Duckweed protein supports the growth and organ development of mice: A feeding study comparison to conventional casein protein.

As global population growth and meat consumption increases, sustainable alternatives to conventional protein-rich fodder crops for livestock are needed to reduce negative environmental impacts. Duckweed, a small floating aquatic plant, can generate 5 to 10 times higher protein yields than conventional land-grown crops. Although some in vivo feeding trials with duckweed have been conducted, those measuring animal weight are limited, and those examining organ development are nonexistent. To secure broad acceptance of new protein sources, such controlled studies are critical. This study measured the food intake, growth, and final organ and adipose tissue mass of male CF-1 mice fed a semi-purified diet containing casein or diets in which 10% or 25% of the casein was replaced with duckweed protein (DWP). Proximate analysis showed that the DWP preparation used contained 39.9% protein (w/w), and contained all of the essential amino acids with Met as the limiting amino acid. The average growth rates were not significantly different among the treatment groups: 0.21 g/day; 0.24 g/day; and 0.25 g/day for the control, 10%, and 25% DWP protein diets, respectively. The daily food intake of both DWP diets was 6.5% to 8.0% higher than the control diet, but feeding efficiency did not differ among diets. The relative weight of the liver, spleen, kidneys, heart, and epidydimal fat, and colon length were not significantly different between treatment groups. The results from this study show that replacement of up to 25% dietary casein with DWP has no adverse effects on the growth rate and final organ and adipose tissue weights of laboratory mice. PRACTICAL APPLICATION: Duckweed can produce 5 to 10 times more protein per area than land-grown crops such as soybean. In this study, up to a 25% replacement of casein with duckweed protein had no observable effect on the growth or organ development of laboratory mice. Thus, duckweed has the potential to be used as a protein supplement for livestock, poultry, and fish, thereby decreasing environmental impacts from land-grown crops used for animal feed.

Flavonoid intake is inversely associated with obesity and C-reactive protein, a marker for inflammation, in US adults.

Recent studies have demonstrated the importance of flavonoid intake and disease risk, however the association between flavonoid intake and obesity has not been evaluated in a nationally representative sample of US adults. The objective of the study was to evaluate the association between flavonoid consumption and established risk factors for obesity and obesity-related inflammation. Data from a nationally representative sample of 9551 adults who participated in the 2005-2008 National Health and Nutrition Examination Survey (NHANES) were analyzed. Flavonoid consumption was inversely associated with obesity in both men and women in multivariate models. Adults in the highest quartile of flavonoid intake had significantly lower body mass index and waist circumference than those in the lowest quartile of flavonoid intake (P<0.03 and P<0.04, respectively), and flavonoid intake was inversely related to C-reactive protein levels in women (p-trend, 0.01). These findings support a growing body of laboratory evidence that flavonoid consumption may be beneficial for disease prevention.

Kinetic analysis of evoked IPSCs discloses mechanism of antagonism of synaptic GABAA receptors by picrotoxin.

BACKGROUND AND PURPOSE: Although picrotoxin is a well-established antagonist of GABA(A) receptors, detailed studies of its action on inhibitory synaptic transmission have not previously been made. EXPERIMENTAL APPROACH: Electrophysiological techniques were used to study the action of picrotoxin on inhibitory postsynaptic currents (IPSCs) evoked in hippocampal neurones, in culture and slice preparations prepared from Wistar rat embryos and juveniles, respectively. KEY RESULTS: Picrotoxin gradually reduced the amplitude of GABA(A) receptor-mediated eIPSCs in a concentration-dependent manner. This was accompanied by a marked acceleration of the eIPSC decay kinetics, which, in contrast to the effect on amplitude, developed immediately and was completely reversed on washing. The decaying phase of the IPSC could be resolved into two components; 30 microM picrotoxin reduced tau(fast) by 34% and increased its relative amplitude, while tau(slow) was reduced by 38%, and its relative amplitude decreased. The area under the decaying phase of the normalized eIPSC showed an immediate reduction by 36% in 30 microM picrotoxin. With increasing concentrations of picrotoxin, this normalized area converged towards 55% of the control, indicating that the rate of relaxation and block has a finite maximum. This implies that picrotoxin does not act by a pore-occluding mechanism (open-channel blocking), and suggests allosteric stabilization of desensitized receptor states as a more likely alternative. This was corroborated by modelling, based on two established microscopic GABA(A) receptor transition schemes. CONCLUSIONS AND IMPLICATIONS: Although the identity of the stabilized state has not been determined unequivocally, picrotoxin effectively traps synaptic GABA(A) receptors in a desensitized state.

tetra-O-methylnordihydroguaiaretic acid inhibits melanoma in vivo.

tetra-O-methylnordihydroguaiaretic acid is a derivative of a naturally-occurring lignan, nordihydroguaiaretic acid, that has previously been shown to inhibit various cancer types in vitro and in vivo. Additionally, nordihydroguaiaretic acid has been shown to have nephrotoxic effects in the rat. Here we show that tetra-O-methylnordihydroguaiaretic acid inhibits the growth of a number of tumor cell lines in vitro by inducing apoptosis in a non-schedule-dependent manner. Further, this compound inhibits the synthesis of DNA by melanoma cells and causes cell cycle arrest in G0/G1 and G2/M phases of the cell cycle. tetra-O-Methylnordihydroguaiaretic acid also inhibits the growth of both murine and human melanomas and human colon cancer in vivo without apparent hepatic or renal toxicity.

Pharmacokinetic analysis by high-performance liquid chromatography of intravenous nordihydroguaiaretic acid in the mouse.

Nordihydroguaiaretic acid (NDGA) has been shown to inhibit both 5-lipoxygenase and ornithine decarboxylase and is active against several cancer cell lines and at least one mouse tumor model. Despite these findings, there have been no reports on the pharmacokinetics of NDGA. A reverse-phase high-performance liquid chromatography (HPLC) method was developed to detect NDGA in mouse plasma. The limit of detection of this method was 0.5 microg/ml. Administration of NDGA (50 mg/kg, i.v.) to mice resulted in a peak plasma concentration of 14.7 microg/ml. The terminal half-life of NDGA was 135.0 min with a clearance of 201.9 ml/min x kg.

Characteristics of AMPA receptor-mediated responses of cultured cortical and spinal cord neurones and their correlation to the expression of glutamate receptor subunits, GluR1-4.

Electrophysiological recordings have been used to characterize responses mediated by AMPA receptors expressed by cultured rat cortical and spinal cord neurones. The EC(50) values for AMPA were 17 and 11 microM, respectively. Responses of cortical neurones to AMPA were inhibited competitively by NBQX (pK(i)=6.6). Lower concentrations of NBQX (< or =1 microM) also potentiated the plateau responses of spinal cord neurones to AMPA, which could be attributed to a depression of desensitization to AMPA. GYKI 52466 inhibited responses of spinal cord neurones to AMPA to about twice the extent of responses of cortical neurones. Blockade of AMPA receptor desensitization by cyclothiazide (CTZ) potentiated responses of spinal cord neurones (6.8 fold) significantly more than responses of cortical neurones (4.8 fold). Responses of cortical neurones to KA were potentiated 3.5 fold by CTZ, while responses of spinal cord neurones were unaffected. Ultra-fast applications of AMPA to outside-out patches showed responses of spinal cord neurones desensitized by 97.5% and exhibit marked inward rectification, whereas cortical neurones desensitized by 91% and exhibited slight outward rectification. The time constants of deactivation and desensitization were about twice as fast in spinal cord than cortical neurones. In cortical neurones, single-cell RT - PCR showed GluR2 and GluR1 accounted for 91% of all subunits and were expressed together in 67% of neurones, predominantly as the flip variants (78%). GluR2 was detected alone in 24% of neurones. GluR3 and GluR4 were present in only 14 and 29% of neurones, respectively. For spinal cord neurones, GluR4(o) was detected in 81% of neurones, whereas predominantly flop versions of GluR1, 2 and 3 were detected in 38, 13 and 13% of neurones, respectively. These expression patterns are related to the respective pharmacological and mechanistic properties.

MAPK signaling by the D quadrant embryonic organizer of the mollusc Ilyanassa obsoleta.

Classical experiments performed on the embryo of the mollusc Ilyanassa obsoleta demonstrate that the 3D macromere acts as an embryonic organizer, by signaling to other cells and inducing them to assume the correct pattern of cell fates. We have discovered that MAP kinase signaling is activated in the cells that require the signal from 3D for normal differentiation. Preventing specification of the D quadrant lineage by removing the polar lobe disrupts the pattern of MAPK activation, as does ablation of the 3D macromere itself. Blocking MAPK activation with the MAP Kinase inhibitor U0126 produces larvae that differentiate the same limited complement of tissues as D quadrant deletions. Our results suggest that the MAP Kinase signaling cascade transduces the inductive signal from 3D and specifies cell fate among the cells that receive the signal.

Developmental increase in asynchronous GABA release in cultured hippocampal neurons.

Developmental changes in GABAergic synaptic transmission were examined in cultured hippocampal neurons using patch-clamp recordings and Ca(2+) imaging. In paired recordings, tetanization of the presynaptic GABAergic neuron with 80 pulses at either 40 or 80Hz was accompanied by tetanic depression of inhibitory postsynaptic responses. In neurons that had been cultured for more than two weeks, asynchronous inhibitory postsynaptic currents often appeared during the tetanus and continued for several seconds following stimulation. There was little asynchronous activity in neurons that had been cultured for shorter times. However, no age-related changes were observed in the amplitude of single synchronous inhibitory postsynaptic currents, paired-pulse depression or post-tetanic potentiation of inhibitory postsynaptic currents. Following equimolar replacement of extracellular Ca(2+) with strontium ions (Sr(2+)), single autaptic inhibitory postsynaptic currents were depressed in amplitude and asynchronous inhibitory postsynaptic currents were present on the decaying phase. Sr(2+)-induced asynchronous inhibitory postsynaptic currents showed no dependence on age in culture. Imaging of Ca(2+) in single GABAergic boutons was performed by including Fluo-3 in the patch pipette. During action potential firing induced by stimulating at 80Hz for 1s, intracellular calcium [Ca(2+)](i) increased rapidly in individual boutons. Following the stimulus, [Ca(2+)](i) decayed back to baseline within 10-15s. The half-time of decay increased from 1. 7+/-0.2s at 15days in vitro to 4.0+/-0.2s at 30days in vitro (P<0. 05), with a developmental profile that closely matched the increase in asynchronous inhibitory postsynaptic currents. We propose that the increase in tetanus-induced asynchronous GABA-release during the first month of synapse maturation in vitro is caused by a slowing of the Ca(2+)-clearing mechanisms in the GABAergic boutons. This results in larger and more prolonged elevations of [Ca(2+)](i) during tetanic stimulation, which leads to enhanced asynchronous transmitter release. We propose that the results of this study demonstrate a potentially important aspect of synapse maturation during development, and also imply that GABA release is up-regulated in conditions of decreased Ca(2+) buffering and clearing.

Larger intercellular variation in (Q/R) editing of GluR6 than GluR5 revealed by single cell RT-PCR.

RNA editing of the pre-mRNA encoding the kainate receptor subtypes determines the Ca2+ permeability and the rectifying properties of the receptors in which these are assembled. GluR6 pre-mRNA contains three characterized editing sites: Q/R, IN and the Y/C, whereas GluR5 pre-mRNA contains only the (Q/R) site. Single cell RT-PCR was used on cultured cortical neurons to determine the relative expression and editing levels of the kainate receptor subunits encoding mRNA. The analysis showed a large intercellular variation in editing efficiency. The overall lower level of GluR5 editing, in the culture, compared to GluR6 editing is a result of an approximately 60% lower editing efficiency of GluR5 pre-mRNA, within single cells, compared with GluR6.

Tetanus-induced asynchronous GABA release in cultured hippocampal neurons.

Asynchronous GABA release was studied in cultured hippocampal neurons using paired whole-cell recordings. Tetanization of the presynaptic GABAergic neuron was accompanied by a train of IPSCs which showed tetanic depression. Asynchronous IPSCs (asIPSCs) also developed during the train and continued for 1.85+/-0.3 s after the stimulation. The threshold frequency for evoking asIPSCs was 10 Hz, while maximal asynchronous activity was achieved at 40 Hz. Perfusion with EGTA-AM blocked asIPSCs. The elevation of [Ca(2+)](i) that accompanies presynaptic action potential firing triggers asynchronous release of GABA vesicles, thereby counteracting tetanic depression of synchronous IPSCs.

The effect of internal GTPgammaS on GABA-release in cultured hippocampal neurons.

The effects of presynaptic guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) on GABAergic inhibitory postsynaptic currents (IPSCs) were studied in cultured hippocampal neurons using whole-cell recordings. Inclusion of GTPgammaS (0.5-1 mM) in the presynaptic electrode reduced both the amplitude and paired-pulse depression of IPSCs, indicating that the probability of GABA-release had been reduced. Presynaptic GTPgammaS increased the depression of IPSCs by the GABA(B)-receptor-agonist baclofen (10 microM), and the effect of baclofen was poorly reversible after washing. Stimulation of the GABAergic neuron at 80 Hz for 1 s was accompanied by tetanic depression of the IPSCs by 52+/-6% and was followed by post-tetanic potentiation (PTP), reaching a peak value of 71+/-21% and lasting about 100 s. IPSCs evoked after tetanic stimulation were depressed and PTP was absent when tetanic stimulation was applied within 3 min after starting injection of GTPgammaS into the presynaptic neuron. At longer times, basal release underlying a single IPSC was depressed. This affected the ratios recorded in response to tetanic stimulations such that tetanic depression was abolished, while PTP increased to 117+/-34%. In conclusion, GTPgammaS reduces the probability of GABA-release in both a use- and time-dependent manner, most likely through an inhibitory action on presynaptic Ca2+-influx through voltage-gated Ca2+ channels or an interaction with small GTP-binding proteins in the nerve terminals.

Post-tetanic potentiation of GABAergic IPSCs in cultured rat hippocampal neurones.

1. Dual whole-cell patch-clamp recording was used to investigate post-tetanic potentiation (PTP) of GABAergic IPSCs evoked between pairs of cultured rat hippocampal neurones. Tetanization of the presynaptic neurone at frequencies (f) ranging from 5 to 100 Hz resulted in PTP of the IPSCs. Maximum PTP had a magnitude of 51.6 % just after the stimulus train, and lasted up to 1 min. PTP was shown to be dependent on the number of stimuli in the train, but independent of f at frequencies > or =5 Hz. 2. Blocking postsynaptic GABAA receptors with bicuculline during the tetanus did not affect the expression of PTP, showing that it is a presynaptic phenomenon. PTP was strongly affected by changing [Ca2+]o during the tetanus: PTP was reduced by lowering [Ca2+]o, and increased by high [Ca2+]o. 3. PTP was still present after presynaptic injection of BAPTA or EGTA, or following perfusion of the membrane-permeable ester EGTA-tetraacetoxymethyl ester (EGTA AM, 50 microM). On the other hand, EGTA AM blocked spontaneous, asynchronous IPSCs (asIPSCs), which were often associated with tetanic stimulation. 4. Tetanic stimulation in the presence of 4-aminopyridine (4-AP), which promotes presynaptic Ca2+ influx, evoked sustained PTP of IPSCs in half of the neurones tested. 5. The results indicate that PTP at inhibitory GABAergic synapses is related to the magnitude of presynaptic Ca2+ influx during the tetanic stimulation, leading to an enhanced probability of vesicle release in the post-tetanic period. The increase in [Ca2+]i occurs despite the presence of high-affinity exogenous and endogenous intracellular Ca2+ buffers. That PTP of IPSCs depends on the number, and not the frequency, of spikes in the GABAergic neurone is in accordance with a slow clearing of intracellular Ca2+ from the presynaptic terminals.

Somatic amplification of distally generated subthreshold EPSPs in rat hippocampal pyramidal neurones.

1. Intracellular recordings from hippocampal CA1 pyramidal neurones revealed that EPSPs evoked by selective stimulation of the isolated afferent input to the distal third of the apical dendrites were relatively insensitive to changes in dendritic membrane potential (Vm) but amplified by depolarizations of the somatic Vm. The amplification was present at potentials depolarized from resting membrane potential (RMP) but was most marked when the EPSPs were close to threshold for action potential generation. The amplification consisted of a uniform component and a variable component which was only present when the EPSPs were threshold straddling. 2. The somatic amplification was caused by an intrinsic membrane current which was blocked by somatic application of tetrodotoxin (TTX, 10 microM), but was insensitive to bath application of NiCl2 (100-200 microM). We therefore suggest that the amplification of the subthreshold EPSP is due primarily to the activation of a non-inactivating Na+ current (INaP). 3. Injection of 4-aminopyridine (4-AP, 25-50 mM) during intradendritic recordings resulted in amplification of the EPSPs in 37% of the dendrites, which was similar to that observed in somatic recordings. However, in the one case in which somatic application of TTX was tested, dendritic amplification was blocked, suggesting that it is a reflection of the somatic amplification. 4. Because the shift to variable amplification was very abrupt and it is present in only a very narrow voltage range close to threshold, we suggest that the variable component is caused by the regenerative activation of INaP. The variability itself is probably due to the simultaneous activation of different outward K+ currents. 5. The present results indicate that the somatic region of CA1 pyramidal neurones can function as a voltage-dependent amplifier of distally evoked EPSPs and that this is due to the activation of a somatic INaP. The presence of this amplifying mechanism will have important functional consequences for the way in which distally generated EPSPs are integrated.

Activity-dependent depression of GABAergic IPSCs in cultured hippocampal neurons.

Short-term depression of monosynaptic GABAergic inhibitory postsynaptic currents (IPSCs) evoked between pairs of cultured rat hippocampal neurons was investigated using dual whole cell patch-clamp recordings. Paired stimuli applied to the GABAergic neuron resulted in paired-pulse depression (PPD) of the second IPSC (IPSC2) at interpulse intervals from 25 to 2,000 ms. CGP 55845A, but not CGP 35348, reduced PPD marginally. Brief paired-pulse applications of exogenous GABA indicated that postsynaptic factors made only minimal contribution to PPD of IPSCs. IPSC1 and PPD was reduced on lowering [Ca2+]o and enhanced on increasing [Ca2+]o. The potassium-channel blocker 4-aminopyridine (4-AP), which increases presynaptic Ca2+ influx, enhanced IPSC1 and PPD. Chelation of residual Ca2+ in the GABAergic boutons with EGTA-AM enhanced PPD. Stimulation of the presynaptic neuron at frequencies (f) ranging from 2.5 to 80 Hz resulted in tetanic depression of IPSCs, which declined rapidly and reached a plateau depending on f and [Ca2+]o. CGP 55845A decreased tetanic depression in the first part of the train, but this could be overcome with continued stimulation. We show that GABAergic IPSCs are robustly depressed by paired-pulse stimulation in cultured hippocampal neurons. The depression of IPSCs is mainly independent of presynaptic GABAB receptors and could be caused by depletion of releasable vesicles. Depleted synapses recover with a slow time course, depending on factors that regulate [Ca2+]i in the GABAergic boutons.

Novel potent AMPA analogues differentially affect desensitisation of AMPA receptors in cultured hippocampal neurons.

The agonist actions of two AMPA receptor analogues, (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) and (RS)-2-amino-3-(3-hydroxy-5-trfluoromethyl-4-isoxazolyl)prop ionic acid (Tri-F-AMPA) have been studied on cultured rat hippocampal neurons. Whole-cell recordings with semi-rapid application of the agonists were used to study steady-state (plateau) responses. ACPA was the most potent agonist (EC50, 1.2 microM), followed by AMPA (4.3 microM) and Tri-F-AMPA (4.6 microM), corresponding to a potency ratio of 4:1:1. Hill coefficients were close to 1 for AMPA and ACPA and close to 2 for Tri-F-AMPA, respectively. Plateau responses to maximal concentrations of the three agonists varied more than 2-fold. ACPA responses were 2.1 times greater and responses to Tri-F-AMPA were 1.6 times greater than responses to AMPA, respectively. Peak responses and desensitization were studied by using a fast piezoelectric device to apply agonists rapidly to outside-out patches. The time constants of desensitization were 8 ms for AMPA, 12 ms for Tri-F-AMPA and 17 ms for ACPA. There were no significant differences in the time-to-peak and 10-90% rise-time of the responses. The results indicate that of the three agonists tested, ACPA is the most potent at AMPA receptors expressed in cultured hippocampal neurons and that the maximum response to the agonists is inversely related to the rate of desensitization.

Role of presynaptic L-type Ca2+ channels in GABAergic synaptic transmission in cultured hippocampal neurons.

Using dual whole cell patch-clamp recordings of monosynaptic GABAergic inhibitory postsynaptic currents (IPSCs) in cultured rat hippocampal neurons, we have previously demonstrated posttetanic potentiation (PTP) of IPSCs. Tetanic stimulation of the GABAergic neuron leads to accumulation of Ca2+ in the presynaptic terminals. This enhances the probability of GABA-vesicle release for up to 1 min, which underlies PTP. In the present study, we have examined the effect of altering the probability of release on PTP of IPSCs. Baclofen (10 microM), which depresses presynaptic Ca2+ entry through N- and P/Q-type voltage-dependent Ca2+ channels (VDCCs), caused a threefold greater enhancement of PTP than did reducing [Ca2+]o to 1.2 mM, which causes a nonspecific reduction in Ca2+ entry. This finding prompted us to investigate whether presynaptic L-type VDCCs contribute to the Ca2+ accumulation in the boutons during spike activity. The L-type VDCC antagonist, nifedipine (10 microM), had no effect on single IPSCs evoked at 0.2 Hz but reduced the PTP evoked by a train of 40 Hz for 2 s by 60%. Another L-type VDCC antagonist, isradipine (5 microM), similarly inhibited PTP by 65%. Both L-type VDCC blockers also depressed IPSCs during the stimulation (i.e., they increased tetanic depression). The L-type VDCC "agonist" (-)BayK 8644 (4 microM) had no effect on PTP evoked by a train of 40 Hz for 2 s, which probably saturated the PTP process, but enhanced PTP evoked by a train of 1 s by 91%. In conclusion, the results indicate that L-type VDCCs do not participate in low-frequency synchronous transmitter release, but contribute to presynaptic Ca2+ accumulation during high-frequency activity. This helps maintain vesicle release during tetanic stimulation and also enhances the probability of transmitter release during the posttetanic period, which is manifest as PTP. Involvement of L-type channels in these processes represents a novel presynaptic regulatory mechanism at fast CNS synapses.

Caregiving as a life course transition among older husbands: a prospective study.

Guided primarily by transitions theory, this study examined changes over two points in time (approximately 5 years apart) in multiple life domains (i.e., household tasks, social life, marital relationship, and well-being) between two groups of husbands aged 60 and older, who indicated that their wives were not in need of care or assistance due to an illness or disability at the initial interview. The two groups included husbands who identified themselves as a provider of care at Time 2 (T2; i.e., they had transitioned into the caregiver role; n = 26), and those married to healthy wives at T2 (i.e., noncaregivers; n = 262). Data came from a national probability sample of U.S. adults who were primary respondents to the National Survey of Families and Households in 1987-88, and who were followed up longitudinally in 1992-93. Findings suggested that husbands who entered the caregiving role demonstrated significant changes in household responsibilities, social integration, marital relationship, and well-being. Implications for practice and future research on the older husband caregiver are highlighted.

Deleterious mutations destabilize ribosomal RNA in endosymbiotic bacteria.

In populations that are small and asexual, mutations with slight negative effects on fitness will drift to fixation more often than in large or sexual populations in which they will be eliminated by selection. If such mutations occur in substantial numbers, the combined effects of long-term asexuality and small population size may result in substantial accumulation of mildly deleterious substitutions. Prokaryotic endosymbionts of animals that are transmitted maternally for very long periods are effectively asexual and experience smaller effective population size than their free-living relatives. The contrast between such endosymbionts and related free-living bacteria allows us to test whether a population structure imposing frequent bottlenecks and asexuality does lead to an accumulation of slightly deleterious substitutions. Here we show that several independently derived insect endosymbionts, each with a long history of maternal transmission, have accumulated destabilizing base substitutions in the highly conserved 16S rRNA. Stabilities of Domain I of this subunit are 15-25% lower in endosymbionts than in closely related free-living bacteria. By mapping destabilizing substitutions onto a reconstructed phylogeny, we show that decreased ribosomal stability has evolved separately in each endosymbiont lineage. Our phylogenetic approach allows us to demonstrate statistical significance for this pattern: becoming endosymbiotic predictably results in decreased stability of rRNA secondary structure.

Factors determining the efficacy of distal excitatory synapses in rat hippocampal CA1 pyramidal neurones.

1. A new preparation of the in vitro rat hippocampal slice has been developed in which the synaptic input to the distal apical dendrites of CA1 pyramidal neurones is isolated. This has been used to investigate the properties of distally evoked synaptic potentials. 2. Distal paired-pulse stimulation (0.1 Hz) evoked a dendritic response consisting of a pair of EPSPs, which showed facilitation. The first EPSP had a rise time (10-90%) of 2.2 +/- 0.05 ms and a half-width of 9.1 +/- 0.13 ms. The EPSPs were greatly reduced by CNQX (10 microM) and the remaining component could be enhanced in Mg(2+)-free Ringer solution and blocked by AP5 (50 microM). In 70% of the dendrites, the EPSPs were followed by a prolonged after-hyperpolarization (AHP) which could be blocked by a selective and potent GABAB antagonist, CGP55845A (2 microM). These results indicate that the EPSPs are primarily mediated by non-NMDA receptors with a small contribution from NMDA receptors, whereas the AHP is a GABAB receptor-mediated slow IPSP. 3. With intrasomatic recordings, the rise time of proximally generated EPSPs (3.4 +/- 0.1 ms) was half that of distally generated EPSPs (6.7 +/- 0.5 ms), whereas the half-widths were similar (19.6 +/- 0.8 ms and 23.8 +/- 1 ms, respectively). These results indicate that propagation through the proximal apical dendrites slows the time-to-peak of distally generated EPSPs. 4. Distal stimulation evoked spikes in 60% of pyramidal neurones. At threshold, the distally evoked spike always appeared on the decaying phase of the dendritic EPSP, indicating that the spike is initiated at some distance proximal to the dendritic recording site. Furthermore, distally and proximally generated threshold spikes had a similar voltage dependency. These results therefore suggest that distally generated threshold spikes are primarily initiated at the initial segment. 5. At threshold, spikes generated by stimulation of distal synapses arose from the decaying phase of the dendritic EPSPs with a latency determined by the time course of the EPSP at the spike initiation zone. With maximal stimulation, however, the spikes arose directly from the peak of the EPSPs with a time-to-spike similar to the time-to-peak of subthreshold dendritic EPSPs. Functionally, this means that the effect of dendritic propagation can be overcome by recruiting more synapses, thereby ensuring a faster response time to distal synaptic inputs. 6. In 42% of the neurones in which distal EPSPs evoked spikes, the relationship between EPSP amplitude and spike latency could be accounted for by a constant dendritic modulation of the EPSP. In the remaining 58%, the change in latency was greater than can be accounted for by a constant dendritic influence. This additional change in latency is best explained by a sudden shift in the spike initiation zone to the proximal dendrites. This would explain the delay observed between the action of somatic application of TTX (10 microM) on antidromically evoked spikes and distally evoked suprathreshold spikes. 7. The present results indicate that full compensation for the electrotonic properties of the main proximal dendrites is not achieved despite the presence of Na+ and Ca2+ currents. Nevertheless, distal excitatory synapses are capable of initiating spiking in most pyramidal neurones, and changes in EPSP amplitude can modulate the spike latency. Furthermore, even though the primary spike initiation zone is in the initial segment, the results suggest that it can move into the proximal apical dendrites under physiological conditions, which has the effect of further shortening the response time to distal excitatory synaptic inputs.

Studies of the antagonist actions of (RS)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl] propionic acid (ATPO) on non-NMDA receptors in cultured rat neurones.

Whole-cell patch-clamp recordings from single cultured cortical neurones have been used to study the action of (RS)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl+ ++]propionic acid (ATPO), which has previously been proposed to be a potent selective antagonist of 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors. ATPO competitively reduced peak responses evoked by semi-rapid applications of AMPA (Ki = 16 microM) but had variable effects on plateau responses, which were on average unchanged. Following blockade of AMPA receptor desensitization by cyclothiazide (CTZ, 100 microM), the plateau responses were reduced by ATPO to a similar extent as the peak responses, indicating that ATPO reduces desensitization of AMPA receptors. Semi-rapid application of kainic acid (KA) and the KA receptor-selective agonist, (2S,4R)-4-methylglutamic acid (MeGlu) evoked non-desensitizing responses which were competitively antagonized by ATPO (Ki values: 27 and 23 microM, respectively). Responses to MeGlu were unaffected by CTZ (100 microM), but potentiated 3 fold following blockade of KA receptor desensitization by concanavalin A (Con A, 300 microg ml(-1)). Responses of spinal cord neurones to MeGlu were blocked by ATPO to a similar extent before and after blockade of KA receptor desensitization by Con A. Although selectively potentiated by Con A, plateau responses to MeGlu were reduced by 69.6% by the AMPA selective antagonist, GYKI 53655 (10 microM). The remaining component was further reduced by ATPO with a Ki of 36 microM, which was not significantly different from that in the absence of GYKI 53655, but was greater than that on responses to AMPA. It is concluded that ATPO is a moderate-potency competitive inhibitor of naturally expressed non-NMDA receptors.