Bath Application Of Serotonin Research Articles

Enhancement of synaptic responses in ascending interneurones following acquisition of social dominance in crayfish.

When crayfish have attained dominant status after agonistic bouts, their avoidance reaction to mechanical stimulation of the tailfan changes from a dart to a turn response. Ascending interneurones originating in the terminal ganglion receive sensory inputs from the tailfan and they affect spike activity of both uropod and abdominal postural motor neurones, which coordinates the uropod and abdominal postural movements. Despite the varying output effects of ascending interneurones, the synaptic responses of all interneurones to sensory stimulation were enhanced when they acquired a dominant state. The number of spikes increased as did a sustained membrane depolarizations. Regardless of social status, the output effects on the uropod motor neurones of all interneurones except VE-1 remained unchanged. VE-1 mainly inhibited the uropod opener motor neurones in naive animals, but tended to excite them in dominant animals. Synaptic enhancement of the sensory response of ascending interneurones was also observed in naive animals treated with bath-applied serotonin. However, subordinate animals or naive animals treated with octopamine had no noticeable effect on the synaptic response of their ascending interneurones to sensory stimulation. Thus, enhancement of the synaptic response is a specific neural event that occurs when crayfish attain social dominance and it is mediated by serotonin.

Serotonergic cerebral cells control activity of cilia in the foregut of the pteropod mollusk Clione limacina

Bilaterally symmetrical pair of serotonergic cells, named C1 in Clione, has been described in the cerebral ganglia of all gastropod species. Here we describe a new role of C1 cells in gastropod mollusks: control of activity of ciliated epithelium in the foregut. Detailed morphological investigation of C1 neurons in the pteropod mollusk Clione limacina revealed that these cells among other destinations send their neurites into foregut where they produce intense arborization with large varicosities along the processes. Intracellular stimulation of a single C1 induced pronounced activation (often followed by inhibition) of cilia lining the foregut. This activation was substantially reduced by serotonin antagonist mianserin. Bath application of serotonin also induced transient increase in ciliary transport rate, followed by inhibition of ciliary activity up to its full cessation in some areas of isolated foregut. These data suggest that C1 in Clione may use serotonin to influence cilia in the foregut. Taking into account high homology of serotonergic cerebral cells across studied species we can speculate that these cells may be involved in the neural control of cilia in the foregut in other gastropod mollusks.

Specificity of Intramuscular Activation During Rhythms Produced by Spinal Patterning Systems in the In Vitro Neonatal Rat With Hindlimb Attached Preparation

In intact adult vertebrates, muscles can be activated with a high degree of specificity, so that even within a single traditionally defined muscle, groups of motor units can be differentially activated. Such differential activation might reflect detailed control by descending systems, potentially resulting from postnatal experience such that activation of motor units is precisely tailored to their mechanical actions. Here we examine the degree to which such specific activation can be seen in the rhythmic patterns produced by isolated spinal motor systems in neonates. We examined motor output produced by the in vitro neonatal rat spinal cord with hindlimb attached. We recorded the activity of different regions within the posterior portion of biceps femoris (BFp; i.e., excluding the anterior/vertebral head). We found that in the rhythms evoked by bath application of serotonin/N-methyl-d-aspartate (5-HT/NMDA), all regions of BFp were active during extension. However, the regions of BFp were activated in a specific sequence, with the activation of rostral regions consistently preceding those of more caudal regions in both afferented and deafferented preparations. In the rhythms evoked by cauda equina (CE) stimulation, rostral and middle regions of BFp remained active in extension, but the caudal region of BFp was usually active in flexion. Stimulation of L5 and S2 dorsal roots typically evoked rhythms with all regions of BFp active during extension; although the same rostral to caudal sequence of activation observed in 5-HT/NMDA evoked rhythms could also be observed in these rhythms, we also observed cases with reversed sequences, with activity proceeding from caudal to rostral. S2 dorsal root stimulation occasionally evoked rhythms with flexor activity in caudal BFp, similar to CE-evoked rhythms. Taken together, these results suggest a high degree of individuated control of muscles by spinal pattern generating networks, even at birth.

Flexibility of Motor Pattern Generation Across Stimulation Conditions by the Neonatal Rat Spinal Cord

Previous studies have demonstrated that "locomotor-like" rhythmic patterns can be evoked in the isolated neonatal rat spinal cord by several means, including pharmacological neuromodulation and electrical stimulation of various pathways. Recent studies have used stimulation of afferent pathways to evoke rhythmic patterns, relying on synaptic activation of interneuronal systems rather than global imposition of neuromodulatory state by pharmacological agents. We use the in vitro neonatal rat spinal cord with attached hindlimb to examine the muscle activation patterns evoked by stimulation of these different pathways and evaluate whether stimulation of these pathways all evoke the same patterns. We find that the patterns evoked by bath application of serotonin (5-HT) and N-methyl-D-aspartic acid (NMDA) consisted of alternation between hip flexors and extensors and similar alternation was observed in the patterns evoked by electrical stimulation of the cauda equina (CE) or contralateral fifth lumbar (L(5)) dorsal nerve root. In contrast, the knee extensor/hip flexor rectus femoris (RF) and knee flexor/hip extensor semitendinosus (ST) were activated differentially across stimulation conditions. In 5-HT/NMDA patterns, RF was active in late flexion and ST in late extension. In CE patterns, these two muscles switched places with RF typically active in late extension and ST active in flexion. In L(5) patterns, ST was activated in extension and RF was silent or weakly active during flexion. There were also systematic differences in the consistency of rhythms evoked by each stimulation method: patterns evoked by electrical stimulation of CE or L(5) were less consistently modulated with the rhythm when compared with 5-HT/NMDA-evoked patterns. All differences were preserved following deafferentation, demonstrating that they reflect intrinsic properties of spinal systems. These results highlight the intrinsic flexibility of motor pattern generation by spinal motor circuitry which is present from birth and provides important information to many studies examining spinal pattern generating networks.

Serotonin excites fast-spiking interneurons in the striatum.

Fast-spiking interneurons (FSIs) control the output of the striatum by mediating feed-forward GABAergic inhibition of projection neurons. Their neuromodulation can therefore critically affect the operation of the basal ganglia. We studied the effects of 5-hydroxytryptamine (5-HT, serotonin), a neurotransmitter released in the striatum by fibres originating in the raphe nuclei, on FSIs recorded with whole-cell techniques in rat brain slices. Bath application of serotonin (30 μm) elicited slow, reversible depolarizations (9 ± 3 mV) in 37/46 FSIs. Similar effects were observed using conventional whole-cell and gramicidin perforated-patch techniques. The serotonin effects persisted in the presence of tetrodotoxin and were mediated by 5-HT2C receptors, as they were reversed by the 5-HT2 receptor antagonist ketanserin and by the selective 5-HT2C receptor antagonist RS 102221. Serotonin-induced depolarizations were not accompanied by a significant change in FSI input resistance. Serotonin caused the appearance of spontaneous firing in a minority (5/35) of responsive FSIs, whereas it strongly increased FSI excitability in each of the remaining responsive FSIs, significantly decreasing the latency of the first spike evoked by a current step and increasing spike frequency. Voltage-clamp experiments revealed that serotonin suppressed a current that reversed around −100 mV and displayed a marked inward rectification, a finding that explains the lack of effects of serotonin on input resistance. Consistently, the effects of serotonin were completely occluded by low concentrations of extracellular barium, which selectively blocks Kir2 channels. We concluded that the excitatory effects of serotonin on FSIs were mediated by 5-HT2C receptors and involved suppression of an inwardly rectifying K+ current.

Transient Enhancement of Spike-Evoked Calcium Signaling by a Serotonergic Interneuron

Enhancement of presynaptic Ca(2+) signals is widely recognized as a potential mechanism for heterosynaptic potentiation of neurotransmitter release. Here we show that stimulation of a serotonergic interneuron increased spike-evoked Ca(2+) in a manner consistent with its neuromodulatory effect on synaptic transmission. In the gastropod mollusk, Tritonia diomedea, stimulation of a serotonergic dorsal swim interneuron (DSI) at physiological rates heterosynaptically enhances the strength of output synapses made by another swim interneuron, C2, onto neurons in the pedal ganglion. Using intracellular electrophysiological recording combined with real-time confocal imaging of C2 (loaded with Oregon Green Bapta 1), it was determined that DSI stimulation increases the amplitude of spike-evoked Ca(2+) signals in C2 without altering basal Ca(2+) signals. This neuromodulatory action was restricted to distal neurites of C2 where synapses with pedal neurons are located. The effect of DSI stimulation on C2 spike-evoked Ca(2+) signals resembled DSI heterosynaptic enhancement of C2 synapses in several measures: both decayed within 15 s, both were abolished by the serotonin receptor antagonist, methysergide, and both were independent of DSI's depolarizing actions on C2. A brief puff of serotonin could mimic the enhancement of spike-evoked Ca(2+) signals in the distal neurites of C2, but larger puffs or bath-applied serotonin elicited nonphysiological effects. These results suggest that DSI heterosynaptic enhancement of C2 synaptic strength may be mediated by a local enhancement of spike-evoked Ca(2+) signals in the distal neurites of C2.

Spatial-specific action of serotonin within the leech midbody ganglion

Serotonin is a conspicuous neuromodulator in the nervous system of many vertebrates and invertebrates. In previous experiments performed in the leech nervous system, we compared the effect of the amine released from endogenous sources [using selective serotonin reuptake inhibitors (SSRIs), e.g. fluoxetine] with that of bath-applied serotonin. The results suggested that the amine does not reach all its targets in a uniform way, but produces the activation of an interneuronal pathway that generated specific synaptic responses on different neurons. Taking into account that the release of the amine is often regulated at the presynaptic level, we have investigated whether autoreceptor antagonists mimic the SSRIs effect. We found that methiothepin (100 microM) produced similar effects than fluoxetine. To further test the hypothesis that endogenous serotonin produce its effect by acting locally at specific sites, we analyzed the effect of iontophoretic applications of serotonin. We found a site in the neuropil of the leech ganglia where serotonin application mimicked the effect of the SSRIs and the 5-HT antagonist. The results further support the view that the effect of serotonin exhibits a spatial specificity that can be relevant to understand its modulatory actions.

Endogenous Serotonin Excites Striatal Cholinergic Interneurons via the Activation of 5-HT 2C, 5-HT6, and 5-HT7 Serotonin Receptors: Implications for Extrapyramidal Side Effects of Serotonin Reuptake Inhibitors

The striatum is richly innervated by serotonergic afferents from the raphe nucleus. We explored the effects of this input on striatal cholinergic interneurons from rat brain slices, by means of both conventional intracellular and whole-cell patch-clamp recordings. Bath-applied serotonin (5-HT, 3-300 microM), induced a dose-dependent membrane depolarization and increased the rate of spiking. This effect was mimicked by the 5-HT reuptake blockers citalopram and fluvoxamine. In voltage-clamped neurons, 5-HT induced an inward current, whose reversal potential was close to the K(+) equilibrium potential. Accordingly, the involvement of K(+) channels was confirmed either by increasing extracellular K(+) concentration and by blockade of K(+) channels with barium. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) profiling demonstrated the presence of 5-HT2C, 5-HT6, and 5-HT7 receptor mRNAs in identified cholinergic interneurons. The depolarization/inward current induced by 5-HT was partially mimicked by the 5-HT2 receptor agonist 2,5-dimethoxy-4-iodoamphetamine and antagonized by both ketanserin and the selective 5-HT2C antagonist RS102221, whereas the selective 5-HT3 and 5-HT4 receptor antagonists tropisetron and RS23597-190 had no effect. The depolarizing response to 5-HT was also reduced by the selective 5-HT6 and 5-HT7 receptor antagonists SB258585 and SB269970, respectively, and mimicked by the 5-HT7 agonist, 5-CT. Accordingly, activation of either 5-HT6 or 5-HT7 receptor induced an inward current. The 5-HT response was attenuated by U73122, blocker of phospholipase C, and by SQ22,536, an inhibitor of adenylyl cyclase. These results suggest that 5-HT released by serotonergic fibers originating in the raphe nuclei has a potent excitatory effect on striatal cholinergic interneurons.

Serotonergic Enhancement of a 4-AP-Sensitive Current Mediates the Synaptic Depression Phase of Spike Timing-Dependent Neuromodulation

The mechanism underlying spike timing-dependent neuromodulation (STDN) was investigated in the opisthobranch mollusc Tritonia diomedea. The serotonergic dorsal swim interneurons (DSIs) dynamically modulated the synaptic output of ventral swim interneuron B (VSI); immediately after DSI stimulation, there was a potentiation of VSI synaptic strength followed by a longer-lasting synaptic depression. The potentiation phase of STDN was unaffected by spike broadening produced by the potassium channel blocker 4-aminopyridine (4-AP). In contrast, the depression phase was eliminated by 4-AP. Bath-applied serotonin (5-HT) decreased VSI spike duration and increased the magnitude of the A-current (IA), a voltage-dependent, transient, outward current. 4-AP preferentially blocked IA and prevented the spike narrowing caused by 5-HT, uncovering the full extent of 5-HT-induced synaptic potentiation. A consistent correlation was observed between IA and spike duration, but the correlation between synaptic strength and spike duration differed between preparations. Conductance-based simulations showed that the magnitude of A-current conductance could affect spike duration and gave an estimation of the change needed to produce spike narrowing. An artificial IA introduced into the VSI in the presence of 4-AP by means of the dynamic-clamp technique restored spike duration and gave a further approximation of the magnitude of modulation needed for spike narrowing. Together, these results suggest a mechanism for STDN: the DSIs release 5-HT, which causes a spike duration-independent enhancement of synaptic strength and a longer-lasting enhancement of IA that narrows the VSI spike and hence decreases VSI synaptic strength. Thus, STDN arises from the dynamics of independent intracellular signaling events.

Excitatory effects of serotonin on rat striatal cholinergic interneurones

We investigated the effects of 5-hydroxytryptamine (5-HT, serotonin) in striatal cholinergic interneurones with gramicidin-perforated whole-cell patch recordings. Bath-application of serotonin (30 microm) significantly and reversibly increased the spontaneous firing rate of 37/45 cholinergic interneurones tested. On average, in the presence of serotonin, firing rate was 273 +/- 193% of control. Selective agonists of 5-HT(1A), 5-HT3, 5-HT4 and 5-HT7 receptors did not affect cholinergic interneurone firing, while the 5-HT2 receptor agonist alpha-methyl-5-HT (30 microm) mimicked the excitatory effects of serotonin. Consistently, the 5-HT2 receptor antagonist ketanserin (10 microm) fully blocked the excitatory effects of serotonin. Two prominent after-hyperpolarizations (AHPs), one of medium duration that was apamin-sensitive and followed individual spikes, and one that was slower and followed trains of spikes, were both strongly and reversibly reduced by serotonin; these effects were fully blocked by ketanserin. Conversely, the depolarizing sags observed during negative current injections and mediated by hyperpolarization-activated cationic currents were not affected. In the presence of apamin and tetrodotoxin, the slow AHP was strongly reduced by 5-HT, and fully abolished by the calcium channel blocker nickel. These results show that 5-HT exerts a powerful excitatory control on cholinergic interneurones via 5-HT2 receptors, by suppressing the AHPs associated with two distinct calcium-activated potassium currents.

Activation of presynaptic 5-HT3 receptors facilitates glutamatergic synaptic inputs to area postrema neurons in rat brain slices

Whole-cell voltage-clamp recordings were performed to investigate the serotonergic modulation of neurotransmitter release onto rat area postrema neurons in vitro. The bath application of serotonin (5-HT; 50 microM) or phenylbiguanide (PBA; 50 microM), a potent 5-HT3 receptor agonist, increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) or miniature EPSCs (mEPSCs) in 35 of 83 neurons (42%). These increases occurred in all electrophysiological cell classes. No cells exhibited a decrease in EPSC frequency. The majority of responding cells showed no inward currents during the application of serotonergic agonists (n = 34/35). However, the amplitude of mEPSCs was increased in 11/11 cells with 5-HT or 3/11 cells with PBA. ICS-205,930, a potent 5-HT3 receptor antagonist, markedly suppressed the 5-HT-induced facilitation of sEPSCs (n = 5) or mEPSCs (n = 5). An increase in the frequency of mEPSCs after PBA exposure was found, even with media containing Cd2+ (50 microM) or zero Ca2+. mEPSCs and evoked EPSCs were completely blocked in media containing the non-NMDA ionotropic receptor antagonist, CNQX (10 microM), indicating that EPSCs were glutamate events. These results suggest that glutamate release is increased in the area postrema by presynaptic 5-HT3 receptor activation. Furthermore, we present evidence that 5-HT3 receptor activation may be able to directly release glutamate from terminals, bypassing a requirement for voltage-dependent calcium entry into terminals. Such a mechanism may contribute to the chemosensitive function of area postrema neurons.

Spike Timing-Dependent Serotonergic Neuromodulation of Synaptic Strength Intrinsic to a Central Pattern Generator Circuit

Neuromodulation is often thought to have a static, gain-setting function in neural circuits. Here we report a counter example: the neuromodulatory effect of a serotonergic neuron is dependent on the interval between its spikes and those of the neuron being modulated. The serotonergic dorsal swim interneurons (DSIs) are members of the escape swim central pattern generator (CPG) in the mollusk Tritonia diomedea. DSI spike trains heterosynaptically enhanced synaptic potentials evoked by another CPG neuron, ventral swim interneuron B (VSI-B), when VSI-B action potentials occurred within 10 sec of a DSI spike train; however, if VSI-B was stimulated 20-120 sec after DSI, then the amplitude of VSI-B synaptic potentials decreased. Consistent with this, VSI-B-evoked synaptic currents exhibited a temporally biphasic and bidirectional change in amplitude after DSI stimulation. Both the DSI-evoked enhancement and decrement were occluded by serotonin and blocked by the serotonin receptor antagonist methysergide, suggesting that both phases are mediated by serotonin. In most preparations, however, bath-applied serotonin caused only a sustained enhancement of VSI-B synaptic strength. The heterosynaptic modulation interacted with short-term homosynaptic plasticity: DSI-evoked depression was offset by VSI-B homosynaptic facilitation. This caused a complicated temporal pattern of neuromodulation when DSI and VSI-B were stimulated to fire in alternating bursts to mimic the natural motor pattern: DSI strongly enhanced summated VSI-B synaptic potentials and suppressed single synaptic potentials after the cessation of the artificial motor pattern. Thus, spike timing-dependent serotonergic neuromodulatory actions can impart temporal information that may be relevant to the operation of the CPG.

Visualization of modulatory effects of serotonin in the silkmoth antennal lobe.

A unique serotonin-immunoreactive neuron innervates every glomerulus of the contralateral antennal lobe (AL), the primary olfactory center, of the male silkmoth Bombyx mori. In order to examine the possible modulatory effects of serotonin in the AL, we utilized high-speed optical imaging with a voltage-sensitive dye combined with bath application of serotonin. We found that serotonin at 10(-4)mol l(-1) caused significant and reversible increases in the optical responses in both the macroglomerular complex (MGC) and the ordinary glomeruli (Gs) evoked by electrical stimulation of the antennal nerve. Optical responses in both the MGC and Gs were also significantly longer lasting following serotonin application. Serotonin exerted a significantly greater enhancing effect in the toroid glomerulus of the MGC than in the cumulus, and the effects of serotonin were also non-homogeneously distributed in the Gs. Our results are evidence that serotonin acts in both the MGC and Gs to modulate the responses of neuronal populations.

Serotonergic modulation of respiratory motoneurons and interneurons in brainstem slices of perinatal rats

Respiration-related membrane potential fluctuations were recorded in hypoglossal (XII) motoneurons and pre-Bötzinger complex (pre-BötC) interneurons in medullary slices from perinatal rats. Bath application of serotonin (5-HT) evoked a ketanserine-sensitive depolarization (∼11 mV) and tonic spike discharge in XII motoneurons, whereas pre-BötC neurons responded with a <6 mV depolarization and no tonic discharge. The membrane effects were accompanied by an increase in respiratory frequency by up to 260% in 64% of preparations. A frequency decrease leading to block of respiratory activity could also occur (20%) as well as an initial acceleration that turned into a frequency depression (16%). In contrast, iontophoresis of 5-HT into the pre-BötC exclusively increased respiratory frequency by 30–220%, whereas iontophoresis into the XII nucleus did not change respiratory frequency but induced tonic nerve discharge. The effects of local iontophoretic administration of 5-HT on membrane properties of XII and pre-BötC cells were very similar to those upon bath application. Bath application and iontophoresis of the 5-HT 2 receptor agonist α-methyl-hydroxytryptamine mimicked the effects of 5-HT. Bath application of the 5-HT 1A receptor agonist 8-hydroxydipropylaminotetralin hydrobromide did not affect XII nerve bursting or pre-BötC neurons. Iontophoresis of 8-hydroxydipropylaminotetralin hydrobromide had almost no effect on respiratory frequency and induced in the interneurons either a depolarization or hyperpolarization (<5 mV) which was blocked by the 5-HT 1A receptor antagonist N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl) N-2-pyridinylcyclohexane carboxamide. In conclusion, 5-HT-evoked tonic excitation of respiratory XII motoneurons is mediated by postsynaptic 5-HT 2 receptors. The excitatory effects on respiratory rhythm are also primarily attributable to postsynaptic 5-HT 2 receptors of pre-BötC neurons. Additional modulatory effects on the interneurons appear to be mediated by postsynaptic 5-HT 1A receptors.

Serotonergic modulation of nonspiking local interneurones in the terminal abdominal ganglion of the crayfish.

The modulatory effect of serotonin on local circuit neurones forming the uropod motor control system of the crayfish Procambarus clarkii Girard was analysed electrophysiologically. Bath application of 10 micro mol l(-1) serotonin caused a decrease in the tonic spike activity of the exopodite reductor motor neurone. The inhibitory effect of serotonin on the motor neurone was dose-dependent and its spike discharge was completely suppressed for long periods by 1 mmol l(-1) serotonin perfusion. Nonspiking local interneurones in the terminal abdominal ganglion showed either a membrane depolarization (N=6) or hyperpolarization (N=9) of 10-30 mV in amplitude when 100 micro mol l(-1) serotonin was perfused for 3-5 min. By contrast, spiking local interneurones and intersegmental ascending interneurones showed no observable excitatory responses to the perfusion of serotonin but instead some showed a small membrane hyperpolarization of 2-5 mV. These results indicate that the nonspiking interneurones could contribute substantially to the level of tonic excitation of the uropod motor neurones. Sensory stimulation elicited depolarizing or hyperpolarizing potentials in the nonspiking interneurones and excitatory postsynaptic potentials (EPSPs) and spikes in the spiking interneurones. The sensory responses of spiking interneurones increased during bath application of serotonin and were reduced after 20-30 min of washing with normal saline. In the nonspiking interneurones, the amplitude of both depolarizing and hyperpolarizing potentials increased without any direct correlation with the serotonin-mediated potential change. This effect of serotonin was long-lasting and continued to enhance the responses of the nonspiking interneurones after washing. This postserotonin enhancement persisted for over 1 h.

Serotonin elicits long-lasting enhancement of rhythmic respiratory activity in turtle brain stems in vitro.

Brain stem preparations from adult turtles were used to determine how bath-applied serotonin (5-HT) alters respiration-related hypoglossal activity in a mature vertebrate. 5-HT (5-20 microM) reversibly decreased integrated burst amplitude by approximately 45% (P < 0.05); burst frequency decreased in a dose-dependent manner with 20 microM abolishing bursts in 9 of 13 preparations (P < 0.05). These 5-HT-dependent effects were mimicked by application of a 5-HT(1A) agonist, but not a 5-HT(1B) agonist, and were abolished by the broad-spectrum 5-HT antagonist, methiothepin. During 5-HT (20 microM) washout, frequency rebounded to levels above the original baseline for 40 min (P < 0.05) and remained above baseline for 2 h. A 5-HT(3) antagonist (tropesitron) blocked the post-5-HT rebound and persistent frequency increase. A 5-HT(3) agonist (phenylbiguanide) increased frequency during and after bath application (P < 0.05). When phenylbiguanide was applied to the brain stem of brain stem/spinal cord preparations, there was a persistent frequency increase (P < 0.05), but neither spinal-expiratory nor -inspiratory burst amplitude were altered. The 5-HT(3) receptor-dependent persistent frequency increase represents a unique model of plasticity in vertebrate rhythm generation.

Serotonin 5-HT 2 receptors activate local GABA inhibitory inputs to serotonergic neurons of the dorsal raphe nucleus

The purpose of the present study was to characterize the synaptic currents induced by bath-applied serotonin (5-HT) in 5-HT cells of the dorsal raphe nucleus (DRN) and to determine which 5-HT receptor subtypes mediate these effects. In rat brain slices, 5-HT induced a concentration-dependent increase in the frequency of inhibitory postsynaptic currents (IPSCs) in 5-HT neurons recorded intracellularly in the ventral part of the DRN (EC 50: 86 μM); 5-HT also increased IPSC amplitude. These effects were blocked by the GABA A receptor antagonist, bicuculline (10 μM) and by the fast sodium channel blocker, TTX, suggesting that 5-HT had increased impulse flow in local GABAergic neurons. DAMGO (300 nM), a selective μ-agonist, markedly suppressed the increase in IPSC frequency induced by 5-HT (100 μM) in the DRN. A near maximal concentration of the selective 5-HT 2A antagonist, MDL100,907 (30 nM), produced a large reduction (∼70%) in the increase in IPSC frequency induced by 100 μM 5-HT; SB242,084 (30 nM), a selective 5-HT 2C antagonist, was less effective (∼24% reduction). Combined drug application suppressed the increase in 5-HT-induced IPSC frequency almost completely, suggesting involvement of both 5-HT 2A and 5-HT 2C receptors. Unexpectedly, the phenethylamine hallucinogen, DOI, a partial agonist at 5-HT 2A/2C receptors, caused a greater increase (+334%) in IPSC frequency than did 5-HT 100 μM (+80%). This result may be explained by an opposing 5-HT 1A inhibitory effect since the selective 5-HT 1A antagonist, WAY-100635, enhanced the 5-HT-induced increase in IPSCs. These results indicate that within the DRN–PAG area there may be a negative feedback loop in which 5-HT induces an increase in IPSC frequency in 5-HT cells by exciting GABAergic interneurons in the DRN via 5-HT 2A and, to a lesser extent, 5-HT 2C receptors. Increased GABA tone may explain the previous observation of an indirect suppression of firing of a subpopulation of 5-HT cells in the DRN induced by phenethylamine hallucinogens in vivo.

Functional partitioning of energy reserves by larvae of the marine bryozoan Bugula neritina (L.).

The effects of extended swimming on short-lived lecithotrophic larvae of the marine bryozoan Bugula neritina (L.) were examined. Larvae were forced to swim for 2 or 24 h by bath application of serotonin. Settlement and metamorphosis success were significantly reduced, larval dimensions were unaffected and ancestrulae were smaller after 24 h of swimming. Larvae settled predominantly on seawater-conditioned glass after 2 h, but became less discriminative after 24 h. Lipid content in intact larvae and dissociated surface ciliated and interior cell fractions was analysed by thin-layer chromatography. Hydrophilic lipids were unaffected by swimming regime. The hydrophobic fraction contained triglyceride, confirmed by proton nuclear magnetic resonance spectroscopy (1H-NMR) analysis and correlation spectroscopy (1H-1H COSY) patterns, which was significantly depleted after 24 h, and diacylglycerol, which was not. NMR spectra suggested no differences in fatty acid chain compositions between larvae swimming for 2 and 24 h. Triglyceride depletion was limited to the ciliated cell fraction. We propose that the functional partitioning of lipid reserves has evolved in association with the costs and benefits linked with larval dispersal.

GABA-receptor-independent dorsal root afferents depolarization in the neonatal rat spinal cord.

Dorsal root afferent depolarization and antidromic firing were studied in isolated spinal cords of neonatal rats. Spontaneous firing accompanied by occasional bursts could be recorded from most dorsal roots in the majority of the cords. The afferent bursts were enhanced after elevation of the extracellular potassium concentration ([K+]e) by 1-2 mM. More substantial afferent bursts were produced when the cords were isolated with intact brain stems. Rhythmic afferent bursts could be recorded from dorsal roots in some of the cords during motor rhythm induced by bath-applied serotonin and N-methyl--aspartate (NMDA). Bilaterally synchronous afferent bursts were produced in pairs of dorsal roots after replacing the NaCl in the perfusate with sodium-2-hydroxyethansulfonate or after application of the gamma-aminobutyric acid-A (GABAA) receptor antagonist bicuculline with or without serotonin (5-HT) and NMDA. Antidromic afferent bursts also could be elicited under these conditions by stimulation of adjacent dorsal roots, ventrolateral funiculus axons, or ventral white commissural (VWC) fibers. The antidromic bursts were superimposed on prolonged dorsal root potentials (DRPs) and accompanied by a prolonged increase in intraspinal afferent excitability. Surgical manipulations of the cord revealed that afferent firing in the presence of bicuculline persisted in the hemicords after hemisection and still was observed after removal of their ventral horns. Cutting the VWC throughout its length did not perturb the bilateral synchronicity of the discharge. These findings suggest that the activity of dorsal horn neurons is sufficient to produce the discharge and that the bilateral synchronicity can be maintained by cross connectivity that is relayed from side to side dorsal to the VWC. Antagonists of GABAB, 5-HT2/5-HT1C, or glutamate metabotropic group II and III receptors could not abolish afferent depolarization in the presence of bicuculline. Depolarization comparable in amplitude to DRPs, could be produced in tetrodotoxin-treated cords by elevation of [K+]e to the levels reported to develop in the neonatal rat spinal cord in response to dorsal root stimulation. A mechanism involving potassium transients produced by neuronal activity therefore is suggested to be the major cause of the GABA-independent afferent depolarization reported in our study. Possible implications of potassium transients in the developing and the adult mammalian spinal cord are discussed.

Whole cell recordings of lumbar motoneurons during locomotor-like activity in the in vitro neonatal rat spinal cord.

Whole cell current- and voltage-clamp recordings were obtained from lumbar motoneurons in the isolated neonatal rat spinal cord to characterize the behavior of motoneurons during neurochemically induced locomotor-like activity. Bath application of serotonin (10-100 muM) in combination with N-methyl-D-aspartate (1-12 muM) initially produced tonic membrane depolarization (mean = 26 mV), increased input resistance, decreased rheobase, and increased spike inactivation in response to depolarizing current pulse injections. After the initial tonic depolarization, rhythmic fluctuations of the motoneuron membrane potential (locomotor drive potentials; LDPs) developed that were modulated phasically in association with ventral root discharge. The peak and trough voltage levels of the LDP fluctuated above and below the membrane potential recorded immediately before the onset of rhythmic activity. Similarly, firing frequency was modulated above and below prelocomotion firing rates (in those motoneurons that displayed neurochemically induced tonic firing immediately before the onset of rhythmic activity). These observations are consistent with an alternation between phasic excitatory and inhibitory synaptic drives. The amplitude of LDPs and rhythmic excitatory drive current increased with membrane depolarization from -80 to -40 mV and then decreased with further depolarization, thus displaying nonlinear voltage-dependence. Faster frequency, small amplitude voltage fluctuations were observed superimposed on the depolarized phase of LDPs. In some motoneurons, the trajectory of these superimposed fluctuations was consistent with a synaptic origin, whereas in other cells, the regular sinusoidal appearance of the fluctuations and the occurrence of superimposed plateau potentials were more compatible with the activation of an intrinsic membrane property. One motoneuron displayed exclusively excitatory phasic drive, and another motoneuron was characterized by inhibitory phasic drive alone, during rhythmic activity. These findings are compatible with the concept of a central pattern generator that is capable of delivering both excitatory and inhibitory drive to motoneurons during locomotion. The data also suggest that the rhythmic excitatory and inhibitory outputs of the hypothetical half-center model can be dissociated and operate in isolation.