Maintenance of bladder innervation in diabetes: a stereological study of streptozotocin-treated female rats.

Neuropathy and cystopathy are two common conditions in patients with chronic diabetes. Despite obvious bladder sensory and motor nerve dysfunction in diabetes, no studies have selectively explored whether sensory or motor innervation is affected in the bladder. In the present study, we tested the hypothesis that loss of bladder sensory and motor fibers is responsible for bladder sensory and motor dysfunction. Parasympathetic and sensory innervation of the bladder dome and neck were examined using immunohistochemistry (IHC) and stereology in adult female rats 12weeks after induction of diabetes by streptozotocin. Naïve and age matched rats were evaluated as controls. Diabetic rats had mean blood glucose level of >400mg/dl, and bladder weights and thicknesses that were more than doubled compared to naïve rats. In naïve rats, parasympathetic vesicular acetylcholine transporter (VAT) and sensory calcitonin gene-related peptide (CGRP) immunopositive nerve fibers were located in bladder smooth muscle and were more densely distributed in the neck compared to the dome. Within the urothelial region, CGRP nerve fibers were densely distributed while VAT nerve fibers were sparsely distributed in the bladder neck and both were virtually absent in the bladder dome. Streptozotocin induced diabetes did not change the total nerve fiber length of either VAT or CGRP stained fibers in either the neck or dome. These studies indicate that hyperglycemia, induced by streptozotocin treatment, does not result in a loss of parasympathetic VAT or CGRP sensory nerve fibers, per se, but the doubling of bladder weight and mass does indicate a decrease in innervation density.

A positive modulator of K Ca 2 and K Ca 3 channels, 4,5-dichloro-1,3-diethyl-1,3-dihydro-benzoimidazol-2-one (NS4591), inhibits bladder afferent firing in vitro and bladder overactivity in vivo.

Calcium-activated potassium channels are attractive targets for the development of therapeutics for overactive bladder. In the current study, we addressed the role of calcium-activated potassium channels of small (SK; K(Ca)2) and intermediate (IK; K(Ca)3) conductance in bladder function pharmacologically. We identified and characterized a novel positive modulator of SK/IK channels, 4,5-dichloro-1,3-diethyl-1,3-dihydro-benzoimidazol-2-one (NS4591). In whole-cell patch-clamp experiments, NS4591 doubled IK-mediated currents at a concentration of 45 +/- 6 nM(n = 16), whereas 530 +/- 100 nM (n = 7) was required for doubling of SK3-mediated currents. In acutely dissociated bladder primary afferent neurons, the presence of SK channels was verified using apamin and 1-ethyl-2-benzimidazolinone. In these neurons, NS4591 (10 microM) inhibited the number of action potentials generated by suprathreshold depolarizing pulses. NS4591 also reduced carbachol-induced twitches in rat bladder detrusor rings in an apamin-sensitive manner. In vivo, NS4591 (30 mg/kg) inhibited bladder overactivity in rats and cats induced by capsaicin and acetic acid, respectively. In conclusion, the present study supports the involvement of calcium-activated potassium channels in bladder function and identifies NS4591 as a potent modulator of IK and SK channels that is effective in animal models of bladder overactivity.

Modulation of BzATP and formalin induced nociception: attenuation by the P2X receptor antagonist, TNP-ATP and enhancement by the P2X(3) allosteric modulator, cibacron blue.

1. Exogenous ATP produces acute and localized pain in humans, and P2X receptor agonists elicit acute nociceptive behaviours in rodents following intradermal administration to the hindpaw. The predominant localization of P2X(3) mRNA in sensory neurones has led to the hypothesis that activation of P2X(3) and/or P2X(2/3) receptors contributes to nociception. 2. The local administration of the P2X receptor agonist, BzATP (100--1000 nmol paw(-1), s.c.) into the rat hindpaw produced an acute (<15 min) paw flinching response that was similar to that observed in the acute phase of the formalin (5%) test. 3. The co-administration of the potent P2X receptor antagonist, TNP-ATP (30--300 nmol paw(-1)), but not an inactive analogue, TNP-AMP, with BzATP into the rat hindpaw attenuated BzATP-induced nociception. Similarly, co-administration of TNP-ATP, but not TNP-AMP, with 5% formalin reduced both acute and persistent nociception in this test. 4. Co-administration of cibacron blue (30 and 100 nmol paw(-1)), a selective allosteric enhancer of P2X(3) and P2X(2/3) receptor activation, with BzATP (30 and 100 nmol paw(-1)) into the rat hindpaw produced significantly greater nociception as compared to the algogenic effects of BzATP alone. Intradermal co-administration of cibacron blue (30 and 100 nmol paw(-1)) with formalin (1 and 2.5%) into the rat hindpaw also produced significantly greater nociceptive behaviour as compared to formalin alone. 5. The ability of TNP-ATP and cibacron blue to respectively attenuate and enhance nociceptive responses elicited by exogenous BzATP and formalin provide further support for the hypothesis that activation of peripheral P2X(3) containing channels contributes specifically to both acute and persistent nociception in the rat.

Competitive antagonism of recombinant P2X(2/3) receptors by 2', 3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP).

TNP-ATP has become widely recognized as a potent and selective P2X receptor antagonist, and is currently being used to discriminate between subtypes of P2X receptors in a variety of tissues. We have investigated the ability of TNP-ATP to inhibit alpha,beta-methylene ATP (alpha,beta-meATP)-evoked responses in 1321N1 human astrocytoma cells expressing recombinant rat or human P2X(2/3) receptors. Pharmacological responses were measured using electrophysiological and calcium imaging techniques. TNP-ATP was a potent inhibitor of P2X(2/3) receptors, blocking both rat and human receptors with IC(50) values of 3 to 6 nM. In competition studies, 10 to 1000 microM alpha,beta-meATP was able to overcome TNP-ATP inhibition. Schild analysis revealed that TNP-ATP was a competitive antagonist with pA(2) values of -8.7 and -8.2. Inhibition of P2X(2/3) receptors by TNP-ATP was rapid in onset, reversible, and did not display use dependence. Although the onset kinetics of inhibition were concentration-dependent, the TNP-ATP off-kinetics were concentration-independent and relatively slow. Full recovery from TNP-ATP inhibition did not occur until >/=5 s after removal of the antagonist. Because of the slow off-kinetics of TNP-ATP, full competition with alpha,beta-meATP for receptor occupancy could be seen only after both ligands had reached a steady-state condition. It is proposed that the slowly desensitizing P2X(2/3) receptor allowed this competitive interaction to be observed over time, whereas the rapid desensitization of other P2X receptors (P2X(3)) may mask the detection of competitive inhibition by TNP-ATP.

Channel properties determine the transient activation kinetics of recombinant GABA(A) receptors.

To understand the mechanisms underlying activation and deactivation of GABA(A) receptor (GABAR) channels, we studied the properties of an identified GABAR isoform under conditions similar to those present at central GABAergic synapses. Recombinant alpha5beta3gamma2L GABARs were expressed in L929 fibroblasts and studied using patch-clamp recording techniques. Brief application of a high GABA concentration to outside-out membrane patches elicited transient currents that resembled those reported for miniature inhibitory postsynaptic currents (mIPSCs), as well as native GABAR currents recorded under similar conditions. Characteristic of these currents was a rapid activation phase followed by a prolonged biphasic deactivation phase that far outlasted GABA application. Single-channel recordings revealed unique patterns of channel activity with two channel conductance states of 22 and 16 pS. The prolonged deactivation phase appeared to be sustained by entry into and reopening from long-lasting closures or desensitized states. Agonist affinity determined the time course of deactivation, indicating that occupied receptors drove the channel activity underlying the decay of current. The time course of deactivation was also longer at depolarized membrane potentials. The similarities between transient activation kinetics of recombinant alpha5beta3gamma2L GABARs to activation of synaptic GABARs (rapid activation and prolonged, voltage-dependent deactivation) suggest that intrinsic channel properties determine much of the response patterns of native GABARs.

Allosteric modulation and accelerated resensitization of human P2X(3) receptors by cibacron blue.

The activity of ATP as a fast neurotransmitter is mediated by the P2X family of ligand-gated ion channels. P2X receptor subtypes are subject to functional modulation by a diverse set of factors, including pH, divalent cations, and temperature. The human P2X(3) (hP2X(3)) receptor subunit is expressed primarily in sensory ganglia where it exists as either a homomultimeric receptor or, in combination with P2X(2), as a heteromultimeric receptor. This article describes the allosteric modulatory effect of the putative P2X receptor antagonist cibacron blue on the activity of recombinant hP2X(3) receptors. In 1321N1 cells expressing the hP2X(3) receptor, cibacron blue mediated a 3- to 7-fold increase in both the magnitude and the potency of ATP-activated Ca(2+) influx and transmembrane currents. The half-maximal concentration of cibacron blue required to mediate maximal potentiation (EC(50) = 1.4 microM) was independent of the agonist used to activate the hP2X(3) receptor. The nonselective P2 receptor antagonist PPADS (pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid) caused a rightward shift of the cibacron blue concentration-effect curve, whereas increasing concentrations of cibacron blue attenuated PPADS antagonism. In addition to potentiating the effects of ATP at the hP2X(3) receptor, cibacron blue also produced a 6-fold increase in the rate of hP2X(3) receptor recovery from desensitization (from T(1/2) = 15.9 to 2.6 min), as evidenced by its ability to restore ATP responsiveness to acutely desensitized receptors. Consistent with the properties of other ligand-gated ion channels, these results suggest that hP2X(3) receptor activity can be allosterically modulated by a ligand distinct from the endogenous agonist.

Molecular and functional characterization of human P2X(2) receptors.

P2X receptors are a family of ATP-gated ion channels. Four cDNAs with a high degree of homology to the rat P2X(2) receptor were isolated from human pituitary and pancreas RNA. Genomic sequence indicated that these cDNAs represent alternatively spliced messages. Northern analysis revealed high levels of human P2X(2) (hP2X(2)) message in the pancreas, and splice variants could be detected in a variety of tissues. Two cDNAs encoded functional ion channels when expressed in Xenopus oocytes, a receptor structurally homologous to the prototype rat P2X(2) receptor (called hP2X(2a)) and a variant containing a deletion within its cytoplasmic C terminus (called hP2X(2b)). Pharmacologically, these functional human P2X(2) receptors were virtually indistinguishable, with the P2X receptor agonists ATP, 2-methylthio-ATP, 2' and 3'-O-(4-benzoylbenzoyl)-ATP, and ATP5'-O-(3-thiotriphosphate) being approximately equipotent (EC(50) = 1 microM) in eliciting extracellular Ca(2+) influx. The P2 receptor agonists alpha,beta-methylene ATP, adenosine, adenosine 5'-O-(2-thiodiphosphate), and UTP were inactive at concentrations up to 100 microM. Both hP2X(2a) and hP2X(2b) receptors were sensitive to the P2 receptor antagonist pyridoxal-5-phosphate-6-azophenyl-2', 4'-disulfonic acid (IC(50) = 3 microM). In contrast to the analogous rat P2X(2) and P2X(2b) receptors, the desensitization rates of the hP2X(2a) and hP2X(2b) receptors were equivalent. Both functional forms of the human P2X(2) receptors formed heteromeric channels with the human P2X(3) receptor. These data demonstrate that the gene structure and mRNA heterogeneity of the P2X(2) receptor subtype are evolutionarily conserved between rat and human, but also suggest that alternative splicing serves a function other than regulating the desensitization rate of the human receptor.

P2X receptor-mediated ionic currents in dorsal root ganglion neurons.

Nociceptive neurons in the dorsal root ganglia (DRG) are activated by extracellular ATP, implicating P2X receptors as potential mediators of painful stimuli. However, the P2X receptor subtype(s) underlying this activity remain in question. Using electrophysiological techniques, the effects of P2X receptor agonists and antagonists were examined on acutely dissociated adult rat lumbar DRG neurons. Putative P2X-expressing nociceptors were identified by labeling neurons with the lectin IB4. These neurons could be grouped into three categories based on response kinetics to extracellularly applied ATP. Some DRG responses (slow DRG) were relatively slowly activating, nondesensitizing, and activated by the ATP analogue alpha,beta-meATP. These responses resembled those recorded from 1321N1 cells expressing recombinant heteromultimeric rat P2X2/3 receptors. Other responses (fast DRG) were rapidly activating and desensitized almost completely during agonist application. These responses had properties similar to those recorded from 1321N1 cells expressing recombinant rat P2X3 receptors. A third group (mixed DRG) activated and desensitized rapidly (P2X3-like), but also had a slow, nondesensitizing component that functionally prolonged the current. Like the fast component, the slow component was activated by both ATP and alpha, beta-meATP and was blocked by the P2X antagonist TNP-ATP. But unlike the fast component, the slow component could follow high-frequency activation by agonist, and its amplitude was potentiated under acidic conditions. These characteristics most closely resemble those of rat P2X2/3 receptors. These data suggest that there are at least two populations of P2X receptors present on adult DRG nociceptive neurons, P2X3 and P2X2/3. These receptors are expressed either separately or together on individual neurons and may play a role in the processing of nociceptive information from the periphery to the spinal cord.

Pharmacological characterization of recombinant human and rat P2X receptor subtypes.

ATP functions as a fast neurotransmitter through the specific activation of a family of ligand-gated ion channels termed P2X receptors. In this report, six distinct recombinant P2X receptor subtypes were pharmacologically characterized in a heterologous expression system devoid of endogenous P2 receptor activity. cDNAs encoding four human P2X receptor subtypes (hP2X1, hP2X3, hP2X4, and hP2X7), and two rat P2X receptor subtypes (rP2X2 and rP2X3), were stably expressed in 1321N1 human astrocytoma cells. Furthermore, the rP2X2 and rP2X3 receptor subtypes were co-expressed in these same cells to form heteromultimeric receptors. Pharmacological profiles were determined for each receptor subtype, based on the activity of putative P2 ligands to stimulate Ca2+ influx. The observed potency and kinetics of each response was receptor subtype-specific and correlated with their respective electrophysiological properties. Each receptor subtype exhibited a distinct pharmacological profile, based on its respective sensitivity to nucleotide analogs, diadenosine polyphosphates and putative P2 receptor antagonists. Alphabeta-methylene ATP (alphabeta-meATP), a putative P2X receptor-selective agonist, was found to exhibit potent agonist activity only at the hP2X1, hP2X3 and rP2X3 receptor subtypes. Benzoylbenzoic ATP (BzATP, 2' and 3' mixed isomers), which has been reported to act as a P2X7 receptor-selective agonist, was least active at the rat and human P2X7 receptors, but was a potent (nM) agonist at hP2X1, rP2X3 and hP2X3 receptors. These data comprise a systematic examination of the functional pharmacology of P2X receptor activation.

Expression of functional GABAA receptors in transfected L929 cells isolated by immunomagnetic bead separation.

Transient cotransfection of fibroblasts, with plasmids encoding individual GABAA receptor (GABAAR) subunits, has provided a model to characterize the pharmacological and kinetic properties of receptor subtype combinations. However, identifying transfected cells for electrophysiological recording is often difficult due to low transfection efficiencies. Selection of transfected cells has required cotransfection with a marker gene and fluorescence microscopic localization prior to recording. To circumvent these problems, two GABAAR subtypes combinations in transfected L929 cells were isolated with a novel biomagnetic separation system. Cell selection was accomplished by cotransfection with a plasmid (pHook-1) encoding a single-stranded cell surface antibody (sFv), which bound to ferromagnetic beads, coated with an antigen (phOx). Bead-covered cells were then magnetically separated from non-transfected cells. Bead-selected cells cotransfected with alpha 6, beta 3 and gamma 2L subtypes, expressed GABAAR currents in 95% (41/43) of cells recorded. Cells cotransfected with alpha 5, beta 3 and gamma 2L subtypes had an EC50 for GABA of 5.4 microM and a Hill slope of 1.4. Membrane patches from cells expressing the alpha 5 beta 3 gamma 2L isoform demonstrated single channel currents with a main conductance state of 23 pS. Magnetic bead immunoselection provides a purified population of transfected cells well suited for whole cell and single channel recording.

Properties of recombinant gamma-aminobutyric acid A receptor isoforms containing the alpha 5 subunit subtype.

The cDNAs encoding alpha 5 and gamma 2L subunit subtypes of the gamma-aminobutyric acid (GABA) type A receptor (GABAR) were transfected into L929 cells together with cDNAs encoding either the beta 1, beta 2, or beta 3 subunit subtype. Properties of expressed recombinant alpha 5 beta X gamma 2L (where X = 1,2, or 3) GABARs were studied with the use of whole-cell, patch-clamp techniques. In cells voltage-clamped at -70 mV with equlvalent bath and pipette chloride concentrations, the application of GABA produced a concentration-dependent inward chloride current with all three alpha 5 beta X gamma 2L isoforms. Minimal or no responses were recorded from cells transfected with only two subunit cDNAs, demonstrating that all three subunits were required for functional receptor assembly in these cells. The GABA concentration producing a half-maximal current was similar for beta 2 and beta 3 subtype-containing receptors (6 microM) but higher for beta 1 subtype-containing receptors (26 microM). alpha 5 beta 3 gamma 2L receptors were zinc and diazepam sensitive but zolpidem insensitive. In response to low GABA concentrations, beta 1 and beta 3 subtype-containing receptors showed outward rectification of the current-voltage relationship, whereas current-voltage responses of beta 2 subtype-containing receptors were relatively linear. Likewise, at high GABA concentrations, beta 1 and beta 3 subtype-containing receptors showed less desensitization at positive than at negative membrane potentials. Beta 2 subtype-containing receptors displayed faster desensitization at depolarized potentials. These voltage-dependent properties were characteristic of alpha 5 but not alpha 1 or alpha 6 subtype-containing receptors and were similar to responses recorded from hippocampal CA1 pyramidal neurons. Based on the pharmacological and biophysical similarities to hippocampal GABAR responses, the alpha 5 beta 3 gamma 2L isoform could represent a native GABAR subtype.

N-methyl-D-aspartate receptor-mediated calcium accumulation in neocortical neurons.

Calcium imaging and patch-clamp recording techniques were used to investigate the relationship between membrane properties and intracellular calcium changes in response to the excitatory amino acid neurotransmitter glutamate. Application of glutamate to cultured neocortical neurons produced concentration-dependent increases in intracellular calcium, membrane depolarization and transmembrane current. At a low concentration (3 microM), glutamate induced only a small depolarization (< 10 mV), yet produced a substantial increase in intracellular calcium. The calcium increase was observed in the presence of extracellular magnesium, was dependent on extracellular calcium, was blocked by an N-methyl-D-aspartate receptor antagonist, and was not affected by manipulation of intracellular calcium stores. This low concentration of glutamate also induced membrane currents that exhibited an N-methyl-D-aspartate-like unconventional voltage dependence. When glutamate was increased to a concentration known to produce excitotoxicity (500 microM), large depolarizations and membrane currents were induced, which rapidly reversed following prolonged glutamate applications. Changes in intracellular calcium in response to 500 microM glutamate had both voltage-sensitive and -insensitive components, and consistently remained elevated following removal of glutamate. These results indicate that low concentrations of glutamate can preferentially activate N-methyl-D-aspartate receptors, leading to increases in intracellular calcium. Functionally this may be involved in N-methyl-D-aspartate receptor responses to ambient extracellular glutamate. In addition, N-methyl-D-aspartate receptor-mediated calcium influx and subsequent depolarization induced by high glutamate concentrations can produce alterations in intracellular calcium homeostasis, which may play an important role in excitotoxicity.

Developmental changes in the voltage-dependence of neocortical NMDA responses.

A change in the response of neocortical neurons to N-methyl-D-aspartate (NMDA) was observed during the first 2 weeks of postnatal development. When NMDA was bath applied, the membrane current-voltage relationship recorded in neurons from postnatal day (PN) 3-5 rats displayed a region of decreased inward current at hyperpolarized membrane potentials. By PN 9-14, the net inward current at hyperpolarized potentials was significantly less than that recorded in PN 3-5 neurons. These results indicate that a developmental increase in the voltage-dependence of NMDA responses exists, which may be due to changes in magnesium sensitivity of the NMDA receptor.

NMDA receptor-mediated components of miniature excitatory synaptic currents in developing rat neocortex.

1. In vitro slices of frontal neocortex were prepared from rat pups at various ages after birth: postnatal days (PN) 3-5, 6-8, and 9-14. Using whole-cell patch-clamp techniques, both spontaneous and evoked excitatory postsynaptic currents (EPSCs) were recorded from voltage-clamped layer II-III pyramidal neurons. Developmental changes in EPSCs were examined. 2. Four properties of miniature EPSCs (mEPSCs) were studied: rise time, amplitude, decay time constant (tau), and frequency. These parameters were not tetrodotoxin sensitive and did not exhibit significant developmental changes during the first two postnatal weeks. 3. mEPSCs occurred approximately every 2-3 s and had peak amplitudes of 25-30 pA. Within each age group, certain parameters of mEPSCs were voltage dependent. mEPSC rise time and decay tau were significantly increased at depolarized potentials (-30 to -45 mV) relative to hyperpolarized (-75 to -90 mV) or resting membrane potential (RMP) (-60 to -70 mV). 4. At threshold stimulation intensity, EPSCs were evoked in an "all-or-none" manner. The amplitude and decay tau of evoked unitary EPSCs and mEPSCs were not significantly different. As stimulation intensity was increased, a late EPSC component appeared that was not seen in mEPSCs. At suprathreshold stimulus intensities, EPSC duration was significantly longer in PN 3-5 than in PN 9-14 neurons. 5. The N-methyl-D-aspartate (NMDA) receptor antagonist D(-)2-amino-5-phosphonovaleric acid (APV, 10 microM) significantly decreased mEPSC decay tau and frequency only at depolarized membrane potentials. Likewise, EPSCs were depressed by APV to a greater extent at depolarized potentials, and the depression was mainly of the late component. mEPSCs recorded at RMP were blocked by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline- 2,3-dione (5 microM). 6. Removal of extracellular Mg2+ reversibly increased the decay tau of mEPSCs at RMP but not at depolarized membrane potentials. The decay tau and duration of evoked EPSCs were also increased in zero Mg2+. These effects were reversible with application of APV. All effects of zero Mg2+ on mEPSCs and EPSCs were observed as early as PN 3-5. 7. These results indicate that the basic kinetic properties of mEPSCs are present by PN 3-5 and do not change significantly over the first two postnatal weeks. NMDA receptor activation contributes to mEPSCs and sensitivity to Mg2+ appears as early as PN 3-5. Unitary EPSCs resemble mEPSCs, but a late NMDA receptor-mediated component appears in EPSCs as stimulus intensity is increased.(ABSTRACT TRUNCATED AT 400 WORDS)

Muscarinic depression of synaptic transmission and blockade of norepinephrine-induced long-lasting potentiation in the dentate gyrus.

Bath application of the muscarinic receptor agonist, muscarine, produced a concentration-dependent depression of synaptic activity in the dentate gyrus of hippocampal slices. A concentration of 10 microM muscarine produced a reversible depression that could be competitively antagonized by the muscarinic receptor antagonist pirenzepine. However, other muscarinic receptor subtype (M1-M3) antagonists could also block the effects of muscarine. The rank order of antagonist potency was: 4-diphenylacetoxy-N-methyl-piperidine methiodide (M3/M1 antagonist) > pirenzepine (M1) > AFDX-116 (M2). The depression produced by 10 microM muscarine was not affected by in vivo pretreatment with pertussis toxin, and therefore was not mediated by a pertussis toxin-sensitive G-protein. In addition, high concentrations of muscarine did not affect either basal or isoproterenol-stimulated accumulation of cyclic AMP from slices of dentate gyrus. Muscarine also produced a concentration-dependent blockade of the induction of norepinephrine-induced long-lasting potentiation in the dentate gyrus. Norepinephrine-induced long-lasting potentiation is a form of long-lasting plasticity induced in medial perforant path synapses by beta-adrenergic agonists such as isoproterenol. The muscarinic blockade of norepinephrine-induced long-lasting potentiation was also prevented by pretreatment with pirenzepine. Based on these pharmacological data, we conclude that muscarinic depression of evoked responses, as well as blockade of norepinephrine-induced long-lasting potentiation, involves activation of either M3 or M1, but not M2, muscarinic receptors. These data also demonstrate that in addition to modulating normal synaptic transmission, muscarinic receptors may also play an important role in modulating synaptic plasticity.

Developmental changes in NMDA and non-NMDA receptor-mediated synaptic potentials in rat neocortex.

1. In vitro slices of frontal neocortex were prepared from rat pups 3-14 days of age. Whole-cell patch-clamp recordings were obtained from layer II-III cortical neurons, and measurements of passive membrane properties were made. The development of evoked synaptic excitation and inhibition was also examined with the use of current- and voltage-clamp techniques. 2. Pharmacological separation of excitatory synaptic activity into both N-methyl-D-aspartate (NMDA) and non-NMDA receptor-mediated components was accomplished by application of D(-)2-amino-5-phosphonovaleric acid (APV), D(-)2-amino-7-phosphonoheptanoic acid (AP7), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Inhibitory synaptic events were described according to their reversal potentials and modulation by the GABAA receptor antagonist bicuculline methiodide (BMI). 3. Pups were grouped into three categories on the basis of age: postnatal day (PN) 3-5, PN 6-8, and PN 9-14. In slices from PN 3-5 pups, neurons exhibited high input resistances (Rn) and relatively low resting membrane potentials (RMP). Rns decreased, and RMPs became more negative with development. At all ages studied, current-voltage relationships measured in current clamp were relatively linear, with inward rectification observed in some neurons at hyperpolarized membrane potentials. Neurons in each group were capable of firing overshooting action potentials. 4. Local stimulation in layer IV-V at 0.033 Hz elicited depolarizing excitatory postsynaptic potentials (EPSPs) in neurons from all three age groups. In PN 3-5 neurons, EPSPs were characterized by a long duration and latency to peak. By PN 6-8, EPSPs had decreased significantly in both duration and latency-to-peak. Some neurons responded with a single-component EPSP, whereas others exhibited multicomponent EPSPs consisting of distinct early and late components. In PN 3-5 neurons, increasing the frequency of stimulation from 0.033 to 1 Hz resulted in an overall decrease in the amplitude of the entire EPSP, whereas in PN 6-8 neurons the main decrease was observed in the late EPSP. 5. Excitatory postsynaptic currents (EPSCs) recorded in both PN 3-5 and PN 6-8 neurons were shorter in duration than corresponding EPSPs and consisted of both early and late components. Early EPSCs routinely increased in amplitude with hyperpolarization at all ages. In PN 3-5 neurons, the voltage dependence of late EPSCs was variable. By PN 6-8, late EPSCs always exhibited a region of reduced amplitude from -45 to -90 mV. The reversal potential for both early and late EPSCs was near +10 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Long-lasting potentiation and epileptiform activity produced by GABAB receptor activation in the dentate gyrus of rat hippocampal slice.

Bath application of the GABAB receptor agonist baclofen produced a concentration-dependent long-lasting potentiation (LLP) of the evoked population spike in the dentate gyrus of rat hippocampal slices. A high concentration of baclofen (5 microM) also produced a loss of inhibition that was manifested as the appearance of epileptiform, multiple evoked population spikes and a decrease in paired-pulse inhibition. Both baclofen-induced potentiation and epileptiform activity could be blocked or significantly reduced in slices from pertussis toxin-treated animals (1 microgram, intradentate) or in slices pretreated with the NMDA receptor antagonist D-(-)-2-amino-5-phosphonovaleric acid (10 microM). At a concentration that had no significant effect on individual evoked responses (0.1 microM) but still produced a loss in paired-pulse inhibition, baclofen facilitated the induction of beta-adrenergic receptor-mediated LLP. LLP was induced in the dentate gyrus by bath application of 1 microM, but not 0.1 microM, isoproterenol. Coapplication of baclofen and isoproterenol, both at a concentration (0.1 microM) that individually had no effect on the population spike, produced a synergistic LLP of the population spike. We propose that baclofen produces a selective disinhibitory effect in the granule cell layer of the dentate gyrus by inhibiting the activity of GABAergic interneurons. At a low concentration, the subtle loss of inhibition can facilitate the induction of isoproterenol-induced LLP. At a high concentration, baclofen can produce an LLP that is probably induced by a loss of inhibition.

Muscarinic receptor activation facilitates the induction of long-term potentiation (LTP) in the rat dentate gyrus.

Bath application of two different concentrations of muscarine produced two different effects on evoked responses in the dentate gyrus of rat hippocampal slices. A concentration of 1 microM muscarine did not affect the evoked population spike or excitatory postsynaptic potential (EPSP), but facilitated the induction of LTP. In contrast, a concentration of 10 microM muscarine depressed both the population spike and EPSP, but had no effect on LTP induction. The M1 muscarinic receptor antagonist pirenzepine (1 microM) blocked the muscarine-induced facilitation of LTP, but had no effect on the depression of evoked responses. These data suggest that activation of M1 receptors can facilitate the induction of LTP.

NMDA receptor antagonists reduce medial, but not lateral, perforant path-evoked EPSPs in dentate gyrus of rat hippocampal slice.

NMDA receptor antagonists produced differential effects on medial and lateral perforant path-evoked excitatory postsynaptic potentials (EPSPs) recorded in the dentate gyrus molecular layer of hippocampal slices. D-(-)-2-amino-5-phosphonovaleric acid (D(-)-APV) and 3[(+/-)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP) significantly reduced the peak amplitude and total area, but not the initial negative slope, of the medial perforant path-evoked EPSP. Neither antagonist affected any component of the lateral perforant path-evoked EPSP. In contrast, population spikes evoked by stimulation of either pathway were depressed.