Electrical activity of caudal neurosecretory neurons in seawater and freshwater-adapted Platichthys flesus, in vivo.

This study examined the electrical firing activity of neuroendocrine Dahlgren cells in the caudal neurosecretory system (CNSS) of the euryhaline flounder in vivo. Intracellular recordings revealed generally similar activity patterns and membrane properties to those previously reported in vitro. To investigate the potential role of the CNSS in osmoregulatory adaptation, extracellular, multiunit, recordings compared the activity patterns of Dahlgren cells in fully seawater- and freshwater-adapted fish. The proportion of cells showing bursting (as opposed to phasic or tonic) activity was greater in seawater-than in freshwater-adapted fish, as was the Correlation Index, a measure of the degree of correlation between firing activities of cells recorded simultaneously from the same preparation. Acute transfer of fish from seawater to freshwater gill perfusion led to recruitment of previously silent Dahlgren cells and a reduction in Correlation Index; freshwater to seawater transfer increased the Correlation Index. Severing the spinal cord anterior to the CNSS led to an increase in overall Dahlgren cell activity. Electrical stimulation of branchial nerve branches providing input to the brainstem, or tactile (pinch) stimulation of lips or fins, led to a reduction in CNSS activity lasting up to 500 s, indicating the presence of descending modulatory pathways from the brain. These results are consistent with a role for CNSS neuropeptides, urotensins, in supporting survival in a hypertonic, seawater, environment.

Voltage- and Ca2+-dependent burst generation in neuroendocrine Dahlgren cells in the teleost Platichthys flesus.

The neuroendocrine Type 1 Dahlgren cells of the caudal neurosecretory system of the flounder display characteristic bursting activity, which may increase secretion efficiency. The firing activity pattern in these cells was voltage-dependent; when progressively depolarized, cells moved from silent (approximately -70 mV), through bursting and phasic to tonic firing (< -65 mV). Brief (10 s) evoked bursts of spikes were followed by a slow after-depolarization (ADP; amplitude up to 10 mV, duration 10-200 s), which was also voltage-dependent and could trigger a prolonged burst. The ADP was significantly reduced in the absence of external Ca(2+) ions or the presence of the L-type Ca(2+) channel blocker, nifedipine. BayK 8644 (which increases L-type channel open times) significantly increased ADP duration, whereas the Ca(2+)-activated nonselective cation channel blocker, flufenamic acid, had no effect. Pharmacological blockade of Ca(2+)-activated K(+) channels, using apamin and charybdotoxin, increased the duration of both ADP and evoked bursts. However, action potential waveform was unaffected by either apamin/charybdotoxin, nifedipine, BayK 8644 or removal of external Ca(2+). The short duration (approximately 100 ms), hyperpolarization-activated, postspike depolarizing afterpotentials (DAP), were significantly reduced by nifedipine. We propose that long duration ADPs underlie bursts and that short duration DAPs play a role in modulation of spike frequency.

Electrical activity of caudal neurosecretory neurons in seawater- and freshwater-adapted flounder: responses to cholinergic agonists.

The caudal neurosecretory system (CNSS) of the euryhaline flounder is involved in osmoregulatory responses underlying adaptation to seawater and freshwater. This study compared electrophysiological activity and responses to cholinergic agonists in the neuroendocrine Dahlgren cells in an in vitro preparation taken from fully seawater- (SWA) or freshwater-adapted (FWA) fish. Resting membrane and action potential parameters showed few differences between SWA and FWA cells. The hyperpolarisation-activated sag potential and depolarising afterpotential were present under both conditions; however, amplitude of the latter was significantly greater in SWA cells. The proportions of cells within the population exhibiting different firing patterns were similar in both adaptation states. However, bursting parameters were more variable in FWA cells, suggesting that bursting activity was less robust. The muscarinic agonist, oxotremorine, was largely inhibitory in Dahlgren cells, but increased activity in a non-Dahlgren cell population, alpha neurons. Nicotine promoted bursting activity in SWA Dahlgren cells, whereas it inhibited over half of FWA cells.

Modification of the philanthotoxin-343 polyamine moiety results in different structure-activity profiles at muscle nicotinic ACh, NMDA and AMPA receptors.

Voltage-dependent, non-competitive inhibition by philanthotoxin-343 (PhTX-343) analogues, with reduced charge or length, of nicotinic acetylcholine receptors (nAChR) of TE671 cells and ionotropic glutamate receptors (N-methyl-D-aspartate receptors (NMDAR) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR)) expressed in Xenopus oocytes from rat brain RNA was investigated. At nAChR, analogues with single amine-to-methylene or amine-to-ether substitutions had similar potencies to PhTX-343 (IC(50)=16.6 microM at -100 mV) whereas PhTX-(12), in which both secondary amino groups of PhTX-343 were replaced by methylenes, was more potent than PhTX-343 (IC(50)=0.93 microM at -100 mV). Truncated analogues of PhTX-343 were less potent. Inhibition by all analogues was voltage-dependent. PhTX-343 (IC(50)=2.01 microM at -80 mV) was the most potent inhibitor of NMDAR. At AMPAR, most analogues were equipotent with PhTX-343 (IC(50)=0.46 microM at -80 mV), apart from PhTX-83, which was more potent (IC(50)=0.032 microM at -80 mV), and PhTX-(12) and 4,9-dioxa-PhTX-(12), which were less potent (IC(50)s>300 microM at -80 mV). These studies show that PhTX-(12) is a selective nAChR inhibitor and PhTX-83 is a selective AMPAR antagonist.

Bursting properties of caudal neurosecretory cells in the flounder Platichthys flesus, in vitro.

Bursting activity in type 1 Dahlgren cells was studied using intra- and extracellular recording from an in vitro preparation of the caudal neurosecretory system of the euryhaline flounder. 45% of cells showed spontaneous bursts of approximately 120s duration and 380s cycle period. Similar bursts were triggered by short duration (<5s) depolarising or hyperpolarising pulses. Cells displayed a characteristic depolarising after potential, following either an action potential with associated afterhyperpolarisation, or a hyperpolarising current pulse. This depolarising after potential was related to a 'sag' potential, which developed during the hyperpolarising pulse. Both the depolarising after potential and the sag potential occurred only in cells at more depolarised (<60 mV) holding potentials. In addition, the amplitude of the depolarising after potential was dependent on the amplitude and the duration of the hyperpolarising pulse. The depolarising after potential following action potentials may provide a mechanism for facilitating repetitive firing during a burst. Extracellular recording revealed similar bursting in individual units which was not, however, synchronised between units. Spontaneous bursting activity recorded both intra- and extracellularly was inhibited by application of a known neuromodulator of the system, 5-hydroxytryptamine. This study provides a basis for investigating the relationship between physiological status, Dahlgren cell activity and neuropeptide secretion.

The caudal neurosecretory system: control and function of a novel neuroendocrine system in fish.

The caudal neurosecretory system (CNSS) of fish was first defined over 70 years ago yet despite much investigation, a clear physiological role has yet to be elucidated. Although the CNSS structure is as yet thought to be confined to piscine species, the secreted peptides, urotensins I and II (UI and UII), have been detected in a number of vertebrate species, most recently illustrated by the isolation of UII in humans. The apparent importance of these peptides, suggested by their relative phylogenetic conservation, is further supported by the complex control mechanisms associated with their secretion. The CNSS in teleosts is known to receive extensive and diverse innervation from the higher central nervous system, with evidence for the presence of cholinergic, noradrenergic, serotonergic, and peptidergic descending inputs. Recent observations also suggest the presence of glucocorticoid receptors in the flounder CNSS, supporting previous evidence for a possible role as a pituitary-independent mechanism controlling cortisol secretion. The most convincing evidence as to a physiological role for the CNSS in fish has stemmed from the direct and indirect influence of the urotensins on osmoregulatory function. Recent advances allowing the measurement of circulating levels of UII in the flounder have supported this. In addition, there is evidence to suggest some seasonal variation in peptide levels supporting the notion that the CNSS may have an integrative role in the control of coordinated changes in the reproductive, osmoregulatory and nutritional systems of migratory euryhaline species.

Solid phase synthesis and biological evaluation of enantiomerically pure wasp toxin analogues PhTX-343 and PhTX-12.

PhTX-343 and PhTX-12, analogues of the natural polyamine wasp toxin PhTX-433, were synthesised in 40-60% yields as pure enantiomers using solid phase synthesis techniques. Capillary electrophoresis procedures were developed for chiral separation and determination of enantiomeric purity (ee) of the enantiomers of PhTX-343 and PhTX-12. The methods were optimised with respect to chiral selector, buffer pH, and temperature around the capillary. Thus, rac-PhTX-343 was resolved using a separation buffer containing 30 mM heptakis-(2, 6-di-O-methyl)-beta-cyclodextrin in 50 mM 6-aminocarproic acid (pH 4. 0) at 15 degrees C. rac-PhTX-12 was not resolvable in this system, but could be resolved using a separation buffer containing 10% w/v of dextrin 10, a linear maltodextrin, in 50 mM 6-aminocaproic acid (pH 4.0) at 15 degrees C. Using these methods, the optical purity of the synthetic enantiomers was determined to be ee > 99%. The enantiomers were also characterised by chiroptical methods. The antagonist potency of the enantiomers was tested on nicotinic acetylcholine receptors (human muscle-type nAChR) expressed in TE671 cells, ionotropic glutamate receptors in Xenopus laevis oocytes (expressing recombinant GluR1flop receptors), and locust muscle ionotropic glutamate receptors sensitive to quisqualate (qGluR). The potencies of each pair of enantiomers were similar (eudismic ratio close to 1).

Analogues of neuroactive polyamine wasp toxins that lack inner basic sites exhibit enhanced antagonism toward a muscle-type mammalian nicotinic acetylcholine receptor.

Philanthotoxin-433 (PhTX-433), a natural polyamine wasp toxin, is a noncompetitive antagonist of certain ionotropic receptors. Six analogues of PhTX-343 (a synthetic analogue of the natural product), in which the secondary amino groups are systematically replaced by oxygen or methylene groups, have been synthesized by coupling of N-(1-oxobutyl)tyrosine with 1,12-dodecanediamine, 4,9-dioxa-1, 12-dodecanediamine, or appropriately protected di- and triamines, the latter being obtained by multistep syntheses. The resulting PhTX-343 analogues were purified and characterized, and their protolytic properties (stepwise macroscopic pK(a) values) were determined by (13)C NMR titrations. All analogues are fully protonated at physiological pH. The effects of these compounds on acetylcholine-induced currents in TE671 cells clamped at various holding potentials were determined. All of the analogues noncompetitively antagonized the nicotinic acetylcholine receptor (nAChR) in a concentration-, time-, and voltage-dependent manner. The amplitudes of acetylcholine-induced currents were compared at their peaks and at the end of a 1 s application in the presence or absence of the analogues. Most of the analogues were equipotent with or more potent than PhTX-343. The dideaza analogue PhTX-12 [IC(50) of 0.3 microM (final current value)] was the most potent, representing the highest potency improvement (about 50-fold) yet achieved by modification of the parent compound (PhTX-343). Thus, the presence of multiple positive charges in the PhTX-343 molecule is not necessary for antagonism of nAChR. In contrast, the compounds were much less potent than PhTX-343 at locust muscle ionotropic glutamate receptors sensitive to quisqualate (qGluR). The results demonstrate that the selectivity for different types of ionotropic receptors can be achieved by manipulating the polyamine moiety of PhTX-343.

Potentiation and inhibition of nicotinic acetylcholine receptors by spermine in the TE671 human muscle cell line.

Nicotinic acetylcholine receptors (nAChR) of the TE671 cell line were investigated using whole-cell and membrane patch recording techniques. At negative holding potentials (VH), pulses of acetylcholine (ACh) elicited whole-cell inward currents that rapidly desensitized. The EC50 value for ACh at VH = -60 mV was 7.8 microM. The ACh-induced current reversed at approximately 0 mV. Desensitization of nAChR by ACh was biphasic and reversible within approximately 20 sec. Spermine (1-100 microM) potentiated responses to ACh (10 microM - 1 mM) by reducing the rate of onset of desensitization; potentiation was inhibited by arcaine (10-100 microM). Spermine (1 mM) noncompetitively antagonized the AChinduced current. Antagonism by 1 to 5 mM spermine was voltage-dependent, increasing with negative VH. In 100 microM arcaine, this antagonism was shown to contain a voltage-independent component. Spermine (10 mM) increased the EC50 values for ACh, suggesting that at this concentration the polyamine is also a competitive antagonist. Single channel openings elicited during application of ACh to outside-out patches had a conductance of 47 pS at VH = -60 mV. At 10 and 100 microM, spermine increased channel open probability (po), but at 1 mM spermine, po was not significantly different from controls. The single channel conductance for ACh was unaffected by 10 and 100 microM spermine, but was decreased by 1 mM spermine. Spermine promoted the occurrence of approximately 27 pS openings. It is proposed that spermine acts at an excitatory modulatory site similar to that present on N-methyl-D-aspartate receptors and at least three inhibitory sites on nAChR of TE671 cells.

Behavioral function of glutamatergic interneurons in the feeding system of Lymnaea: plateauing properties and synaptic connections with motor neurons.

Intracellular recording techniques were used to examine the electrical properties and behavioral function of a novel type of retraction phase interneuron, the N2 ventral (N2v) cells in the feeding network of the snail Lymnaea. The N2vs were compared with the previously identified N2 cells that now are renamed the N2 dorsal (N2d) cells. The N2vs are a bilaterally symmetrical pair of electrotonically coupled plateauing interneurons that are located on the ventral surfaces of the buccal ganglia. Their main axons project to the opposite buccal ganglion, but they have an additional fine process in the postbuccal nerve. N2v plateaus that outlast the duration of the stimulus can be triggered by depolarizing current pulses and prematurely terminated by applied hyperpolarizing pulses. Gradually increasing the amplitude of depolarizing pulses reveals a clear threshold for plateau initiation. N2v plateauing persists in a high Mg2+/nominally zero Ca2+ saline that blocks chemical synaptic connections, suggesting an endogenous mechanism for plateau generation. The N2vs fire sustained bursts of action potentials throughout the N2/rasp phase of the fictive feeding cycle and control the retraction phase feeding motor neurons. The N2vs excite the B3 and B9 feeding motor neurons to fire during the rasp phase of the feeding cycle. They also inhibit the B7 and B8 feeding motor neurons. The B8 cells recover from inhibition and fire during the following swallowing phase. These synaptic connections appear to be monosynaptic as they persist in high Mg2+/high Ca2+ (HiDi) saline that blocks polysynaptic pathways. Strong current-induced plateaus in the N2vs generate brief inhibitory postsynaptic responses in the B4CL rasp phase motor neurons, but this was due to the indirect N2v --> N2d --> B4CL pathway. The N2vs are coupled electrotonically to the N2d cells, and triggering plateau in a N2v usually induced one or two spikes in a N2d. Previous experiments showed that the N2ds generate plateau potentials during a fictive feeding cycle. Here we show that the main component of the "plateauing" waveform is due to the electrotonic coupling with the N2v cells. The differential synaptic connections of the N2v and N2d cells with retraction phase motor neurons results in a sequence of motor neuron burst activity B9 --> B4CL --> B8 that produces the full retraction (rasp --> swallow) movements of the feeding apparatus (buccal mass). We conclude that the N2v cells are an essential component of the interneuronal network required to produce feeding motor neuron activity.

Glutamatergic N2v cells are central pattern generator interneurons of the lymnaea feeding system: new model for rhythm generation.

We aimed to show that the paired N2v (N2 ventral) plateauing cells of the buccal ganglia are important central pattern generator (CPG) interneurons of the Lymnaea feeding system. N2v plateauing is phase-locked to the rest of the CPG network in a slow oscillator (SO)-driven fictive feeding rhythm. The phase of the rhythm is reset by artificially evoked N2v bursts, a characteristic of CPG neurons. N2v cells have extensive input and output synaptic connections with the rest of the CPG network and the modulatory SO cell and cerebral giant cells (CGCs). Synaptic input from the protraction phase interneurons N1M (excitatory), N1L (inhibitory), and SO (inhibitory-excitatory) are likely to contribute to a ramp-shaped prepotential that triggers the N2v plateau. The prepotential has a highly complex waveform due to progressive changes in the amplitude of the component synaptic potentials. Most significant is the facilitation of the excitatory component of the SO --> N2v monosynaptic connection. None of the other CPG interneurons has the appropriate input synaptic connections to terminate the N2v plateaus. The modulatory function of acetylcholine (ACh), the transmitter of the SO and N1M/N1Ls, was examined. Focal application of ACh (50-ms pulses) onto the N2v cells reproduced the SO --> N2v biphasic synaptic response but also induced long-term plateauing (20-60 s). N2d cells show no endogenous ability to plateau, but this can be induced by focal applications of ACh. The N2v cells inhibit the N3 tonic (N3t) but not the N3 phasic (N3p) CPG interneurons. The N2v --> N3t inhibitory synaptic connection is important in timing N3t activity. The N3t cells recover from this inhibition and fire during the swallow phase of the feeding pattern. Feedback N2v inhibition to the SO, N1L protraction phase interneurons prevents them firing during the retraction phase of the feeding cycle. The N2v --> N1M synaptic connection was weak and only found in 50% of preparations. A weak N2v --> CGC inhibitory connection prevents the CGCs firing during the rasp (N2) phase of the feeding cycle. These data allowed a new model for the Lymnaea feeding CPG to be proposed. This emphasizes that each of the six types of CPG interneuron has a unique set of synaptic connections, all of which contribute to the generation of a full CPG pattern.

Glutamate is the transmitter for N2v retraction phase interneurons of the Lymnaea feeding system.

Electrophysiological and pharmacological methods were used to examine the role of glutamate in mediating the excitatory and inhibitory responses produced by the N2v rasp phase neurons on postsynaptic cells of the Lymnaea feeding network. The N2v --> B3 motor neuron excitatory synaptic response could be mimicked by focal or bath application of -glutamate at concentrations of >/=10(-3) M. Quisqualate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were potent agonists for the B3 excitatory glutamate receptor (10(-3) M), whereas kainate only produced very weak responses at the same concentration. This suggested that non-N-methyl--aspartate (NMDA), AMPA/quisqualate receptors were present on the B3 cell. The specific non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10(-5) M) blocked 85% of the excitatory effects on the B3 cell produced by focal application of glutamate (10(-3) M), confirming the presence of non-NMDA receptors. CNQX also blocked the major part of the excitatory postsynaptic potentials on the B3 cell produced by spontaneous or current-evoked bursts of spikes in the N2v cell. As with focal application of glutamate, a small delayed component remained that was CNQX insensitive. This provided direct evidence that glutamate acting via receptors of the non-NMDA, AMPA/quisqualate type were responsible for mediating the main N2v --> B3 cell excitatory response. NMDA at 10(-2) M also excited the B3 cell, but the effects were much more variable in size and absent in one-third of the 25 B3 cells tested. NMDA effects on B3 cells were not enhanced by bath application of glycine at 10(-4) M or reduction of Mg2+ concentration in the saline to zero, suggesting the absence of typical NMDA receptors. The variability of the B3 cell responses to NMDA suggested these receptors were unlikely to be the main receptor type involved with N2v --> B3 excitation. Quisqualate and AMPA at 10(-3) M also mimicked N2v inhibitory effects on the B7 and B8 feeding motor neurons and the modulatory slow oscillator (SO) interneuron, providing further evidence for the role of AMPA/quisqualate receptors. Similar effects were seen with glutamate at the same concentration. However, CNQX could not block either glutamate or N2v inhibitory postsynaptic responses on the B7, B8, or SO cells, suggesting a different glutamate receptor subtype for inhibitory responses compared with those responsible for N2v --> B3 excitation. We conclude that glutamate is a strong candidate transmitter for the N2v cells and that AMPA/quisquate receptors of different subtypes are likely to be responsible for the excitatory and inhibitory postsynaptic responses.

Functional expression of a recombinant unitary glutamate receptor from Xenopus, which contains N-methyl-D-aspartate (NMDA) and non-NMDA receptor subunits.

A cDNA encoding a 100-kDa subunit (XenNR1) of the N-methyl-D-aspartate (NMDA) glutamate receptor type has been cloned from Xenopus central nervous system. When XenNR1 is coexpressed in a mammalian cell line with a recently cloned 51-kDa non-NMDA receptor subunit (XenU1), also from Xenopus, it forms a functional unitary receptor exhibiting the pharmacological properties characteristic of both NMDA and non-NMDA receptors. Firstly, XenU1 can replace NR2 subunits, in complementing XenNR1 to introduce the ligand binding properties of a complete NMDA receptor. Second, responses to both NMDA and non-NMDA receptor agonists and antagonists were obtained in patch-clamp recordings from the cotransfected cells, but no significant responses were recorded when the cells were singly transfected. Third, from solubilized cell membranes from the cotransfected cells, an antibody to the NR1 subunit coprecipitated the binding sites of the non-NMDA receptor subunit. The unitary glutamate receptor has a unique set of properties that denote intersubunit interaction, including a glycine requirement for the responses to non-NMDA as well as to NMDA receptor agonists and voltage-dependent block by Mg2+ of the non-NMDA agonist responses.

Central pattern generator interneurons are targets for the modulatory serotonergic cerebral giant cells in the feeding system of Lymnaea.

1. The objective of the experiments was to explore the modulatory functions of the serotonergic cerebral giant cells (CGCs) of the Lymnaea feeding system by examining their synaptic connections with the central pattern generator (CPG) interneurons and the modulatory slow oscillator (SO) interneuron. 2. One type of modulatory function, "gating," requires that the CGCs fire tonically at a minimum of 7 spikes/min. Above this minimum level the CGCs control the frequency of CPG interneuron oscillation-- "frequency control," a second type of modulation. In an SO-driven fictive feeding rhythm, an increase in the frequency of the rhythm, with increased CGC firing rate, resulted from a reduction in the duration of the N1 (protraction) and N2 (rasp) phases of the feeding cycle with little effect on the N3 (swallow) phase. 3. The CGCs excited the N1 phase interneurons SO and N1M (N1 medial) cells but had no consistent effects on the N1 lateral cells. The CGC-->SO postsynaptic response was probably monosynaptic (< or = 200 ms in duration) with unitary 1:1 excitatory postsynaptic potentials (EPSPs) following each CGC spike. The CGC-->N1M excitatory response was slow and nonunitary, and a burst of CGC spikes evoked a depolarization of the N1M cells that lasted up to 10 s and triggered N1M cell bursts. Both CGC-->SO and CGC-->N1M excitatory responses could be mimicked by the focal application of serotonin (5-HT). 4. Both CGC-->SO and CGC-->N1M excitatory connections systematically increased the N1M cell firing rate within the CGCs' physiological firing range (0-40 spikes/min). This was due to both the direct (CGC-->N1M) and indirect (CGC-->SO-->N1M) excitatory synaptic pathways. The CGC-induced increase in N1M cell firing rate probably accounted for the reduced duration of the N1M cell feeding burst by causing a more rapid reversal of the feeding cycle from the N1 phase to the N2 phase. This phase reversal was due to the previously described recurrent inhibitory pathway (N1-->N2 excitation followed by N2-->N1 inhibition). 5. The CGCs' ability to provide a depolarizing drive to the N1M cells meant that this excitatory connection was also likely to be important for gating. 6. Activity in the CGCs produced nonunitary, long-lasting, excitatory postsynaptic responses on the N2 ventral (N2v) CPG interneurons, and these were likely to be involved in both the gating and the frequency control by the CGCs on the N2 phase of the feeding rhythm. Suppressing CGC tonic firing initially increased the duration of the N2v plateau (which determines the duration of the N2 phase of the feeding cycle, frequency function) but eventually led to a loss of N2v plateauing (gating function). 7. Nonunitary, weakly inhibitory CGC-->N2 dorsal responses were recorded that could be mimicked by the application of 5-HT. 8. Spikes in the CGCs evoked 1:1 monosynaptic EPSPs in the N3 tonic (N3t) CPG interneurons. This excitatory effect could be mimicked by the application of 5-HT. Within the physiological range of CGC firing, this excitation did not appear to influence the firing rate of the N3t cells. 9. N3 phasic (N3p) CPG interneurons showed biphasic (hyperpolarizing followed by depolarizing) unitary responses to spikes evoked in the CGCs. The inhibitory synaptic response was maintained in a high-Ca2+/high-Mg2+ (Hi-Di) saline and was mimicked by the focal application of 5-HT, indicating that it was probably monosynaptic. The excitatory component was, however, reduced in a Hi-Di saline, indicating that it was probably polysynaptic. Suppressing the CGCs during an SO-driven feeding rhythm caused the N3p cells to fire less, suggesting that the removal of the excitatory component of the response might be significant. 10. We conclude that the general depolarizing effects of the CGCs on a number of the CP

A molecularly defined cardiorespiratory interneuron expressing SDPFLRFamide/GDPFLRFamide in the snail Lymnaea: monosynaptic connections and pharmacology.

1. Neuron-specific expression of alternately spliced exons of the gene encoding the Phe-Met-Arg-Phe-NH2 (FMRFamide) family of neuropeptides and the role of encoded peptides in synaptic transmission were examined in an identified cardiorespiratory interneuron, the visceral white interneuron (VWI), in the snail Lymnaea. 2. In situ hybridization using exon-specific probes showed VWI cytoplasmic expression of the exon encoding the Lymnaea heptapeptides Gly-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (GDPFLRF amide) Ser-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (SDPFLRF amide) but not the exon encoding the tetrapeptides FMRFamide and Phe-Leu-Arg-Phe-NH2 (FLRFamide). 3. The absence of the tetrapeptides (FMRFamide and FLRFamide) in the VWI was indirectly confirmed by the lack of immunoreactivity to a specific antibody raised against the sequence Leu-Tyr. This sequence is present in the Lymnaea tetrapeptide precursor, but not the heptapeptide precursor. 4. The VWI has monosynaptic connections with many identifiable neurons in the CNS. These were excitatory on three clusters of identified neurons [B group (Bgp), E group (Egp), and F group (Fgp)], inhibitory on another cluster [A group (Agp)] or biphasic (excitation followed by inhibition) on a single giant neuron [right pedal dorsal 1 (RPeD1)]. 5. The role of GDPFLRFamide/SDPFLRFamide as putative neurotransmitters was examined by comparing neuronally evoked postsynaptic responses with the effects of focal peptide application. 6. The heptapeptides closely mimicked the inhibitory responses (threshold pressure pipette concentration 10(-9) M) on the Agp cells and RPeD1, including an increase in membrane conductance. FMRFamide was 1 order of magnitude less potent. GDPFLRFamide/SDPFLRFamide, applied either alone or in "cocktails" (combinations of GDPFLRFamide, SDPFLRFamide, FMRFamide, and FLRFamide), did not reproduce the excitatory effect of the VWI on the Bgp, Egp, and Fgp cells. These peptides, applied either together or separately, inhibited the cells. 7. FMRFamide or FLRFamide, but not GDPFLRFamide or SDPFLRFamide, could reproduce the initial depolarizing component of the biphasic response on RPeD1. This only occurred at concentrations of > or = 10(-4) M (10(-3) M was necessary to get spikes on RPeD1) and may not be physiologically significant. 8. We conclude that at least one so far unidentified co-transmitter must be present in the VWI to account for its full range of synaptic responses.