Organization and properties of GABAergic neurons in solitary tract nucleus (NTS)

Cranial visceral afferents enter the brain at the solitary tract nucleus (NTS). GABAergic neurons are scattered throughout the NTS, but their relation to solitary tract (ST) afferent pathways is imprecisely known. We hypothesized that most GABAergic NTS neurons would be connected only indirectly to the ST. We identified GABAergic neurons in brain stem horizontal slices using transgenic mice in which enhanced green fluorescent protein (EGFP) expression was linked to glutamic acid decarboxylase expression (GAD(+)). Finely graded electrical shocks to ST recruit ST-synchronized synaptic events with all-or-none thresholds and individual waveforms did not change with greater suprathreshold intensities--evidence consistent with initiation by single afferent axons. Most (approximately 70%) GAD(+) neurons received ST-evoked excitatory postsynaptic currents (EPSCs) that had minimally variant latencies (jitter, SD of latency <200 micros) and waveforms consistent with single, direct ST connections (i.e., monosynaptic). Increasing stimulus intensity evoked additional ST-synchronized synaptic responses with jitters >200 micros including inhibitory postsynaptic currents (IPSCs), indicating indirect connections (polysynaptic). Shocks of suprathreshold intensity delivered adjacent (50-300 microm) to the ST failed to excite non-ST inputs to second-order neurons, suggesting a paucity of axons passing near to ST that connected to these neurons. Despite expectations, we found similar ST synaptic patterns in GAD(+) and unlabeled neurons. Generally, ST information that arrived indirectly had small amplitudes (EPSCs and IPSCs) and frequency-dependent failures that reached >50% for IPSCs to bursts of stimuli. This ST afferent pathway organization is strongly use-dependent--a property that may tune signal propagation within and beyond NTS.     

Excitatory synaptic currents in lumbosacral parasympathetic preganglionic neurons elicited from the lateral funiculus

Excitatory postsynaptic currents (EPSCs) in parasympathetic preganglionic neurons (PGNs) were examined using the whole cell patch-clamp recording technique in L6 and S1 spinal cord slices from neonatal rats (6-16 days old). PGNs were identified by labeling with retrograde axonal transport of a fluorescent dye (Fast Blue) injected into the intraperitoneal space 3-7 days before the experiment. Synaptic responses were evoked in PGNs by field stimulation of the lateral funiculus (LF) in the presence of bicuculline methiodide (10 microM) and strychnine (1 microM). In approximately 40% of the cells (total, 100), single-shock electrical stimulation of the LF elicited short, relatively constant latency [3.0 +/- 0.1 (SE) ms] fast EPSCs consistent with a monosynaptic pathway. The remainder of the cells did not respond to stimulation. At low intensities of stimulation, the EPSCs often occurred in an all-or-none manner, indicating that they were mediated by a single axonal input. Most cells (n = 33) exhibited only fast EPSCs (type 1), but some cells (n = 8) had fast EPSCs with longer, more variable latency polysynaptic EPSCs superimposed on a slow inward current (type 2). Type 1 fast synaptic EPSCs were pharmacologically dissected into two components: a transient component that was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 microM), a non-NMDA glutamatergic antagonist, and a slow decaying component that was blocked by 2-amino-5-phosphonovalerate (APV, 50 microM), a NMDA antagonist. Type 2 polysynaptic currents were reduced by 5 microM CNQX and completely blocked by combined application of 5 microM CNQX and 50 microM APV. The fast monosynaptic component of type 1 EPSCs had a linear current-voltage relationship and reversed at a membrane potential of 5.0 +/- 5.9 mV (n = 5), whereas the slow component exhibited a negative slope conductance at holding potentials greater than -20 mV. The type 1, fast synaptic EPSCs had a time to peak of 1.4 +/- 0.1 ms and exhibited a biexponential decay (time constants, 5.7 +/- 0.6 and 38.8 +/- 4.0 ms). In the majority of PGNs (n = 11 of 15 cells), EPSCs evoked by electrical stimulation of LF exhibited paired-pulse inhibition (range; 25-33% depression) at interstimulus intervals ranging from 50 to 120 ms. These results indicate that PGNs receive monosynaptic and polysynaptic glutamatergic excitatory inputs from axons in the lateral funiculus.     

Excitatory synaptic currents in lumbosacral parasympathetic preganglionic neurons evoked by stimulation of the dorsal commissure

Excitatory pathways from the dorsal commissure (DCM) to L(6)-S(1) parasympathetic preganglionic neurons (PGN) were examined using whole-cell patch-clamp recording techniques in spinal cord slices from neonatal rats. PGN were identified by retrograde axonal transport of a fluorescent dye injected into the intraperitoneal space. Excitatory postsynaptic currents (EPSCs) were evoked in PGN by stimulation of DCM in the presence of bicuculline methiodide (10 microM) and strychnine (1 microM) to block inhibitory pathways. Electrical stimulation of DCM evoked two types of inward currents. In the majority of PGN (n = 66), currents (mean amplitude, 47.9 +/- 4.7 pA) occurred at a short and relatively constant latency (3.8 +/- 0.1 ms) and presumably represent monosynaptic EPSCs (Type 1). However, in other neurons (n = 20), a different type of EPSC (Type 2) was noted, consisting of a fast monosynaptic component followed by a prolonged inward current with superimposed fast transients presumably representing excitatory inputs mediated by polysynaptic pathways. Type 1 EPSCs were pharmacologically dissected into two components. A fast component was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 microM) and a slowly decaying component was blocked by 2-amino-5-phosphonovalerate (APV, 50 microM). The fast component of Type 1 EPSCs had a linear current-voltage relationship and reversed at a membrane potential of -7.6 +/- 1.3 mV (n = 5). The fast component of Type 2 EPSCs was also blocked by 5 microM CNQX and the remaining slower component was blocked by 50 microM APV. When the DCM was stimulated in the presence of 50 microM APV, the time to peak and decay time constant in Type 1 EPSCs were 1.9 +/- 0.2 and 4.1 +/- 0.8 ms, respectively. Examination of the NMDA receptor-mediated component of the EPSCs in the presence of 5 microM CNQX revealed a current-voltage relationship that had a region of negative slope conductance (from -20 to -80 mV), which was abolished in Mg(2+)-free external solution. The time to peak and decay time constant of this component were 14.2 +/- 2.0 and 91.0 +/- 12.4 ms, respectively. Type 1 EPSCs in some PGN responded in an all-or-none manner and presumably represented unitary synaptic responses; whereas Type 2 EPSCs always exhibited a graded stimulus intensity-response relationship. Paired-pulse facilitation (50-ms interstimulus intervals; 141 +/- 5.6% increase, n = 8) of EPSCs was observed. These results indicate that PGN receive monosynaptic and polysynaptic glutamatergic excitatory inputs from neurons and/or axonal pathways in the DCM.     

Dopamine modulates synaptic transmission in the nucleus of the solitary tract

10.1152/jn.00224.2002. Dopamine (DA) modulates the cardiorespiratory reflex by peripheral and central mechanisms. The aim of this study was to examine the role of DA in synaptic transmission of the nucleus tractus solitarius (NTS), the major integration site for cardiopulmonary reflexes. To examine DA's role, we used whole cell, voltage-clamp recordings in a rat horizontal brain stem slice. Solitary tract stimulation evoked excitatory postsynaptic currents (EPSCs) that were reduced to 70 +/- 5% of control by DA (100 microM). The reduction in EPSCs by DA was accompanied by a decrease in the paired pulse depression ratio with little or no change in input resistance or EPSC decay, suggesting a presynaptic mechanism. The D1-like agonist SKF 38393 Br (30 microM) did not alter EPSC amplitude, whereas the D2-like agonist, quinpirole HCl (30 microM), depressed EPSCs to 73 +/- 4% of control. The D2-like receptor antagonist, sulpiride (20 microM), abolished DA modulation of EPSCs. Most importantly, sulpiride alone increased EPSCs to 131 +/- 10% of control, suggesting a tonic D2-like modulation of synaptic transmission in the NTS. Examination of spontaneous EPSCs revealed DA reversibly decreased the frequency of events from 9.4 +/- 2.2 to 6.2 +/- 1.4 Hz. Sulpiride, however, did not alter spontaneous events. Immunohistochemistry of NTS slices demonstrated that D2 receptors colocalized with synaptophysin and substance P, confirming a presynaptic distribution. D2 receptors also localized to cultured petrosal neurons, the soma of presynaptic afferent fibers. In the petrosal neurons, D2 was found in cells that were TH-immunopositive, suggesting they were chemoreceptor afferent fibers. These results demonstrate that DA tonically modulates synaptic activity between afferent sensory fibers and secondary relay neurons in the NTS via a presynaptic D2-like mechanism.     

Modulation of synaptic transmission in the rat nucleus of the solitary tract by endomorphin-1

Activation of opioid receptors in the periphery and centrally in the brain results in inhibition of gastric and other vagally mediated functions. The aim of this study was to examine the role of the endogenous opioid agonist endomorphin 1 (EM-1) in regulating synaptic transmission within the nucleus tractus solitarius (NTS), an integration site for autonomic functions. We performed whole cell patch-clamp recordings from coronal brain slices of the rat medulla. A subset of the neurons studied was prelabeled with a stomach injection of the transsynaptic retrograde virus expressing EGFP, PRV-152. Solitary tract stimulation resulted in constant latency excitatory postsynaptic currents (EPSCs) that were decreased in amplitude by EM-1 (0.01-10 microM). The paired-pulse ratio was increased with little change in input resistance, suggesting a presynaptic mechanism. Spontaneous EPSCs were decreased in both frequency and amplitude by EM-1, and miniature EPSCs were reduced in frequency but not amplitude, suggesting a presynaptic mechanism for the effect. Spontaneous inhibitory postsynaptic currents (IPSCs) were also reduced in frequency by EM-1, but the effect was blocked by TTX, suggesting activity at receptors on the somata of local inhibitory neurons. Synaptic input arising from local NTS neurons, which were activated by focal photolysis of caged glutamate, was inhibited by EM-1. The actions of EM-1 were similar to those of D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) and were blocked by naltrexone, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), or D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP). These results suggest that EM-1 acts at mu-opioid receptors to modulate viscerosensory input and specific components of local synaptic circuitry in the NTS.     

Hydrogen sulfide augments synaptic neurotransmission in the nucleus of the solitary tract

Within the brain stem, the nucleus tractus solitarii (NTS) serves as a principal central site for sensory afferent integration from the cardiovascular and respiratory reflexes. Neuronal activity and synaptic transmission in the NTS are highly pliable and subject to neuromodulation. In the central nervous system, hydrogen sulfide (H?S) is a gasotransmitter generated primarily by the enzyme cystathionine-β-synthase (CBS). We sought to determine the role of H?S, and its generation by CBS, in NTS excitability. Real-time RT-PCR, immunoblot, and immunohistochemistry analysis identified the presence of CBS in the NTS. Patch-clamp electrophysiology in brain stem slices examined excitatory postsynaptic currents (EPSCs) and membrane properties in monosynaptically driven NTS neurons. Confocal imaging of labeled afferent synaptic terminals in NTS slices monitored intracellular calcium. Exogenous H?S significantly increased the amplitude of evoked solitary tract (TS)-EPSCs, frequency of miniature (m)EPSCs, and presynaptic terminal calcium fluorescence in the NTS. H?S did not alter action potential discharge or postsynaptic properties. On the other hand, the CBS inhibitor aminooxyacetate (AOA) significantly reduced the amplitude of TS-EPSCs and presynaptic terminal calcium fluorescence in the NTS without altering postsynaptic properties. Taken together, these data support a presynaptic role for endogenous H?S in modulation of excitatory neurotransmission in the NTS.     

Endomorphin-1 modulates intrinsic inhibition in the dorsal vagal complex

Mu-opioid receptor (MOR) agonists profoundly influence digestive and other autonomic functions by modulating neurons in nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMV). Whole cell recordings were made from NTS and DMV neurons in brain stem slices from rats and transgenic mice that expressed enhanced green fluorescent protein (EGFP) under the control of a GAD67 promoter (EGFP-GABA neurons) to identify opioid-mediated effects on GABAergic circuitry. Synaptic and membrane properties of EGFP-GABA neurons were assessed. The endogenous selective MOR agonist endomorphin-1 (EM-1) reduced spontaneous and evoked excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic currents (IPSCs) in both rat and mouse DMV neurons. Electrical stimulation of the solitary tract evoked constant-latency EPSCs in approximately 50% of EGFP-GABA neurons, and the responses were reduced by EM-1 application. EM-1 reduced action potential firing, the frequency and amplitude of synaptic inputs in EGFP-GABA neurons and responses to direct glutamate stimulation. A subset of EGFP-GABA neurons colocalized mRFP1 after retrograde, transneuronal infection after gastric inoculation with PRV-614, indicating that they synapsed with gastric-projecting DMV neurons. Glutamate photolysis stimulation of intact NTS projections evoked IPSCs in DMV neurons, and EM-1 reduced the evoked response, most likely by activation of MOR on the soma of premotor GABA neurons in NTS. Naltrexone or H-d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), MOR antagonists, blocked the effects of EM-1. Our results show that GABA neurons in the NTS receive direct vagal afferent input and project to gastric-related DMV neurons. Furthermore, modulation by EM-1 of specific components of the vagal complex differentially suppresses excitatory and inhibitory synaptic input to the DMV by acting at different receptor locations.     

Ammonium ions abolish excitatory synaptic transmission between cerebellar neurons in primary dissociated tissue culture.

1. Transmitter glutamate is thought to be derived from glutamine via cleavage by glutaminase. NH+4 inhibits glutaminase. Therefore the decrease of glutamatergic excitatory synaptic transmission by NH+4 was thought to be due to the inability of glutamine to serve as precursor for glutamate. However, in cat spinal cord, NH+4 abolished excitatory synaptic transmission by a conduction block for action potentials in presynaptic terminals. The conduction block prevented inferences as to the effects of NH+4 on the availability of glutamate for synaptic transmission. This study reexamines the effects of NH+4 on glutamatergic excitatory synaptic transmission in cerebellar neurons in tissue culture. 2. Whole-cell patch voltage-clamp recordings were obtained from presumed Purkinje cells. Extracellular stimulation of presumed granule cells produced mono- and polysynaptic excitatory postsynaptic currents (EPSCs). In addition, presumed Purkinje cells showed spontaneous EPSCs that occurred independently of the addition of tetrodotoxin (TTX) or Cd2+ to the extracellular solution. 3. NH+4 (5-10 mM) abolished evoked mono- and polysynaptic EPSCs without abolishing spontaneous EPSCs and without significant effects on action currents in the Purkinje cell soma. 4. Increase of K+ in the extracellular solution to 10-12 from 5 mM abolished evoked EPSCs without abolishing spontaneous EPSCs and without significant effects on action currents in the Purkinje cell soma. 5. Mixtures of NH+4 and K+, with each ion in a concentration insufficient to affect evoked EPSCs when given alone, abolished evoked EPSCs when the sum of NH+4 and K+ exceeded 10-12 mM. 6. Increase of intracellular pH by trimethylamine had no effect on evoked and spontaneous EPSCs.(ABSTRACT TRUNCATED AT 250 WORDS)     

GABA(B)-mediated inhibition of multiple modes of glutamate release in the nucleus of the solitary tract

In the caudal portions of the solitary tract (ST) nucleus, primary sensory afferents fall into two broad classes based on the expression of transient receptor potential vanilloid type 1 (TRPV1) receptors. Both afferent classes (TRPV1+/-) have indistinguishable glutamate release mechanisms for ST-evoked excitatory postsynaptic currents (EPSCs). However, TRPV1+ terminals release additional glutamate from a unique, TRPV1-operated vesicle pool that is temperature sensitive and facilitated by ST activity to generate asynchronous EPSCs. This study tested whether presynaptic γ-aminobutyric acid (GABA)(B) receptors inhibit both the evoked and TRPV1-operated release mechanisms on second-order ST nucleus neurons. In horizontal slices, shocks activated single ST axons and evoked the time-invariant (latency jitter <200 μs), glutamatergic EPSCs, which identified second-order neurons. Gabazine eliminated GABA(A) responses in all recordings. The GABA(B) agonist baclofen inhibited the amplitude of ST-EPSCs from both TRPV1+ and TRPV1- afferents with a similar EC(50) (～1.2 μM). In TTX, GABA(B) activation decreased miniature EPSC (mEPSC) rates but not amplitudes, suggesting presynaptic actions downstream from terminal excitability. With calcium entry through voltage-activated calcium channels blocked by cadmium, baclofen reduced mEPSC frequency, indicating that GABA(B) reduced vesicle release by TRPV1-dependent calcium entry. GABA(B) activation also reduced temperature-evoked increases in mEPSC frequency, which relies on TRPV1. Our studies indicate that GABA(B) G protein-coupled receptors are uniformly distributed across all ST primary afferent terminals and act at multiple stages of the excitation-release cascades to suppress both action potential-triggered and TRPV1-coupled glutamate transmission pathways. Moreover, the segregated release cascades within TRPV1+ ST primary afferents represent independent, potential targets for differential modulation.     

Morphology and physiology of lamina I neurons of the caudal part of the trigeminal nucleus

It has been suggested that in mammals, trigeminal lamina I neurons play a role in the processing and transmission of sensory information from the orofacial region. We investigated the physiological and morphological properties of trigeminal subnucleus caudalis (Sp5C) lamina I neurons in slices prepared from the medulla oblongata of 13- to 15-day-old postnatal rats using patch-clamp recordings and subsequent biocytin-streptavidin-Alexa labeling. Twenty-five neurons were recorded and immunohistochemically stained. The Sp5C lamina I consisted of several types of neurons which, on the basis of their responses to somatic current injection, can be classified into four groups: tonic neurons, which fired throughout the depolarizing pulse; phasic neurons, which expressed an initial burst of action potentials; delayed onset neurons, which showed a significant delay of the first action potential; and single spike neurons, characterized by only one to five action potentials at the very beginning of the depolarizing pulse even at high levels of stimulation intensity. Electrical stimulation of the spinal trigeminal tract evoked AMPA receptor-mediated excitatory postsynaptic currents (EPSC) exhibiting a strong polysynaptic component. AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSC) were characterized by a 10-90% rise time of 0.50+/-0.06 ms and a decay time constant of 2.5+/-0.5 ms. The kinetic properties of NMDA receptor-mediated EPSCs were measured at +40 mV. The 10-90% rise time was 8+/-2 ms and the deactivation time constants were 94+/-31 and 339+/-72 ms, respectively. Intracellular staining and morphological analysis revealed three groups of neurons: fusiform, pyramidal, and multipolar. Statistical analysis indicated that the electrophysiological properties and morphological characteristics are correlated. Tonic and phasic neurons were fusiform or pyramidal and delayed onset and single spike neurons were multipolar. Our results show that both the physiological and morphological properties of Sp5C lamina I neurons exhibit significant differences, indicating their specific integration in the processing and transmission of sensory information from the orofacial region.     

Dual-component miniature excitatory synaptic currents in rat hippocampal CA3 pyramidal neurons.

1. Spontaneous miniature synaptic events were studied with tight-seal whole-cell recordings from CA3 neurons maintained in the hippocampal slice from immature rats (3-15 days). CA3 neurons suffer a constant, high-frequency barrage of inhibitory synaptic input. When inhibitory postsynaptic currents were suppressed by bicuculline, a smaller contribution from excitatory synapses was revealed. 2. Addition of tetrodotoxin (TTX) removed a persistent inward current and substantially reduced the baseline noise facilitating the detection of "miniature" excitatory currents. Addition of hyperosmotic media increased the frequency of spontaneous excitatory postsynaptic currents (EPSCs). 3. Under both physiological and elevated potassium conditions, individual spontaneous miniature EPSCs (10-30 pA amplitude) were composed of components mediated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors as determined by their voltage dependence, time course, and sensitivity to selective antagonists. 6-Cyano-7-nitro-quinoxaline-2,3-dione (CNQX) or D-2-amino-5-phosphonovaleric acid (D-APV) shifted the amplitude distribution of miniature EPSCs to a smaller mode at both +40 mV and -40 mV. Similar to EPSCs recorded in CA1 neurons, the rise and decay times of the NMDA receptor component were slower than those of the non-NMDA component. The time course of the non-NMDA component was voltage independent. 4. In 13 of 21 neurons, no correlation existed between individual EPSC rise times and their corresponding halfwidth, peak amplitude, or decay time constant. This suggests that the large range of EPSC kinetics observed in each individual neuron was not due solely to cable attenuation of EPSCs widely distributed over the dendritic tree. Plots of the mean EPSC rise time against mean halfwidth for each cell, however, revealed a striking correlation, suggesting that in neonates, active synapses may be grouped in a restricted region of the dendritic tree and as such are subject to similar amounts of dendritic filtering. 5. The electrotonic length of CA3 neurons (L = 0.52) predicted that at this maturity the electrotonic compactness of the neuron facilitated voltage control over all but the most distal synapses. The reversal potential of the fast component of spontaneous events was close to 0 mV, whereas the reversal potential of exogenously applied kainate and NMDA was more positive. This discrepancy likely reflects a compromise of the voltage clamp by the activation of conductances distributed over the entire cell.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synaptic physiology in the cochlear nucleus angularis of the chick

Nucleus angularis (NA), one of the two cochlear nuclei in birds, is important for processing sound intensity for localization and most likely has role in sound recognition and other auditory tasks. Because the synaptic properties of auditory nerve inputs to the cochlear nuclei are fundamental to the transformation of auditory information, we studied the properties of these synapses onto NA neurons using whole cell patch-clamp recordings from auditory brain stem slices from embryonic chickens (E16-E20). We measured spontaneous excitatory postsynaptic currents (EPSCs), and evoked EPSCs and excitatory postsynaptic potentials (EPSPs) by using extracellular stimulation of the auditory nerve. These excitatory EPSCs were mediated by AMPA and N-methyl-D-aspartate (NMDA) receptors. The spontaneous EPSCs mediated by AMPA receptors had submillisecond decay kinetics (556 micros at E19), comparable with those of other auditory brain stem areas. The spontaneous EPSCs increased in amplitude and became faster with developmental age. Evoked EPSC and EPSP amplitudes were graded with stimulus intensity. The average amplitude of the EPSC evoked by minimal stimulation was twice as large as the average spontaneous EPSC amplitude (approximately 110 vs. approximately 55 pA), suggesting that single fibers make multiple contacts onto each postsynaptic NA neuron. Because of their small size, minimal EPSPs were subthreshold, and we estimate at least three to five inputs were required to reach threshold. In contrast to the fast EPSCs, EPSPs in NA had a decay time constant of approximately 12.5 ms, which was heavily influenced by the membrane time constant. Thus NA neurons spatially and temporally integrate auditory information arriving from multiple auditory nerve afferents.     

Wheat germ agglutinin enhances EPSCs in cultured postnatal rat hippocampal neurons by blocking ionotropic quisqualate receptor desensitization.

1. The effect of the lectin wheat germ agglutinin (WGA), an inhibitor of ionotropic quisqualate receptor desensitization, on both evoked and spontaneous fast excitatory postsynaptic events was examined in cultured postnatal rat hippocampal neurons with the use of whole cell recordings. 2. WGA, at 580 nM, potentiated evoked fast excitatory postsynaptic currents (EPSCs) by increasing the amplitudes by 100 +/- 27% (mean +/- SE) and the time constant of decay from 5.8 +/- 0.6 to 7.9 +/- 0.5 ms. The increases in these parameters were not accompanied by changes in the current-voltage (I-V) relationship or pharmacological profile of the fast EPSCs. 3. WGA did not alter the amplitude or time course of decay of inhibitory postsynaptic currents (IPSCs), and it did not alter neuronal input resistance or action potentials. 4. WGA increased the amplitude of spontaneous fast miniature EPSCs (MEPSCs), defined as spontaneous EPSCs recorded in the presence of tetrodotoxin, by 53 +/- 11% and increased the time required to decay to 50% of the peak amplitude by 48 +/- 23%. These changes were not associated with a change in the rate of MEPSC occurrence. 5. These results suggest that WGA augments hippocampal excitatory postsynaptic events via a postsynaptic mechanism. The results further imply that ionotropic quisqualate receptor desensitization can modulate the amplitude and time course of decay of fast excitatory synaptic events. Thus desensitization may be one factor that regulates fast excitatory synaptic transmission.     

Selective enhancement of excitatory synaptic activity in the rat nucleus tractus solitarius by hypocretin 2

Hypocretin 2 (orexin B) is a hypothalamic neuropeptide thought to be involved in regulating energy homeostasis, autonomic function, arousal, and sensory processing. Neural circuits in the caudal nucleus tractus solitarius (NTS) integrate viscerosensory inputs, and are therefore implicated in aspects of all these functions. We tested the hypothesis that hypocretin 2 modulates fast synaptic activity in caudal NTS areas that are generally associated with visceral sensation from cardiorespiratory and gastrointestinal systems. Hypocretin 2-immunoreactive fibers were observed throughout the caudal NTS. In whole-cell recordings from neurons in acute slices, hypocretin 2 depolarized 48% and hyperpolarized 10% of caudal NTS neurons, effects that were not observed when Cs(+) was used as the primary cation carrier. Hypocretin 2 also increased the amplitude of tractus solitarius-evoked excitatory postsynaptic currents (EPSCs) in 36% of neurons and significantly enhanced the frequency of spontaneous EPSCs in most (59%) neurons. Spontaneous inhibitory postsynaptic currents (IPSCs) were relatively unaffected by the peptide. The increase in EPSC frequency persisted in the presence of tetrodotoxin, suggesting a role for the peptide in regulating glutamate release in the NTS by acting at presynaptic terminals.These data suggest that hypocretin 2 modulates excitatory, but not inhibitory, synapses in caudal NTS neurons, including viscerosensory inputs. The selective nature of the effect supports the hypothesis that hypocretin 2 plays a role in modulating autonomic sensory signaling in the NTS.     

Intracortical circuits in rat anterior cingulate cortex are activated by nociceptive inputs mediated by medial thalamus

We investigated the afferents and intracortical synaptic organization of the anterior cingulate cortex (ACC) during noxious electrical stimulation. Extracellular field potentials were recorded simultaneously from 16 electrodes spanning all layers of the ACC in male Sprague-Dawley rats anesthetized by halothane inhalation. Laminar-specific transmembrane currents were calculated with the current source density analysis method. Two major groups of evoked sink currents were identified: an early group (latency = 54.04 +/- 2.12 ms; 0.63 +/- 0.07 mV/mm(2)) in layers V-VI and a more intense late group (latency = 80.07 +/- 4.85 ms; 2.16 +/- 0.22 mV/mm(2)) in layer II/III and layer V. Multiunit activities were evoked mainly in layer V and deep layer II/III with latencies similar to that of the early and late sink groups. The evoked EPSP latencies of pyramidal neurons in layers II/III and V related closely with the sink currents. The sink currents were inhibited by intracortical injection of CNQX (1 mM, 1 microl), a glutaminergic receptor antagonist, and enhanced by intraperitoneal (5 mg/kg) and intracortical (10 microg/microl, 1 microl) injection of morphine, a mu-opioid receptor agonist. Paired-pulse depression was observed with interpulse intervals of 50 to 1,000 ms. High-frequency stimulation (100 Hz, 11 pulses) enhanced evoked responses in the ACC and evoked medial thalamic (MT) unit activities. MT lesions blocked evoked responses in the ACC. Our results demonstrated that two distinct synaptic circuits in the ACC were activated by noxious stimuli and that the MT is the major thalamic relay that transmits nociceptive information to the ACC.     

Selective blockade of Ca2+ permeable AMPA receptors in CA1 area of rat hippocampus

Using whole cell patch-clamp recording from pyramidal cells and interneurons in the CA1 area of hippocampal slices, the effect of IEM-1460, a selective channel blocker of Ca2+ permeable AMPA receptors (AMPARs), on postsynaptic currents (PSCs) was studied. Excitatory postsynaptic currents (EPSCs) were evoked by stimulation of Schaffer collaterals (SCs) in the presence of APV and bicuculline to pharmacologically isolate the EPSCs mediated by AMPAR activation. IEM-1460 (50 microM) did not affect the amplitude of EPSCs in CA1 pyramidal cells but reversibly decreased their amplitude in interneurons of pyramidal layer (15 cells), radiatum (37 cells) and border radiatum-lacunosum-moleculare (R-LM) (55 cells) layers. The ability of IEM-1460 to decrease EPSC amplitude correlated with EPSC rectification properties in CA1 interneurons, providing evidence for synaptic localization of Ca2+ permeable AMPARs at the SC synaptic input. Independent of their localization, the majority of interneurons studied exhibited only modest sensitivity to IEM-1460 (EPSC amplitude decreased by less than 30%), while in 15% of interneurons IEM-1460 induced more than 50% reduction in EPSC amplitude. To reveal possible afferent-specific localization of Ca2+ permeable AMPARs on R-LM interneurons, the effect of IEM-1460 on EPSCs evoked by stimulation of SC was compared with that of perforant path (PP). Although average sensitivities did not differ significantly, in 61% of R-LM layer interneurons, the SC-evoked EPSCs exhibited higher sensitivity to IEM-1460 than the PP-evoked EPSCs. Moreover, in 54% of R-LM layer interneurons the EPSCs evoked by SC stimulation were complex, having an initial peak followed by one or several late components. Kinetics, latency distribution and reversal potential of late components suggest di- and polysynaptic origin of the late components. Late EPSCs were strongly and reversibly inhibited by IEM-1460 indicating that Ca2+ permeable AMPARs are involved in the indirect excitation of R-LM layer interneurons. Despite the ability to decrease the excitatory synaptic input to interneurons, IEM-1460 did not affect interneuron-mediated inhibitory postsynaptic currents (IPSCs) evoked in pyramidal neurons by SC stimulation. These data suggest that interneurons with a synaptic input highly sensitive to IEM-1460 do not contribute specifically to the feed-forward inhibition of hippocampal pyramidal neurons.     

Evans blue reduces macroscopic desensitization of non-NMDA receptor mediated currents and prolongs excitatory postsynaptic currents in cultured rat thalamic neurons.

Fast application of L-glutamate, AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) or kainate to cultured rat thalamic neurons revealed properties of non-NMDA (N-methyl-D-aspartate) receptors similar to those described in hippocampal neurons. The kinetics of non-NMDA receptor-mediated currents were altered by the addition of the dye Evans Blue (EB). Macroscopic desensitization was reduced and activation and deactivation kinetics were slowed. Delayed addition of EB, after desensitization of non-NMDA receptors, resulted in reactivation of desensitized receptors. Thus, both ion channel gating and entry into the desensitized state were affected. Evans blue also slowed the activation and the decay of glutamatergic miniature EPSCs (excitatory postsynaptic currents), demonstrating that receptor kinetics determine the time course of the synaptic response.     

Prostaglandin E2 depresses solitary tract-mediated synaptic transmission in the nucleus tractus solitarius

Prostaglandin E(2) (PGE(2)) is a prototypical inflammatory mediator that excites and sensitizes cell bodies [Kwong K, Lee LY (2002) PGE(2) sensitizes cultured pulmonary vagal sensory neurons to chemical and electrical stimuli. J Appl Physiol 93:1419-1428; Kwong K, Lee LY (2005) Prostaglandin E(2) potentiates a tetrodotoxin (TTX)-resistant sodium current in rat capsaicin-sensitive vagal pulmonary sensory neurons. J Physiol 56:437-450] and peripheral nerve terminals [Ho CY, Gu Q, Hong JL, Lee LY (2000) Prostaglandin E (2) enhances chemical and mechanical sensitivities of pulmonary C fibers in the rat. Am J Respir Crit Care Med 162:528-533] of primary vagal sensory neurons. Nearly all central nerve terminals of vagal afferents are in the nucleus tractus solitarius (NTS), where they operate with a high probability of release [Doyle MW, Andresen MC (2001) Reliability of monosynaptic sensory transmission in brain stem neurons in vitro. J Neurophysiol 85:2213-2223]. We studied the effect of PGE(2) on synaptic transmission between tractus solitarius afferent nerve terminals and the second-order NTS neurons in brain stem slices of Sprague-Dawley rats. Whole-cell patch recording in voltage clamp mode was used to study evoked excitatory postsynaptic glutamatergic currents (evEPSCs) from NTS neurons elicited by electrical stimulation of the solitary tract (ST). In 34 neurons, bath-applied PGE(2) (200 nM) decreased the evEPSC amplitude by 49+/-5%. In 22 neurons, however, PGE(2) had no effect. We also tested 15 NTS neurons for capsaicin sensitivity. Seven neurons generated evEPSCs that were equally unaffected by PGE(2) and capsaicin. Conversely, evEPSCs of the other eight neurons, which were PGE(2)-responsive, were abolished by 200 nM capsaicin. Furthermore, the PGE(2-)induced depression of evEPSCs was associated with an increase in the paired pulse ratio and a decrease in both the frequency and amplitude of the spontaneous excitatory postsynaptic currents (sEPSCs) and TTX-independent spontaneous miniature excitatory postsynaptic currents (mEPSCs). These results suggest that PGE(2) acts both presynaptically on nerve terminals and postsynaptically on NTS neurons to reduce glutamatergic responses.     

Long-term potentiation of synaptic transmission in kitten visual cortex

1. Potentiation of synaptic transmission in visual cortex (areas 17 and 18) of kittens was investigated by extracellular recording of field potentials (FPs) and cortical units in cortical slices and whole-animal preparations. Responses to test stimulation (0.05 Hz) of the white matter (WM), lateral geniculate nucleus (LGN), and optic chiasm (OC) were documented before and after conditioning stimulation (2 Hz for 1 h). 2. In slice preparations of area 17, the FPs were always depressed during conditioning stimulation and were usually potentiated immediately after conditioning stimulation. Long-term potentiation (LTP) of FPs developed rapidly during the initial 1-2 h and continued to increase slowly for several hours after conditioning. 3. LTP of FPs was age dependent: LTP occurred most frequently (43/53) at the ages of 21-34 days, less frequently (4/7 and 5/11) at 14-20 and 35-41 days, and never (0/5 and 0/5) at 7-13 and 42-49 days. LTP age relationship determined as a ratio of the amplitudes of FPs after conditioning to that before conditioning was greater at 21-34 days (mean potentiation, 2.4 +/- 0.6) than at 14-20 or 35-41 days (1.7 +/- 0.5). 4. LTP was also documented by the shortening in latencies of orthodromic responses of cortical units sampled from 10 pairs of conditioned and unconditioned control slices. Unit responses were classified into mono- and polysynaptic groups according to the central delay, defined as the time required for their activation after the arrival of afferent impulses. The monosynaptic central delays were 0.22 ms shorter in conditioned (0.60 +/- 0.17 ms, n = 56) than in control slices (0.82 +/- 0.22 ms, n = 57); similarly, polysynaptic central delays were 0.66 ms smaller (1.70 +/- 0.43 ms, n = 51; and 2.36 +/- 0.79 ms, n = 51). Both differences were statistically significant (P less than 0.001). 5. There were laminar differences in LTP of mono- and polysynaptic transmission. LTP of monosynaptic transmission occurred throughout layers II-V (central delays shortened about 0.2 ms), whereas LTP of polysynaptic transmission was greatest in layer II (1.17 ms), moderate in layer III (0.66 ms), and slight in layer IV (0.3 ms). The time course of shortening in orthodromic latency in five polysynaptic units agreed with the time course of LTP of FP. 6. Location of synapses involved in LTP of synaptic transmission was studied by current source-density (CSD) analysis in slice preparations of area 17 during test stimulation of WM. CSD analysis demonstrated two components of current sinks (early and late), probably representing mono- and polysynaptic transmission.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of synaptic inputs to paraventricular-spinal output neurons by alpha2 adrenergic receptors

Neurons in the paraventricular nucleus (PVN) that project to the brain stem and spinal cord are important for autonomic regulation. The excitability of preautonomic PVN neurons is controlled by the noradrenergic input from the brain stem. In this study, we determined the role of alpha(2) adrenergic receptors in the regulation of excitatory and inhibitory synaptic inputs to spinally projecting PVN neurons. Excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) were recorded using whole cell voltage-clamp techniques on PVN neurons labeled by a retrograde fluorescence tracer injected into the thoracic spinal cord of rats. Bath application of 5-20 muM clonidine, an alpha(2) receptor agonist, significantly reduced the amplitude of evoked GABAergic IPSCs in a dose-dependent manner. Also, 10 microM clonidine significantly decreased the frequency (from 2.68 +/- 0.41 to 1.22 +/- 0.40 Hz) but not the amplitude of miniature IPSCs (mIPSCs), and this effect was blocked by the alpha(2) receptor antagonist yohimbine. Furthermore, clonidine increased the paired-pulse ratio of evoked IPSCs from 1.25 +/- 0.05 to 1.61 +/- 0.08 (P < 0.05). On the other hand, clonidine had little effect on evoked glutamatergic EPSCs, mEPSCs, and the paired-pulse ratio of evoked EPSCs in most labeled cells examined. Additionally, immunofluorescence labeling revealed that the alpha(2A) receptor and GABA immunoreactivities were co-localized in close apposition to labeled PVN neurons. Collectively, these data suggest that stimulation of alpha(2) adrenergic receptors primarily attenuates GABAergic inputs to PVN output neurons to the spinal cord. The presynaptic alpha(2) receptors function as heteroreceptors to modulate synaptic GABA release and contribute to the hypothalamic regulation of sympathetic outflow.