepts of cellular and synaptic function. Interictal discharges appear to be due to a combination of synaptic events and intrinsic currents, the exact proportion of which in any given neuron may vary according to the anatomic and functional substrate involved in the epileptic discharge and the epileptogenic agent used in a given model. The transition to seizure appears to be due to simultaneous increments in excitatory influences and decrements in inhibitory processes--both related to frequency-dependent neuronal events. A variety of specific hypotheses have been proposed to account for the increased excitability that occurs during epileptiform activity. Although each of the proposed mechanisms is likely to contribute significantly to the epileptic process, no single hypothesis provides an exclusive unifying framework within which all kinds of focal epilepsy can be understood. The spread of epileptic activity throughout the brain, the development of primary generalized epilepsy, the existence of "gating" mechanisms in specific anatomic locations, and the extrapolation of hypotheses derived from simple models of focal epilepsy to explain more complex forms of human epilepsy, all are not yet fully understood.

Coexistence of acetylcholinesterase and somatostatin-immunoreactivity in neurons cultured from rat cerebrum.

Cultures derived from rat cerebral hemispheres were sequentially stained for acetylcholinesterase activity and for either somatostatin-like immunoreactivity or cholecystokinin-like immunoreactivity. Somatostatin-like immunoreactivity was found to coexist with acetylcholinesterase activity in individual neurons of several morphological subtypes, but cholecystokinin-like immunoreactivity and acetycholinesterase activity were never seen in the same neurons. These findings suggest a specific anatomical association, perhaps even an overlap, of the cholinergic and somatostatinergic systems in the mammalian cerebrum, and indicate that the combined deficiencies of somatostatin and cholinergic markers in Alzheimer's dementia and senile dementia of the Alzheimer type may be of pathophysiological importance.

Neural properties of cultured human endocrine tumor cells of proposed neural crest origin.

Cells from human endocrine tumors of proposed neural crest origin--five pheochromocytomas, two medullary carcinomas of the thyroid, and two bronchial carcinoids--were grown in monolayer culture. Cells from all nine tumors, including epithelial forms of medullary carcinoma of the thyroid and bronchial carcinoid cells, and epithelial and neuron-like pheochromocytoma cells demonstrated all-or-nothing, short-duration action potentials.

Characterization of polyamines having agonist, antagonist, and inverse agonist effects at the polyamine recognition site of the NMDA receptor.

The endogenous polyamines spermine and spermidine increase the binding of [3H]MK-801 to NMDA receptors. This effect is antagonized by diethylenetriamine (DET). We report here that spermine increases the rates of both association and dissociation of binding of [3H]MK-801, suggesting that it increases the accessibility of the binding site for MK-801 within the ion channel of the receptor complex. 1,10-Diaminodecane (DA10) inhibited the binding of [3H]MK-801. This effect was due to a decrease in the rate of association with no change in the rate of dissociation of [3H]MK-801. The effect of DA10 was not mediated by an action of DA10 at the binding sites for glutamate, glycine, Mg2+, or Zn2+, and was attenuated by DET. This suggests that DA10 acts at the polyamine recognition site. In hippocampal neurons the NMDA-elicited current was decreased by DA10, an effect opposite to that of spermine. The effects of spermine and DA10 were selectively blocked by DET. It is concluded that DA10 acts as a negative allosteric modulator or inverse agonist at the polyamine recognition site of the NMDA receptor.

Basal expression and induction of glutamate decarboxylase and GABA in excitatory granule cells of the rat and monkey hippocampal dentate gyrus.

The excitatory, glutamatergic granule cells of the hippocampal dentate gyrus are presumed to play central roles in normal learning and memory, and in the genesis of spontaneous seizure discharges that originate within the temporal lobe. In localizing the two GABA-producing forms of glutamate decarboxylase (GAD65 and GAD67) in the normal hippocampus as a prelude to experimental epilepsy studies, we unexpectedly discovered that, in addition to its presence in hippocampal nonprincipal cells, GAD67-like immunoreactivity (LI) was present in the excitatory axons (the mossy fibers) of normal dentate granule cells of rats, mice, and the monkey Macaca nemestrina. Using improved immunocytochemical methods, we were also able to detect GABA-LI in normal granule cell somata and processes. Conversely, GAD65-LI was undetectable in normal granule cells. Perforant pathway stimulation for 24 hours, which evoked population spikes and epileptiform discharges in both dentate granule cells and hippocampal pyramidal neurons, induced GAD65-, GAD67-, and GABA-LI only in granule cells. Despite prolonged excitation, normally GAD- and GABA-negative dentate hilar neurons and hippocampal pyramidal cells remained immunonegative. Induced granule cell GAD65-, GAD67-, and GABA-LI remained elevated above control immunoreactivity for at least 4 days after the end of stimulation. Pre-embedding immunocytochemical electron microscopy confirmed that GAD67- and GABA-LI were induced selectively within granule cells; granule cell layer glia and endothelial cells were GAD- and GABA-immunonegative. In situ hybridization after stimulation revealed a similarly selective induction of GAD65 and GAD67 mRNA in dentate granule cells. Neurochemical analysis of the microdissected dentate gyrus and area CA1 determined whether changes in GAD- and GABA-LI reflect changes in the concentrations of chemically identified GAD and GABA. Stimulation for 24 hours increased GAD67 and GABA concentrations sixfold in the dentate gyrus, and decreased the concentrations of the GABA precursors glutamate and glutamine. No significant change in GAD65 concentration was detected in the microdissected dentate gyrus despite the induction of GAD65-LI. The concentrations of GAD65, GAD67, GABA, glutamate and glutamine in area CA1 were not significantly different from control concentrations. These results indicate that dentate granule cells normally contain two "fast-acting" amino acid neurotransmitters, one excitatory and one inhibitory, and may therefore produce both excitatory and inhibitory effects. Although the physiological role of granule cell GABA is unknown, the discovery of both basal and activity-dependent GAD and GABA expression in glutamatergic dentate granule cells may have fundamental implications for physiological plasticity presumed to underlie normal learning and memory. Furthermore, the induction of granule cell GAD and GABA by afferent excitation may constitute a mechanism by which epileptic seizures trigger compensatory interictal network inhibition or GABA-mediated neurotrophic effects.

Antioxidant therapy in neurologic disease.

Free radical or oxidative injury may be a fundamental mechanism underlying a number of human neurologic diseases. Therapy using free radical scavengers (antioxidants) has the potential to prevent, delay, or ameliorate many neurologic disorders. However, the biochemistry of oxidative pathobiology is complex, and optimum antioxidant therapeutic options may vary and need to be tailored to individual diseases. In vitro and animal model studies support the potential beneficial role of various antioxidant compounds in neurologic disease. However, the results of clinical trials using various antioxidants, including vitamin E, tirilazad, N-acetylcysteine, and ebselen, have been mixed. Potential reasons for these mixed results include lack of pretrial dose-finding studies and failure to appreciate and characterize the individual unique oxidative processes occurring in different diseases. Moreover, therapy with antioxidants may need to be given early in chronic insidious neurologic disorders to achieve an appreciable clinical benefit. Predisease screening and intervention in at-risk individuals may also need to be considered in the near future.

Effects of ethosuximide and tetramethylsuccinimide on cultured cortical neurons.

In cultured cortical neurons, ethosuximide (ESM) had unexpected actions for an antiepileptic drug; it slightly diminished the effects of inhibitory neurotransmitters, GABA, and glycine. ESM had no direct membrane effects, did not change action potential characteristics or alter spontaneous activity, and did not reverse the effects of convulsants that are GABA antagonists. The structurally related convulsant tetramethylsuccinimide (TMSM) reduced the amplitude of GABA-mediated inhibitory postsynaptic potentials and antagonized responses to applied GABA, effects not reversed by ESM. The convulsant effects of TMSM include a blockade of postsynaptic GABA actions, but the antiepileptic effects of ESM are not due to an enhancement of GABA-mediated inhibition.

Effects of valproic acid in cultured mammalian neurons.

Valproic acid (VPA) has been postulated to exert its anticonvulsant effects by interaction with the postsynaptic GABA receptor. To test that hypothesis, we applied VPA in clinically appropriate concentrations to cortical neurons in dissociated cell culture. VPA did not enhance the postsynaptic effect of GABA, but did decrease the generation of sodium-dependent action potentials. VPA may exert anticonvulsant effects by inhibiting spike generation, independent of the GABAergic system.

Sexual dysfunction in partial epilepsy: a deficit in physiologic sexual arousal.

Men and women with epilepsy frequently complain of sexual dysfunction. We studied the sexual response in men and women with partial epilepsy of temporal lobe origin (TLE) by measuring genital blood flow (GBF) during sexual arousal. Nine women and eight men with TLE and 12 women and seven men as controls completed inventories for symptoms of depression, sexual experience, and sexual attitude and underwent measurement of digital pulse and GBF during alternating segments of sexually neutral and erotic videotape. Subjective ratings of arousal to the videotape were obtained. We calculated digital pulse and GBF response as the percentage increase in pulse amplitude during the erotic compared with the preceding sexually neutral film. No subject group reported symptoms of significant depression on the inventory. However, men and women with epilepsy had fewer sexual experiences than subjects without epilepsy, and women with epilepsy imagined specific sexual activities to be more anxiety-producing and less arousing than did women without epilepsy. Men and women with TLE had a diminished GBF response. The mean increase in GBF in men with TLE was 184% versus 660% for controls (p = 0.01). Women with TLE had a mean increase of 117% versus 161% for controls (p < 0.01). Digital pulse did not vary across stimulus conditions. Subjective ratings for all groups indicated moderate sexual arousal. We conclude that there is a diminution in one aspect of physiologic sexual arousal in some men and women with TLE.

Plasmapheresis in Rasmussen's encephalitis.

Rasmussen's encephalitis (RE) is a progressive childhood disorder characterized by intractable focal seizures, hemiplegia, dementia, and inflammatory histopathology. The process is typically limited to one cerebral hemisphere. We report four patients with pathologically confirmed RE who were treated with repeated plasmapheresis. Three patients exhibited repeated, dramatic, transient responses to plasmapheresis, manifested by reduced seizure frequency and improved neurologic function. One patient exhibited marginal improvement after treatment with plasmapheresis. These observations indicate that circulating factors, likely autoantibodies, are pathogenic in at least some patients with RE and suggest that RE is an autoimmune disease. Plasmapheresis may be a useful adjunctive therapy in status epilepticus, and can also aid in assessment of residual function in the diseased hemisphere before surgical resection.

Future directions for epilepsy research.

The authors propose that epilepsy research embark on a revitalized effort to move from targeting control of symptoms to strategies for prevention and cure. The recent advances that make this a realistic goal include identification of genes mutated in inherited epilepsy syndromes, molecular characterization of brain networks, better imaging of sites of seizure origin, and developments in seizure prediction by quantitative EEG analysis. Research directions include determination of mechanisms of epilepsy development, identification of genes for common epilepsy syndromes through linkage analysis and gene chip technology, and validation of new models of epilepsy and epileptogenesis. Directions for therapeutics include identification of new molecular targets, focal methods of drug delivery tied to EEG activity, gene and cell therapy, and surgical and nonablative therapies. Integrated approaches, such as coupling imaging with electrophysiology, are central to progress in localizing regions of epilepsy development in people at risk and better seizure prediction and treatment for people with epilepsy.

Desensitization of GABA-induced currents in cultured rat hippocampal neurons.

The basic characteristics of desensitization of the GABAA receptor were investigated in cultured rat hippocampal neurons (three days to four weeks in vitro) using whole cell patch clamp techniques. GABA at 10-500 microM was perfused on to neurons for 30 or 60 s, with 60 s intervals of wash with control bath solution between perfusions. Desensitization, evaluated by peak-to-plateau ratio and time constants of current decay (tau), was dose-dependent and culture age-dependent. Desensitization was observed as early as three days in culture, the earliest time tested. At all ages, higher concentrations of GABA induced both larger and faster desensitization. Desensitization was markedly voltage-dependent and decreased with depolarization; peak-to-plateau ratio went from 6.3 to 1.4 and tau went from 4.6 to 26.8 s when holding potentials were changed from -80 mV to +30 mV. Low concentrations of GABA (1-2 microM) perfused for 2-60 s, which did not induce any current, had no effect on the maximal response nor desensitization produced by a subsequent application of 100 microM GABA. This finding suggests that GABA receptors were not desensitized without first being activated.

Evidence that a novel somatostatin receptor couples to an inward rectifier potassium current in AtT-20 cells.

The recent cloning of five somatostatin receptors has made it possible to begin screening for selective ligands in order to begin characterization of these receptor subtypes expressed endogenously. We have recently reported the characterization of ligands selective for SSTR2 and SSTR5 [Raynor K. et al. (1993) Molec. Pharmac. 43, 838-844; 44, 385-392]. Both of these somatostatin receptor subtypes are endogenously expressed in the mouse pituitary cell line AtT-20 [O'Carroll A.-M. et al. (1992) Molec. Pharmac. 42, 939-946; Patel Y. C. et al. (1994) J. biol. Chem. 269, 1506-1509; Tallent M. et al. (1996) Neuroscience 71, 1073-1081]. Using these selective ligands, as well as other somatostatin analogs, we have characterized the somatostatin receptor which couples to the inward rectifier K+ current in AtT-20 cells. This receptor is sensitive to hexapeptide analogs of somatostatin, but insensitive to octapeptide analogs. This pharmacological profile is distinct from any of the cloned somatostatin receptors and therefore may represent a novel receptor. Somatostatin has been shown to potentiate an inward rectifying K+ channel in many different types of neuronal and non-neuronal cells. The activation of this current is thought to be an important mechanism by which somatostatin inhibits neuronal firing and decreases neurotransmitter and hormone release [Mihara S. et al. (1987) J. Physiol. 390, 335-355]. Therefore, the novel somatostatin receptor coupling to the inward rectifier in AtT-20 cells may be important in somatostatin's role in regulating neurotransmission and hormone release.

Differential regulation of the cloned kappa and mu opioid receptors.

To directly compare the regulation of the cloned kappa and mu opioid receptor, we expressed them in the same cells, the mouse anterior pituitary cell line AtT-20. The coupling of an endogenous somatostatin receptor to adenylyl cyclase and an inward rectifier K+ current has been well characterized in these cells, enabling us to do parallel studies comparing the regulation of both the kappa and the mu receptor to this somatostatin receptor. We show that the kappa receptor readily uncoupled from the K+ current and from adenylyl cyclase after a 1 h pretreatment with agonist, as indicated by the loss in the ability of the agonist to induce a functional response. The desensitization of the kappa receptor was homologous, as the ability of somatostatin to mediate inhibition of adenylyl cyclase or potentiation of the K+ current was not altered by kappa receptor desensitization. The mu receptor uncoupled from the K+ current but not adenylyl cyclase after a 1 h pretreatment with agonist. Somatostatin was no longer able to potentiate the K+ current after mu receptor desensitization, thus this desensitization was heterologous. Interestingly, pretreatment with a somatostatin agonist caused uncoupling of the mu receptor but not the kappa receptor from the K+ current. These results show that in the same cell line, after a 1 h pretreatment with agonist, the kappa receptor displays homologous regulation, whereas the mu receptor undergoes only a heterologous form of desensitization. mu receptor desensitization may lead to the alterations of diverse downstream events, whereas kappa receptor regulation apparently occurs at the level of the receptor itself. Broad alterations of non-opioid systems by the mu receptor could be relevant to the addictive properties of mu agonists. Comparison of kappa and mu receptor regulation may help define the properties of the mu receptor which are important in the development of addiction, tolerance, and withdrawal to opioid drugs. These are the first studies to directly compare the coupling of the kappa and mu receptors to two different effectors in the same mammalian expression system.

Different patterns of synaptic transmission revealed between hippocampal CA3 stratum oriens and stratum lucidum interneurons and their pyramidal cell targets.

Stratum lucidum (SL) interneurons likely mediate feedforward inhibition between the dentate gyrus mossy fibers and CA3 pyramidal cells, while stratum oriens (SO) interneurons likely provide both feedforward and feedback inhibition within the CA3 commissural/associational network. Using dual whole-cell patch-clamp recordings between interneurons and CA3 pyramidal cells, we have examined SL and SO interneurons and their synapses within organotypic hippocampal slice cultures. Biocytin staining revealed different morphologies between these interneuron groups, both being very similar to those found previously in acute slices. The kinetics of IPSCs were similar between the two groups, but the reliability of synaptic transmission of SL interneuron (SL-INT) IPSCs was significantly lower than the virtually 100% reliability (non-existent failure rates) of SO-INT IPSCs. The SL-INT IPSCs also had a lower quantal content than the SO-INT IPSCs. In addition, SL-INTs were less likely than SO-INTs to innervate or to be innervated by nearby CA3 pyramidal cells. Paired-pulse stimulation at 100 ms interstimulus intervals produced similar paired-pulse depression in both interneuron synapses, despite the significantly higher failure rate of IPSCs produced by the SL-INTs compared with SO-INTs. CV analysis supported the hypothesis that paired-pulse depression was presynaptic. During repetitive, high frequency stimulation (>10 Hz for 500 ms) the two different synapses exhibited distinctly different forms of short-term plasticity: all SL interneurons displayed significant short-term facilitation (mean 113% facilitation, n=4), while, by contrast, SO interneuron synapses displayed either short-term depression (mean 42% depression, n=5 of 8) or no net facilitation or depression (n=3 of 8). These results indicate that the synaptic properties of interneurons can be quite different for interneurons in different hippocampal circuits.

The cloned kappa opioid receptor couples to an N-type calcium current in undifferentiated PC-12 cells.

We have recently reported the cloning of a mouse kappa opioid receptor cDNA. Following transfection of the kappa receptor cDNA into COS-1 cells, a receptor is expressed with the pharmacological specificity of a kappa opioid receptor. To further analyse its functional properties, we have stably expressed the kappa opioid receptor in undifferentiated PC-12 cells, a pheochromocytoma clonal cell line, which do not endogenously express this receptor. We have previously shown that kappa opioid agonists selectively bind to these PC-12 membranes with high affinity. Here we show that kappa selective agonists are able to inhibit accumulation of cyclic adenosine monophosphate in a stereoselective manner. Further, the kappa agonist U-50,488 is able to inhibit an N-type calcium current in a pertussis toxin sensitive manner; this inhibition is blocked by the kappa-selective antagonist norbinaltorphimine. Inhibition of the calcium current via the kappa receptor is stereoselective as the agonist levorphanol is able to mediate inhibition whereas in the same cells dextrorphan is ineffective. This is the first demonstration that the cloned kappa opioid receptor functionally couples to a calcium current, as has been reported for kappa receptors expressed endogenously in the nervous system. Kappa opioid receptors are thought to be important in pain pathways, learning and memory deficits, and seizure activity. A major physiological action of the dynorphins, the endogenous ligands of the kappa receptor, is thought to be inhibition of neurotransmitter release at presynaptic terminals. N-type calcium channels may be important in neurotransmitter release.(ABSTRACT TRUNCATED AT 250 WORDS)

Non-classical nuclear localization signal peptides for high efficiency lipofection of primary neurons and neuronal cell lines.

Gene transfer into CNS is critical for potential therapeutic applications as well as for the study of the genetic basis of neural development and nerve function. Unfortunately, lipid-based gene transfer to CNS cells is extremely inefficient since the nucleus of these post-mitotic cells presents a significant barrier to transfection. We report the development of a simple and highly efficient lipofection method for primary embryonic rat hippocampal neurons (up to 25% transfection) that exploits the M9 sequence of the non-classical nuclear localization signal of heterogeneous nuclear ribonucleoprotein A1 for targeting beta(2)-karyopherin (transportin-1). M9-assistant lipofection resulted in 20-100-fold enhancement of transfection over lipofection alone for embryonic-derived retinal ganglion cells, rat pheochromocytoma (PC12) cells, embryonic rat ventral mesencephalon neurons, as well as the clinically relevant human NT2 cells or retinoic acid-differentiated NT2 neurons. This technique can facilitate the implementation of promoter construct experiments in post-mitotic cells, stable transformant generation, and dominant-negative mutant expression techniques in CNS cells.

Characterization of desensitization in recombinant N-methyl-D-aspartate receptors: comparison with native receptors in cultured hippocampal neurons.

In the present study we have characterized the effect of Ca2+, glycine, and agonist concentration on inactivation and desensitization in native and recombinant N-methyl-d-aspartate (NMDA) receptors. In agreement with earlier studies on neurons, we found that in the presence of saturating glycine concentrations, lowering [Ca2+]o, will decrease inactivation of NMDA receptors in cultured hippocampal neurons. However, unlike native NMDA receptors under the same recording conditions, recombinant receptors did not exhibit Ca2+-dependent inactivation. We also show that the glycine-insensitive desensitization observed in the recombinant receptors is subunit dependent, as NR1a2A and NR1a2B receptors significantly desensitized while the NR1a2C combination did not. Furthermore, we show this form of desensitization in NR1a2A receptors is due to classic agonist-induced desensitization. In addition, we demonstrate the presence of glycine-dependent desensitization in recombinant receptors. The ability of glycine to inhibit desensitization correlates to the rank order of glycine's affinity for potentiating the peak response for each subtype. Finally, using ifenprodil in the presence of high and low glycine concentrations, we present evidence that both 2A-like and 2B-like subtypes of receptors can independently coexist in single neurons.

Electrophysiological effects of somatostatin-14 and somatostatin-28 on mammalian central nervous system neurons.

Somatostatin (SOM) exists in at least two active forms in the central nervous system (CNS): SOM-14 and SOM-28. These peptides have multiple actions on neurons in the CNS and these actions appear to be mediated by different receptors. Thus, SOM-14 can enhance voltage-dependent K currents, whereas SOM-28 inhibits these same currents, sometimes even in the same neurons. These effects are not mediated via cAMP, but do seem mediated by GTP-binding proteins. On the other hand, both forms of SOM inhibit a voltage-dependent Ca current, again via a GTP-binding protein. SOM can also interact with the GABA(A) receptor to modulate responses to this inhibitory transmitter. The physiological effects of SOM in an integrated circuit within the CNS will depend on the form of SOM released, the kinds and numbers of receptors present on the postsynaptic neurons, and the presence of other neurotransmitters.