Amplification of tumor immunity by gene transfer of the co-stimulatory 4-1BB ligand: synergy with the CD28 co-stimulatory pathway

The superficial cells of the entorhinal cortex (EC), main input to the hippocampus, receive a serotonergic input from the raphe nuclei and express 5-hydroxytryptamine creatine sulfate complex (5-HT) receptors at high density. With the use of intracellular recordings, we investigated the effects of serotonin on synaptic inhibition of layer II and III neurons of the EC. Serotonin reduced both polysynaptic fast and slow inhibitory postsynaptic potentials (IPSPs) in projection neurons of the superficial EC. Polysynaptic fast and slow IPSPs were depressed by serotonin in a dose-dependent manner (0.1-100 microM). Serotonin in a concentration of 1 microM reduced the amplitudes of polysynaptic fast and slow IPSPs by approximately 40 and 50%, respectively. To identify the subtype of the 5-HT-receptor mediating the effects on polysynaptic IPSPs, we applied various 5-HT-receptor agonists and antagonists. Although the serotonin agonists for the 5-HT1B,2C,3 receptors were ineffective, the effects were mimicked by the 5-HT1A-receptor agonists (8-OH-DPAT, 5-CT) and prevented by the 5-HT1A-receptor antagonist NAN-190. To look at the direct effects of 5-HT on inhibitory interneurons, we elicited monosynaptic IPSPs in the absence of excitatory synaptic transmission. In contrast to the polysynaptic IPSPs, monosynaptic IPSPs were not significantly affected by serotonin. Recordings from putative inhibitory interneurons revealed that their excitatory postsynaptic potentials (EPSPs) were reversibly reduced by serotonin. We conclude that serotonin suppresses polysynaptic inhibition in projection neurons of layers II and III of the EC by depression of EPSPs on inhibitory interneurons via 5-HT1A receptors.     

A postsynaptic interaction between dopamine D1 and NMDA receptors promotes presynaptic inhibition in the rat nucleus accumbens via adenosine release

The mechanism underlying dopamine D1 receptor-mediated attenuation of glutamatergic synaptic input to nucleus accumbens (NAcc) neurons was investigated in slices of rat forebrain, using whole-cell patch-clamp recording. The depression by dopamine of EPSCs evoked by single-shock cortical stimulation was stimulus-dependent. Synaptic activation of NMDA-type glutamate receptors was critical for this effect, because dopamine-induced EPSC depressions were blocked by the competitive NMDA receptor antagonist D/L-2-amino-5-phosphonopentanoate (AP5). Application of NMDA also depressed the EPSC, and both this effect and the dopamine depressions were blocked by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), implicating adenosine release in the EPSC depression. A1 receptor agonists also depressed EPSCs by a presynaptic action, causing increased paired-pulse facilitation, but this was insensitive to AP5. Activation of D1 receptors enhanced both postsynaptic inward currents evoked by NMDA application and the isolated NMDA receptor-mediated component of synaptic transmission. The biochemical processes underlying the dopamine-induced EPSC depression did not involve either protein kinase A or the production of cAMP and its metabolites, because this effect was resistant to the protein kinase inhibitors H89 and H7 and the cAMP-specific phosphodiesterase inhibitor rolipram. We conclude that activation of postsynaptic D1 receptors enhances the synaptic activation of NMDA receptors in nucleus accumbens neurons, thereby promoting a transsynaptic feedback inhibition of glutamatergic synaptic transmission via release of adenosine. Unusually for D1 receptors, this phenomenon occurs independently of adenylyl cyclase stimulation. This process may contribute to the locomotor stimulant action of dopaminergic agents in the NAcc.     

Neurobiological mechanisms involved in antidepressant therapies

Selective serotonin reuptake inhibitors (SSRIs) are effective in alleviating the symptoms of depression. However, clinical improvement is only obtained after several weeks of treatment. SSRIs, when administered acutely to animals, have little effect on synaptic levels of serotonin. This suggests the existence of one or more regulatory mechanisms controlling serotonergic neurotransmission. The firing rate of dorsal raphe serotonergic neurons is under the control of somatodendritic 5-hydroxytryptamine 1A (5-HT1A) autoreceptors, the release of serotonin from nerve terminals is under the control of 5-HT autoreceptors (5-HT1B subtype in rodents, 5-HT1D in other species), whereas the control of the activity of tryptophan hydroxylase, the rate-limiting enzyme of serotonin synthesis, is complex, involving 5-HT1A but possibly other 5-HT receptors including the 5-HT1B/D subtype. During prolonged administration with a SSRI, these three feedback systems become desensitized and their regulatory effects on serotonergic neurotransmission are weakened or lost. This has the effect of allowing the synaptic levels of serotonin to rise with a consequently increased stimulation of one or more types of postsynaptic 5-HT receptor. Thus, it is only after prolonged administration that the pharmacological activity of SSRI is fully expressed in terms of synaptic serotonin levels. This may explain the latency of antidepressant action seen with these drugs in humans. Various other classes of antidepressant therapies (tricyclic antidepressants and monoamine oxidase inhibitor drugs, electroconvulsive therapy) have long-term effects on one or more of the feedback mechanisms such that an increase in synaptic concentrations of serotonin may be a common mechanism of many antidepressant therapies.     

The effects of prenatal exposure to cocaine on the dopaminergic cells in the rat retina. An immunocytochemical and neurochemical study

5-Hydroxytryptamine1A (5-HT1A) receptors have been visualized at the electron microscopic level in selected areas (dorsal raphe nucleus, hippocampus, septum) of the rat brain using specific anti-peptide antibodies. 5-HT1A receptor immunoreactivity was found almost exclusively in the somatodendritic compartment of neurons and was very rarely observed within processes possibly belonging to glial cells. The immunoenzymatic reaction product was associated exclusively with dendritic spines in the dorsal hippocampus, whereas in the dorsal raphe nucleus and the septal complex, immunoreactivity was found in both dendritic processes and somata. Although some immunolabeling was observed within the cytoplasm of cell bodies, 5-HT1A receptor immunoreactivity was essentially confined to the plasma membrane where it was unevenly distributed. It was frequently associated with synapses (except in the dorsal raphe nucleus), but was also found extrasynaptically in both somata and dendrites. These data suggest that the action of serotonin via 5-HT1A receptor could occur through junctional as well as nonjunctional transmission.     

Dopamine neurons make glutamatergic synapses in vitro

Interactions between dopamine and glutamate play prominent roles in memory, addiction, and schizophrenia. Several lines of evidence have suggested that the ventral midbrain dopamine neurons that give rise to the major CNS dopaminergic projections may also be glutamatergic. To examine this possibility, we double immunostained ventral midbrain sections from rat and monkey for the dopamine-synthetic enzyme tyrosine hydroxylase and for glutamate; we found that most dopamine neurons immunostained for glutamate, both in rat and monkey. We then used postnatal cell culture to examine individual dopamine neurons. Again, most dopamine neurons immunostained for glutamate; they were also immunoreactive for phosphate-activated glutaminase, the major source of neurotransmitter glutamate. Inhibition of glutaminase reduced glutamate staining. In single-cell microculture, dopamine neurons gave rise to varicosities immunoreactive for both tyrosine hydroxylase and glutamate and others immunoreactive mainly for glutamate, which were found near the cell body. At the ultrastructural level, dopamine neurons formed occasional dopaminergic varicosities with symmetric synaptic specializations, but they more commonly formed nondopaminergic varicosities with asymmetric synaptic specializations. Stimulation of individual dopamine neurons evoked a fast glutamatergic autaptic EPSC that showed presynaptic inhibition caused by concomitant dopamine release. Thus, dopamine neurons may exert rapid synaptic actions via their glutamatergic synapses and slower modulatory actions via their dopaminergic synapses. Together with evidence for glutamate cotransmission in serotonergic raphe neurons and noradrenergic locus coeruleus neurons, the present results suggest that glutamatergic cotransmission may be the rule for central monoaminergic neurons.     

Serotonin induces excitatory postsynaptic potentials in apical dendrites of neocortical pyramidal cells

By intracellular and whole cell recording in rat brain slices, it was found that bath-applied serotonin (5-HT) produces an increase in the frequency and amplitude of spontaneous excitatory postsynaptic potentials/currents (EPSPs/EPSCs) in layer V pyramidal cells of neocortex and transitional cortex (e.g. medial prefrontal, cigulate and frontoparietal). The EPSCs were suppressed by LY293558, an antagonist selective for the AMPA subtype of excitatory amino acid receptor, and by two selective 5-HT2A receptor antagonists, MDL 100907 and SR 46349B. In addition, the EPSCs were suppressed by the fast sodium channel blocker tetrodotoxin (TTX) and were dependent upon external calcium. However, despite being TTX-sensitive and calcium dependent, there was no evidence that the EPSPs resulted from an increase in impulse flow in excitatory neuronal afferents to layer V pyramidal cells. The EPSCs could be induced rapidly by the microiontophoresis of 5-HT directly to "hot spots" within the apical (but not basilar) dendritic field of recorded neurons, indicating that excitatory amino acids may be released by a TTX-sensitive focal action of 5-HT on a subset of glutamatergic terminals in this region. Consistent with such a presynaptic action, the inhibitory metabotropic glutamate receptor agonist (1S,3S)-aminocyclopentane-1,3-dicarboxylate markedly reduced the induction of EPSPs by 5-HT. Postsynaptically, 5-HT enhanced a subthreshold TTX-sensitive sodium current, potentially contributing to an amplification of EPSC amplitudes. These data suggest 5-HT. via 5-HT2A receptors, enhances spontaneous EPSPs/EPSCs in neocortical layer V pyramidal cells through a TTX-sensitive focal action in the apical dendritic field which may involve both pre- and postsynaptic mechanisms.     

A purinergic component of the excitatory postsynaptic current mediated by P2X receptors in the CA1 neurons of the rat hippocampus

The pyramidal neurons in the CA1 area of hippocampal slices from 17- to 19-day-old rats have been investigated by means of patch clamp. Excitatory postsynaptic currents (EPSCs) were elicited by stimulating the Schaffer collateral at a frequency below 0.2 Hz. It was found that inhibition of glutamatergic transmission by 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 100 microM 2-amino-5-phosphonovaleric acid (D-APV) left a small component of the EPSC uninhibited. The amplitude of this residual EPSC (rEPSC) comprised 25 +/- 11% of the total EPSC when measured at a holding potential of -50 mV. The rEPSC was blocked by selective P2 blocker pyridoxal phosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS) 10 microM and bath incubation with non-hydrolysable ATP analogues, ATP-gamma-S and alpha, beta-methylene-ATP at 50 and 20 microM, respectively. The rEPSC was dramatically potentiated by external Zn2+ (10 microM). In another series of experiments exogenous ATP was applied to the CA1 neurons in situ. An inward current evoked by ATP was inhibited by PPADS to the same extent as the rEPSC. It is concluded that, depending on membrane voltage, about one-fifth to one-quarter of the EPSC generated by the excitatory synaptic input to the hippocampal CA1 neurons of rat is due to the activity of P2X receptors.     

Preferential potentiation of the effects of serotonin uptake inhibitors by 5-HT1A receptor antagonists in the dorsal raphe pathway: role of somatodendritic autoreceptors

5-HT1A autoreceptor antagonists enhance the effects of antidepressants by preventing a negative feedback of serotonin (5-HT) at somatodendritic level. The maximal elevations of extracellular concentration of 5-HT (5-HT(ext)) induced by the 5-HT uptake inhibitor paroxetine in forebrain were potentiated by the 5-HT1A antagonist WAY-100635 (1 mg/kg s.c.) in a regionally dependent manner (striatum > frontal cortex > dorsal hippocampus). Paroxetine (3 mg/kg s.c.) decreased forebrain 5-HT(ext) during local blockade of uptake. This reduction was greater in striatum and frontal cortex than in dorsal hippocampus and was counteracted by the local and systemic administration of WAY-100635. The perfusion of 50 micromol/L citalopram in the dorsal or median raphe nucleus reduced 5-HT(ext) in frontal cortex or dorsal hippocampus to 40 and 65% of baseline, respectively. The reduction of cortical 5-HT(ext) induced by perfusion of citalopram in midbrain raphe was fully reversed by WAY-100635 (1 mg/kg s.c.). Together, these data suggest that dorsal raphe neurons projecting to striatum and frontal cortex are more sensitive to self-inhibition mediated by 5-HT1A autoreceptors than median raphe neurons projecting to the hippocampus. Therefore, potentiation by 5-HT1A antagonists occurs preferentially in forebrain areas innervated by serotonergic neurons of the dorsal raphe nucleus.     

Hippocampal interneurons are excited via serotonin-gated ion channels

Hippocampal interneurons are excited via serotonin-gated ion channels. J. Neurophysiol. 78: 2493-2502, 1997. Serotonergic neurons of the median raphe nucleus heavily innervate hippocampal GABAergic interneurons located in stratum radiatum of area CA1, suggesting that this strong subcortical projection may modulate interneuron excitability. Using whole cell patch-clamp recording from interneurons in brain slices, we tested the effects of serotonin (5-HT) on the physiological properties of these interneurons. Serotonin produces a rapid inward current that persists when synaptic transmission is blocked by tetrodotoxin and cobalt, and is unaffected by ionotropic glutamate and gamma-aminobutyric acid (GABA) receptor antagonists. The 5-HT-induced current was independent of G-protein activation. Pharmacological evidence indicates that 5-HT directly excites these interneurons through activation of 5-HT3 receptors. At membrane potentials negative to -55 mV, the current-voltage (I-V) relationship of the 5-HT current displays a region of negative slope conductance. Therefore the response of interneurons to 5-HT strongly depends on membrane potential and increases greatly as cells are depolarized. Removal of extracellular calcium, but not magnesium, increases the amplitude of 5-HT-induced currents and removes the region of negative slope conductance, thereby linearizing the I-V relationship. The axons of 5-HT-responsive interneurons ramify widely within CA1; some of these interneurons also project to and arborize extensively in the dentate gyrus. The organization of these inhibitory connections strongly suggests that these cells regulate excitability of both CA1 pyramidal cells and dentate granule cells. As our results indicate that 5-HT may mediate fast excitatory synaptic transmission onto these interneurons, serotonergic inputs can simultaneously modulate the output of both hippocampus and dentate gyrus.     

A competitive enzyme-linked immunosorbent assay for measuring the levels of serum antibody to Haemophilus influenzae type b

1. The protein family of the neurotrophins, consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and Neurotrophin-3, -4/5, and -6 (NT-3; NT-4/5; NT-6) is well known to enhance the survival and to stabilize the phenotype of different populations of neurons in the central and the peripheral nervous system. These effects are mediated via binding to specific tyrosine kinase receptors (Trks) and to the low-affinity p75 neurotrophin receptor. 2. The neurotrophins NGF, BDNF, and NT-3 and the BDNF and NT-3 selective receptors (TrkB, TrkC) are expressed at high levels in neurons of the basal forebrain, the hippocampus, and the neocortex of the mammalian brain. The expression and secretion of NGF and BDNF in these brain areas is regulated by (physiological levels of) neuronal activity. 3. Exogenous application of the neurotrophins to hippocampal and neocortical neurons can enhance excitatory glutamatergic synaptic transmission via activation of Trk receptors. In addition, long-term potentiation (a potential cellular correlate for learning and memory formation in mammals) in the rodent hippocampus depends on endogenous supply of neurons with BDNF. 4. Judged by the analysis of electrophysiological data, the BDNF- and NT-3-induced enhancement of glutamatergic synapses is mediated by increasing the efficacy of glutamate release from the presynaptic neuron. However, neurotrophin-dependent postsynaptic enhancement of NMDA (but not AMPA) receptor responsiveness has also been shown. 5. On the molecular level, neither the pre- nor the postsynaptic modulation of glutamatergic synapses by neurotrophins is well understood. However, neurotrophins were shown to acutely affect intraneuronal Ca2+ levels and to influence molecular components of the transmitter release machinery, which could underly the presynaptic modifications, whereas BDNF-induced phosphorylation of NMDA-type glutamate receptors could account for the postsynaptic effects. 6. Taken together, these results suggest that the activity-dependent release of neurotrophins at frequently used synapses could modulate the synaptic efficacy at these junctions. Thus, neurotrophins might operate as locally released feedback modulators of synaptic transmission, and this could be a cellular correlate for certain aspects of information processing in the mammalian brain.     

Quantitative evaluation of 5-hydroxytryptamine (serotonin) neuronal release and uptake: an investigation of extrasynaptic transmission

Whether neurotransmitters are restricted to the synaptic cleft (participating only in hard-wired neurotransmission) or diffuse to remote receptor sites (participating in what has been termed volume or paracrine transmission) depends on a number of factors. These include (1) the location of release sites with respect to the receptors, (2) the number of molecules released, (3) the diffusional rate away from the release site, determined by both the geometry near the release site as well as binding interactions, and (4) the removal of transmitter by the relevant transporter. Fast-scan cyclic voltammetry allows for the detection of extrasynaptic concentrations of many biogenic amines, permitting direct access to many of these parameters. In this study the hypothesis that 5-hydroxytryptamine (5-HT) transmission is primarily extrasynaptic in the substantia nigra reticulata, a terminal region with identified synaptic contacts, and the dorsal raphe nucleus, a somatodendritic region with rare synaptic incidence, was tested in brain slices prepared from the rat. Using carbon fiber microelectrodes, we found the concentration of 5-HT released per stimulus pulse in both regions to be identical when elicited by single pulse stimulations or trains at high frequency. 5-HT efflux elicited by a single stimulus pulse was unaffected by uptake inhibition or receptor antagonism. Thus, synaptic efflux is not restricted by binding to intrasynaptic receptors or transporters. The number of 5-HT molecules released per terminal was estimated in the substantia nigra reticulata and was considerably less than the number of 5-HT transporter and receptor sites, reinforcing the hypothesis that these sites are extrasynaptic. Furthermore, the detected extrasynaptic concentrations closely match the affinity for the predominant 5-HT receptor in each region. Although they do not disprove the existence of classical synaptic transmission, our results support the existence of paracrine neurotransmission in both serotonergic regions.     

Comparison of the effects of serotonin in the hippocampus and the entorhinal cortex

Among the molecular, cellular, and systemic events that have been proposed to modulate the function of the hippocampus and the entorhinal cortex (EC), one of the most frequently cited possibilities is the activation of the serotonergic system. Neurons in the hippocampus and in the EC receive a strong serotonergic projection from the raphe nuclei and express serotonin (5-HT) receptors at high density. Here we review the various effects of 5-HT on intrinsic and synaptic properties of neurons in the hippocampus and the EC. Although similar membrane-potential changes following 5-HT application have been reported for neurons of the entorhinal cortex and the hippocampus, the effects of serotonin on synaptic transmission are contrary in both areas. Serotonin mainly depresses fast and slow inhibition of the principal output cells of the hippocampus, whereas it selectively suppresses the excitation in the entorhinal cortex. On the basis of these data, we discuss the possible role of serotonin under physiological and pathophysiological circumstances.     

Ascending afferent regulation of rat midbrain dopamine neurons

Standard, extracellular single-unit recording techniques were used to examine the electrophysiological and pharmacological responsiveness of midbrain dopamine (DA) neurons to selected, ascending afferent inputs. Sciatic nerve stimulation-induced inhibition of nigrostriatal DA (NSDA) neurons was blocked by both PCPA (5-HT synthesis inhibitor) and 5,7-DHT (5-HT neurotoxin), suggesting mediation by a serotonergic (5-HT) system. Direct stimulation of the dorsal raphe (which utilizes 5-HT as a neurotransmitter and inhibits slowly firing NSDA neurons) inhibited all mesoaccumbens DA (MADA) neurons tested. Paradoxically, DPAT, a 5-HT1A agonist which inhibits 5-HT cell firing, enhanced sciatic nerve stimulation-induced inhibition of NSDA neurons. MADA neurons were not inhibited by sciatic nerve stimulation and, therefore, could not be tested in this paradigm. In contrast to the dorsal raphe, electrical stimulation of the pedunculopontine tegmental nucleus preferentially excited slowly firing NSDA and MADA neurons. Thus, both excitatory and inhibitory ascending afferents influence the activity of midbrain DA neurons, and intact 5-HT systems are necessary for sciatic nerve stimulation to alter DA cell activity. However, the role that 5-HT plays in mediating peripheral sensory input remains unclear.     

Effects of corticotropin-releasing factor on brain serotonergic activity

The serotonergic dorsal raphe nucleus is innervated by corticotropin-releasing factor (CRF) and expresses CRF receptors, suggesting that endogenous CRF impacts on this system. The present study characterized interactions between CRF and the dorsal raphe serotonin (5-HT) system. The effects of intracerebroventricularly (i.c.v.) administered CRF on microdialysate concentrations of 5-HT in the lateral striatum of freely moving rats were determined. CRF had biphasic effects, with 0.1 and 0.3 microgram decreasing, and 3.0 micrograms increasing 5-HT dialysate concentrations. i.c.v. administration of CRF inhibited neuronal activity of the majority of dorsal raphe neurons at both low (0.3 microgram) and high (3 micrograms) doses. Likewise, intraraphe administration of CRF (0.3 and 1.0 ng) had predominantly inhibitory effects on discharge rate. Together, these results suggest that CRF is positioned to regulate the function of the dorsal raphe serotonergic system via actions within the cell body region. This regulation may play a role in stress-related psychiatric disorders in which 5-HT has been implicated.     

5-Hydroxytryptamine2 receptor facilitates GABAergic neurotransmission in rat hippocampus

5-Hydroxytryptamine (5-HT; serotonin) administration enhances GABAergic synaptic activity recorded in pyramidal neurons of the CA1 region of hippocampus. Previous studies have attributed this effect to the activation of HT-5(3) receptors located on GABAergic interneurons. During unrelated experiments, we noticed that under our recording conditions, 5-HT can still increase GABAergic synaptic activity after the complete blockade of 5-HT3 receptors. This indicated the involvement of an additional 5-HT receptor subtype. Therefore, we reinvestigated the effects of 5-HT on GABAergic synaptic activity recorded in pyramidal cells of the CA1 region. The ability of 5-HT to increase GABAergic synaptic activity in the presence of 5-HT3 receptor blockade was mimicked by the selective 5-HT2 agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane and blocked by the selective 5-HT2 antagonist ketanserin. This indicated that the additional 5-HT receptor belongs to 5-HT2 receptor family. 5-HT2 receptor activation resulted in an increase in the frequency of spontaneous inhibitory postsynaptic currents as well as a shift in their amplitude distribution toward larger sizes. These effects were absent in the presence of tetrodotoxin. We interpret these results to indicate that 5-HT2 receptors activate GABAergic interneurons in the slice, leading to an increase in GABAergic synaptic activity onto pyramidal cells of the CA1 region.     

Cellular and synaptic modulation underlying substance P-mediated plasticity of the lamprey locomotor network

The tachykinin substance P modulates the lamprey locomotor network by increasing the frequency of NMDA-evoked ventral root bursts and by making the burst activity more regular. These effects can last in excess of 24 hr. In this paper, the effects of substance P on the synaptic and cellular properties of motor neurons and identified network interneurons have been examined. Substance P potentiated the amplitude of monosynaptic glutamatergic inputs from excitatory interneurons and reticulospinal axons. The amplitude and frequency of miniature EPSPs was increased, suggesting that the synaptic modulation was mediated presynaptically and postsynaptically. The postsynaptic modulation was caused by a specific effect of substance P on the NMDA component of the synaptic input, whereas the presynaptic component was calcium-independent. Substance P did not affect monosynaptic glycinergic inputs from lateral interneurons, crossed inhibitory interneurons, or ipsilateral segmental interneurons or postsynaptic GABAA or GABAB responses, suggesting that it has little effect on inhibitory synaptic transmission. At the cellular level, substance P increased synaptic inputs, resulting in membrane potential oscillations in motor neurons, crossed caudal interneurons, lateral interneurons, and excitatory interneurons. The spiking in response to depolarizing current pulses was increased in motor neurons, lateral interneurons, and excitatory interneurons, but usually was reduced in crossed inhibitory interneurons. Substance P reduced the calcium-dependent afterhyperpolarization after an action potential in motor neurons and lateral interneurons, but did not affect this conductance in excitatory or crossed inhibitory interneurons. The relevance of these cellular and synaptic changes to the modulation of the locomotor network is discussed.     

Serotonin modulation of sensory inputs to the lateral amygdala: dependency on corticosterone

The lateral nucleus of the amygdala (LA) receives excitatory (glutamatergic) inputs from thalamic and cortical sensory processing areas and is believed to be involved in evaluation of the affective significance of sensory events. We examined whether serotonin (5-HT) affects excitatory transmission in auditory afferents to the LA and, if so, whether this modulation of sensory transmission is regulated by the stress hormone corticosterone (CORT). Neuronal activity in the LA was elicited via iontophoretic ejection of L-glutamate or synaptically via electrical stimulation of auditory afferent pathways. In the intact rat, iontophoretically applied 5-HT inhibited both synaptically and glutamate-evoked action potentials in most neurons examined. However, after adrenalectomy (ADX), which eliminates endogenous CORT, 5-HT no longer inhibited evoked activity in the LA. High-CORT doses given to ADX animals reinstated the inhibition of excitatory transmission of 5-HT, whereas low-CORT doses had little effect. Immunocytochemical labeling of the glucocorticoid receptor in the intact rat demonstrated nuclear staining throughout several amygdala regions, including the LA. However, after ADX, no nuclear labeling was visible. With a high replacement dose of CORT (5 or 10 mg) after ADX, dense nuclear staining returned, but with a low replacement dose (1 mg/kg), there was only light nuclear staining. Thus, the ability of 5-HT to modulate glutamatergic activity in auditory pathways to the amygdala is dependent on the presence of CORT and possibly glucocorticoid activation. Via this mechanism, 5-HT modulates the processing of sensory information within the LA and thus may regulate amygdala-related functions.     

Mechanism and kinetics of heterosynaptic depression at a cerebellar synapse

High levels of activity at a synapse can lead to spillover of neurotransmitter from the synaptic cleft. This extrasynaptic neurotransmitter can diffuse to neighboring synapses and modulate transmission via presynaptic receptors. We studied such modulation at the synapse between granule cells and Purkinje cells in rat cerebellar slices. Brief tetanic stimulation of granule cell parallel fibers activated inhibitory neurons, leading to a transient elevation of extracellular GABA, which in turn caused a short-lived heterosynaptic depression of the parallel fiber to Purkinje cell EPSC. Fluorometric calcium measurements revealed that this synaptic inhibition was associated with a decrease in presynaptic calcium influx. Heterosynaptic inhibition of synaptic currents and calcium influx was eliminated by antagonists of the GABAB receptor. The magnitude and time course of the depression of calcium influx were mimicked by the rapid release of an estimated 10 microM GABA using the technique of flash photolysis. We found that inhibition of presynaptic calcium influx peaked within 300 msec and decayed in <3 sec at 32 degrees C. These results indicate that presynaptic GABAB receptors can sense extrasynaptic GABA increases of several micromolar and that they rapidly regulate the release of neurotransmitter primarily by modulating voltage-gated calcium channels.     

Serotonergic neurons differentially modulate the efficacy of two motor neurons innervating the same muscle fibers in Aplysia

Feeding behavior in Aplysia shows substantial plasticity. An important site for the generation of this plasticity is the modulation of synaptic transmission between motor neurons and the buccal muscles that generate feeding movements. We have been studying this modulation in the anterior portion of intrinsic buccal muscle 3 (I3a), which is innervated by two excitatory motor neurons and identified serotonergic modulatory neurons, the metacerebral cells (MCCs). We have shown previously that serotonin (5-HT) applied selectively to the muscle potently modulates excitatory junction potentials (EJPs) and contractions. All the effects of 5-HT were persistent, lasting many hours after wash out. We examined whether the release of endogenous 5-HT from the MCC could produce effects similar to the application of 5-HT. Stimulation of the MCCs did produce similar short-term effects to the application of 5-HT. MCC stimulation facilitates EJPs, potentiates contractions, and decreases the latency between the onset of a motor neuron burst and the onset of the evoked contraction. The effects of MCC stimulation reached a maximum at quite low firing frequencies, which were in the range of those previously recorded during feeding behavior. The maximal effects were similar to those produced by superfusion with approximately 0.1 microM 5-HT. Although the effects of MCC stimulation on EJPs were persistent, they were less persistent than the effects of 0.1 microM 5-HT. Mechanisms that may account for differences in the persistence between released and superfused 5-HT are discussed. Thus activity in the MCCs has dramatic short-term effects on the behavioral output of motor neurons, increasing the amplitude and relaxation rate of contractions evoked by both B3 and B38 and shifting the temporal relationship between B38 bursts and evoked contractions.     

ATP P2x receptors and sensory synaptic transmission between primary afferent fibers and spinal dorsal horn neurons in rats

ATP P2x receptors and sensory synaptic transmission between primary afferent fibers and spinal dorsal horn neurons in rats. J. Neurophysiol. 80: 3356-3360, 1998. Glutamate is a major fast transmitter between primary afferent fibers and dorsal horn neurons in the spinal cord. Recent evidence indicates that ATP acts as another fast transmitter at the rat cervical spinal cord and is proposed to serve as a transmitter for nociception and pain. Sensory synaptic transmission between dorsal root afferent fibers and neurons in the superficial dorsal horn of the lumbar spinal cord were examined by whole cell patch-clamp recording techniques. Experiments were designed to test if ATP could serve as a transmitter at the lumbar spinal cord. Monosynaptic excitatory postsynaptic currents (EPSCs) were completely abolished after the blockade of both glutamatergic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate and N-methyl--aspartate receptors. No residual current was detected, indicating that glutamate but not ATP is a fast transmitter at the dorsal horn of the lumbar spinal cord. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a selective P2x receptor antagonist, produced an inhibitory modulatory effect on fast EPSCs and altered responses to paired-pulse stimulation, suggesting the involvement of a presynaptic mechanism. Intrathecal administration of PPADS did not produce any antinociceptive effect in two different types of behavioral nociceptive tests. The present results suggest that ATP P2x2 receptors modulate excitatory synaptic transmission in the superficial dorsal horn of the lumbar spinal cord by a presynaptic mechanism, and such a mechanism does not play an important role in behavioral responses to noxious heating. The involvement of other P2x subtype receptors, which is are less sensitive to PPADS, in acute nociceptive modulation and persistent pain remains to be investigated.