Biomedical subjects
M A Dichter
Publications and source records attributed to M A Dichter.
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.
Heterologous modulation of inhibitory synaptic transmission by metabotropic glutamate receptors in cultured hippocampal neurons.
1. Whole cell patch-clamp recordings of monosynaptically connected pairs of hippocampal neurons in very low-density culture were performed to determine the effects of the activation of metabotropic glutamate receptors (mGluRs) on inhibitory terminals. The mGluR agonist (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S, 3R)-ACPD] and the recently described mGluR antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG) were used. In addition, the glutamate uptake inhibitors L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC) and D,L-beta-threo-hydroxyaspartate (THA) were used to determine whether endogenous agents (presumably glutamate) could activate mGluRs at inhibitory terminals. Previous reports of the role of mGluRs on inhibitory terminals were performed in slice preparations; our use of patch-clamp recordings from isolated pairs of hippocampal neurons is uniquely useful for the study of inhibitory synaptic transmission in the absence of polysynaptic connectivity. 2. The mGluR agonist (1S, 3R)-ACPD (100 microM) reversibly decreased the amplitude of evoked inhibitory postsynaptic currents (IPSCs) in all pairs tested; this effect was completely blocked by coapplication of the mGluR antagonist MCPG (500 microM) with (1S, 3R)-ACPD. MCPG (500 microM) alone had no effect on IPSC amplitude. These results indicate that all inhibitory neurons in our cultures express functional mGluRs in their terminals. 3. Examination of the frequency and the distribution of amplitudes of miniature IPSCs (mIPSCs) provide indications of changes in the sensitivity of postsynaptic receptors and/or of changes in the process of presynaptic transmitter release. Recordings of miniature currents from hippocampal neurons cultured at very low density makes possible the analysis of mIPSCs that arise from a single input, whereas in high density or slice preparations, spontaneous miniature currents reflect numerous synaptic inputs. No change in the amplitudes or frequency of the mIPSCs were observed upon application of (1S, 3R)-ACPD (100 microM). Thus we conclude that the depression of the evoked IPSC amplitude by (1S, 3R)-ACPD is mediated by a presynaptic mechanism in these isolated pairs of hippocampal neurons. 4. The glutamate uptake inhibitor L-trans-PDC also reduced IPSC amplitude in 8 of 13 pairs. In these eight pairs, an increase in N-methyl-D-aspartate (NMDA) receptor-mediated membrane noise indicated an increase in ambient concentrations of glutamate induced by L-trans-PDC. In the remaining five pairs, membrane noise remained unaffected by L-trans-PDC, and IPSCs were not attenuated. Similar results were observed with the use of the uptake inhibitor THA. The mGluR antagonist MCPG blocked the effects of L-trans-PDC and THA on IPSC amplitude. We propose that inhibition of glutamate uptake mechanisms results in activation of mGluRs on GABAergic terminals via endogenous sources of glutamate and that the uptake inhibitors (L-trans-PDC and THA) do not directly activate the metabotropic receptor. 5. Presynaptic receptors and active modulation of uptake mechanisms are clearly involved in a wide range of physiological and pathological synaptic events. The data presented here suggest that heterosynaptic modulation of inhibitory synaptic transmission by metabotropic glutamate receptors may be important for the maintenance and plasticity of the balances between excitatory and inhibitory synaptic transmission in the CNS.
Glycine regulation of synaptic NMDA receptors in hippocampal neurons.
1. Although glycine has been identified as a required coagonist with glutamate at N-methyl-D-aspartate (NMDA) receptors, the understanding of glycine's role in excitatory synaptic neurotransmission is quite limited. In the present study, we used the whole cell patch-clamp technique to examine the ability of glycine to regulate current flow through synaptic NMDA receptors at excitatory synapses between cultured hippocampal neurons and in acutely isolated hippocampal slices. 2. These studies demonstrate that the glycine modulatory site on the synaptic NMDA receptor is not saturated under baseline conditions and that increased glycine concentrations can markedly increased NMDA-receptor-mediated excitatory postsynaptic currents (EPSCs) in hippocampal neurons in both dissociated cell culture and in slice. Saturation of the maximal effect of glycine takes place at different concentrations for different cells in culture, suggesting the presence of heterogenous NMDA receptor subunit compositions. 3. Bath-applied glycine had no effect on the time course of EPSCs in either brain slice or culture, indicating that desensitization of the NMDA receptor is not prevented by glycine over the time course of an EPSC. 4. When extracellular glycine concentration is high, all miniature EPSCs recorded in the cultured hippocampal neurons contained NMDA components, indicating that segregation of non-NMDA receptors at individual synaptic boutons does not occur.
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.
Integrated use of old and new antiepileptic drugs.
Three new antiepileptic drugs have been approved for use in the USA in the past year and a half and several others are available in Europe and Japan. Each of these drugs has been efficacious against partial seizures without or with secondary generalization and some may also be efficacious against primary generalized seizures. None of the new drugs appears to be a quantum leap in therapy over the others that are already available. How these new drugs will be integrated into a rational scheme for the treatment of individuals with epilepsy remains to be determined. Side-effect profiles, pharmacokinetic issues, and cost will likely be significantly issues.
Frequency-dependent depression of excitatory synaptic transmission is independent of activation of MCPG-sensitive presynaptic metabotropic glutamate receptors in cultured hippocampal neurons.
1. A paired-pulse paradigm, and a high-frequency train followed by a test pulse, were used to investigate the possible role of presynaptic metabotropic glutamate receptors (mGluRs) in frequency-dependent modulation of the amplitude of excitatory post-synaptic currents (EPSCs). Paired whole cell patch-clamp recordings from monosynaptically connected hippocampal neurons maintained in very low-density cultures were performed, using the mGluR antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG, 500 microM) and the mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD, 100 microM]. 2. Paired-pulse depression (PPD) was observed in all the excitatory pairs recorded. The average PPD ratio (amplitude of the 2nd EPSC divided by the amplitude of the 1st EPSC) was 0.80 +/- 0.1 (SD) (n = 8). Application of the mGluR antagonist MCPG had no effect on the amplitude of the EPSCs and did not affect the ratio of the two EPSCs (PPD ratio 0.79 +/- 0.2). 3. The amplitudes of 10 successive EPSCs stimulated at a high frequency (20 Hz) decremented on average in both 4 mM extracellular Ca2+ (n = 5) and in 1 mM extracellular Ca2+ (n = 6). In all pairs tested, posttetanic depression (PTD) was observed (PTD ratio 0.7 +/- 0.2). Bath application of MCPG (500 microM) did not affect the amplitudes of the EPSCs during the train; MCPG also did not affect PTD. 4. The mGluR agonist (1S,3R)-ACPD depressed the amplitudes of the EPSCs in both the paired-pulse (1st EPSC, 35 +/- 9%; 2nd EPSC, 36 +/- 10%) and posttetanic pulse (1 and 4 mM extracellular Ca2+) paradigms. The amount of depression observed, both PPD and PTD, remained unaffected by application of (1S,3R)-ACPD. Coapplication of the antagonist MCPG (500 microM) blocked the effects of (1S,3R)-ACPD (100 microM). 5. We conclude that frequency-dependent depression of EPSC amplitudes occurs independent of endogenous activation of MCPG-sensitive mGluRs in cultured hippocampal neurons. Moreover, we demonstrate that exogenous activation of mGluRs by the agonist (1S,3R)-ACPD can produce additional EPSC depression above that already present due to frequency-dependent mechanisms.
Properties of inhibitory and excitatory synapses between hippocampal neurons in very low density cultures.
The whole cell patch clamp technique was used to examine the electrophysiological properties of embryonic hippocampal neurons maintained in a very low density (VLD) culture preparation. The goal of these experiments was to establish the viability of the VLD culture as a model system in which to study regulation of neurotransmission at single monosynaptic connections, in the absence of polysynaptic innervation. Depolarization of neurons in the VLD culture revealed voltage-dependent sodium, calcium, and potassium currents which were blocked with, respectively, tetrodotoxin (TTX), cobalt, and tetraethylammonium and 4-aminopyridine. When pairs of neurons were simultaneously recorded, action potentials evoked in presynaptic neurons elicited either excitatory or inhibitory postsynaptic currents (EPSCs or IPSCs, respectively). The dual component EPSCs were due to the activation of both types of postsynaptic, ionotropic glutamate receptors: N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Evoked IPSCs were due to the activation of postsynaptic gamma-aminobutyric acid (GABA) receptors. Both excitatory and inhibitory synapses exhibited short term depression in response to high frequency stimulation, although IPSCs were routinely decreased to a much greater degree than EPSCs. Spontaneous miniature EPSCs and IPSCs were found to persist in TTX, were blocked by the same pharmacological antagonists which blocked evoked responses, increased in frequency in response to hypersomotic solution, and were unaffected by changes in extracellular calcium concentration. mIPSCS were found to occur at a significantly lower frequency than mEPSCs. These experiments indicated that neurotransmission in the VLD cultures occurs in a manner consistent with the quantal hypothesis and, therefore, the VLD culture is a good model for studying excitatory and inhibitory neurotransmission between isolated pairs of neurons. In addition, these experiments, performed under comparable physiological conditions, demonstrated that there are fundamental differences underlying neurotransmitter release between excitatory and inhibitory neurons.
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)
Emerging insights into mechanisms of epilepsy: implications for new antiepileptic drug development.
Most currently available antiepileptic drugs (AEDs) were developed by testing new compounds in animal models of seizures. Increased knowledge of the cellular and molecular mechanisms underlying normal CNS function and seizure phenomena is now being used to design new AEDs specifically to interfere with epileptic mechanisms. Focal epilepsy develops in areas of cortex that are damaged and in which aberrant recurrent excitatory circuits develop, producing spike discharges in the EEG. Occasionally, normal membrane conductances and inhibitory synaptic currents break down and excess excitability spreads, either locally to produce a focal seizure or more widely to produce a generalized seizure. Both original synchronous activation and seizure spread appear to utilize normal synaptic pathways and mechanisms. Much new development of AEDs is targeted at modulating these excitatory and inhibitory synaptic effects, focusing directly on multiple components of glutamate and GABA receptors. Intrinsic, voltage-dependent currents are also involved in the pathophysiology of epileptic processes. Calcium currents act to amplify excess neuronal depolarization during hypersynchronous activation, are involved in neurotransmitter release, and play a role in the development of longer-term changes in synaptic efficacy, which may be involved in some seizure phenomena. They also appear to be involved in some forms of primary generalized epilepsy, in which burst discharges due to calcium currents in deep diencephalic neurons with widely ramifying axons may act as synchronizing influences. Neuromodulatory agents, including purines, peptides, cytokines, and steroid hormones, also play important roles in regulating brain excitability. Adenosine in some experimental models act as an endogenous antiepileptic substance, and agents that enhance the actions of adenosine are often antiepileptic in animal models.(ABSTRACT TRUNCATED AT 250 WORDS)
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.
The glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate depresses excitatory synaptic transmission via a presynaptic mechanism in cultured hippocampal neurons.
Sodium-dependent high-affinity uptake of glutamate is thought to play a major role in the maintenance of very low extracellular concentrations of excitatory amino acids (EAA), and may modulate the actions of released transmitter at G-protein-coupled receptors and extrasynaptic receptors that are activated over a longer distance and time course. We have examined the effects of the recently developed uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC) on monosynaptically evoked excitatory postsynaptic currents (EPSCs) in very-low-density cultures of hippocampal neurons. L-Trans-PDC produced a decreased amplitude of both the non-NMDA and NMDA receptor-mediated components of monosynaptically evoked EPSCs. Examination of miniature EPSCs (mEPSCs) indicated that changes in the sensitivity of postsynaptic non-NMDA receptors did not underline the decrease in evoked EPSC amplitudes. The metabotropic receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) also depressed both components of the EPSC. The competitive metabotropic receptor antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG) blocked the depression of EPSC amplitude induced by 1S,3R-ACPD and also blocked the effects of L-trans-PDC. Finally, low concentrations of L-glutamate (2 microM) mimicked the effects of L-trans-PDC on EPSC amplitude. From these results we conclude that the application of L-trans-PDC to cultured hippocampal neurons results in the activation of presynaptic metabotropic receptors, leading to a decrease in synaptic transmission. We propose that this effect is due to an increase in ambient glutamate concentrations following inhibition of glutamate uptake, resulting in presynaptic inhibition of excitatory synaptic transmission.
Paired pulse depression in cultured hippocampal neurons is due to a presynaptic mechanism independent of GABAB autoreceptor activation.
Most rapid synaptic inhibition in the vertebrate forebrain is mediated by GABA acting via GABAA and GABAB postsynaptic receptors. GABAergic neurotransmission exhibits frequency-dependent modulation; sequential inhibitory post-synaptic currents (IPSCs) evoked with interstimulus intervals between 25 msec and 4 sec routinely result in the attenuation of the amplitude of the second IPSC. This form of synaptic plasticity is known as paired pulse depression (PPD). The mechanism of PPD is presently unknown and the experiments performed in this study were designed to determine directly the location of the mechanism of PPD in hippocampal neurons maintained in low-density tissue culture. Evoked IPSCs were recorded between pairs of cultured neurons grown in relative isolation that were simultaneously being recorded with the whole-cell, patch-clamp technique. It was therefore possible to measure miniature IPSCs (mIPSCs) originating from the same synapses that were being stimulated to evoke release. PPD occurred routinely in this system, but the amplitudes of mIPSCs following IPSCs were unchanged. These results indicate that a presynaptic mechanism mediates PPD. The inability of GABAB receptor antagonists to block PPD revealed that this form of presynaptic plasticity was not due to autoinhibition of transmitter release via activation of presynaptic GABAB receptors. However, manipulations that significantly lowered the probability of release of neurotransmitter during the first action potential of a trial (e.g., lower calcium or baclofen) prevented the development of PPD. These results indicate that, under baseline conditions, the quantal content for IPSCs is relatively large for a single action potential, but the quantal content rapidly decreases, such that subsequent action potentials consistently result in much smaller IPSCs for periods as long as 4 sec.
Old and new mechanisms of antiepileptic drug actions.
At the present time, between 30% and 50% of individuals with epilepsy continue to have seizures and among those who are considered "controlled", a good many unpleasant side effects are endured. It is clear that we need more and better antiepileptic drugs. The virtual explosion in knowledge about the cellular and molecular mechanisms underlying both normal and pathological reactions of the brain provides encouragement that we are on the right path towards developing new, effective and safe agents. Whether we continue to use "model-based" screening or "mechanism-based screening", or both, we must not go another 14 years without the introduction of a new antiepileptic drug. The problem is too large, our patients are too impatient and our responsibilities are clear.
Deciding to discontinue antiepileptic medication.
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Non-uniform distribution of GABA activated chloride channels in cultured cortical neurons.
Several different neurotransmitter receptors and ion channels exist in non-random distributions in the membrane of nerve and muscle cells, a pattern which appears to have functional implications. The data presented here, based on the distribution of single channels in outside-out patches, directly demonstrate that receptors for gamma-aminobutyric acid (GABA) and their associated chloride channels (GABAA receptors) exist almost exclusively in clusters in the membranes of cultured rat cortical neurons. This non-uniform distribution is present both in innervated and uninnervated neurons.
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.
Up-regulation of N-methyl-D-aspartate receptors on cultured cortical neurons after exposure to antagonists.
The density of N-methyl-D-aspartate (NMDA) receptors on membranes prepared from cultured cortical neurons was determined using binding assays with [125I]I-MK-801 after exposure of cultures to antagonists of the NMDA receptor complex. The density of binding sites for [125I]I-MK-801 was increased by 40-80% after exposure to D-2-amino-5-phosphonopentanoic acid (D-AP5), with no change in the number or viability of neurons. The effect of D-AP5 was concentration dependent, with an EC50 of 10 microM. Up-regulation of NMDA receptors was observed after 2-7 days but not after 1 day of exposure to 100 microM D-AP5. The density of NMDA receptors was also increased after exposure of cells to CGS 19755 and MK-801 but not after exposure to the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. The binding of [3H]AMPA was unaltered after exposure to D-AP5. These results demonstrate that the density of NMDA receptors on cultured neurons can be selectively up-regulated by exposure to NMDA receptor antagonists. Increases in the density of NMDA receptors occurring in vivo could complicate therapeutic approaches to the treatment of neurological disorders.