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.     

Reliability of monosynaptic sensory transmission in brain stem neurons in vitro

The timing of events within the nervous system is a critical feature of signal processing and integration. In neurotransmission, the synaptic latency, the time between stimulus delivery and appearance of the synaptic event, is generally thought to be directly related to the complexity of that pathway. In horizontal brain stem slices, we examined synaptic latency and its shock-to-shock variability (synaptic jitter) in medial nucleus tractus solitarius (NTS) neurons in response to solitary tract (ST) electrical activation. Using a visualized patch recording approach, we activated ST 1-3 mm from the recorded neuron with short trains (50-200 Hz) and measured synaptic currents under voltage clamp. Latencies ranged from 1.5 to 8.6 ms, and jitter values (SD of intraneuronal latency) ranged from 26 to 764 micros (n = 49). Surprisingly, frequency of synaptic failure was not correlated with either latency or jitter (P > 0.147; n = 49). Despite conventional expectations, no clear divisions in latency were found from the earliest arriving excitatory postsynaptic currents (EPSCs) to late pharmacologically polysynaptic responses. Shortest latency EPSCs (<3 ms) were mediated by non-N-methyl-D-aspartate (non-NMDA) glutamate receptors. Longer latency responses were a mix of excitatory and inhibitory currents including non-NMDA EPSCs and GABAa receptor-mediated currents (IPSC). All synaptic responses exhibited prominent frequency-dependent depression. In a subset of neurons, we labeled sensory boutons by the anterograde fluorescent tracer, DiA, from aortic nerve baroreceptors and then recorded from anatomically identified second-order neurons. In identified second-order NTS neurons, ST activation evoked EPSCs with short to moderate latency (1.9-4.8 ms) but uniformly minimal jitter (31 to 61 micros) that were mediated by non-NMDA receptors but had failure rates as high as 39%. These monosynaptic EPSCs in identified second-order neurons were significantly different in latency and jitter than GABAergic IPSCs (latency, 2.95 +/- 0.71 vs. 5.56 +/- 0.74 ms, mean +/- SE, P = 0.027; jitter, 42.3 +/- 6.5 vs. 416.3 +/- 94.4 micros, P = 0.013, n = 4, 6, respectively), but failure rates were similar (27.8 +/- 9.0 vs. 9.7 +/- 4.4%, P = 0.08, respectively). Such results suggest that jitter and not absolute latency or failure rate is the most reliable discriminator of mono- versus polysynaptic pathways. The results suggest that brain stem sensory pathways may differ in their principles of integration compared with cortical models and that this importantly impacts synaptic performance. The unique performance properties of the sensory-NTS pathway may reflect stronger axosomatic synaptic processing in brain stem compared with dendritically weighted models typical in cortical structures and thus may reflect very different strategies of spatio-temporal integration in this NTS region and for autonomic regulation.     

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.     

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.     

Somatostatin presynaptically inhibits both GABA and glutamate release onto rat basal forebrain cholinergic neurons

A whole cell patch-clamp study was carried out in slices obtained from young rat brain to elucidate the roles of somatostatin in the modulation of synaptic transmission onto cholinergic neurons in the basal forebrain (BF), a region that contains cholinergic and GABAergic corticopetal neurons and somatostatin (SS)-containing local circuit neurons. Cholinergic neurons within the BF were identified by in vivo prelabeling with Cy3 IgG. Because in many cases SS is contained in GABAergic neurons in the CNS, we investigated whether exogenously applied SS can influence GABAergic transmission onto cholinergic neurons. Bath application of somatostatin (1 muM) reduced the amplitude of the evoked GABAergic inhibitory presynaptic currents (IPSCs) in cholinergic neurons. SS also reduced the frequency of miniature IPSCs (mIPSCs) without affecting their amplitude distribution. SS-induced effect on the mIPSC frequency was significantly larger in the solution containing 7.2 mM Ca(2+) than in the standard (2.4 mM Ca(2+)) external solution. Similar effects were observed in the case of non-NMDA glutamatergic excitatory postsynaptic currents (EPSCs). SS inhibited the amplitude of evoked EPSCs and reduced the frequency of miniature EPSCs dependent on the external Ca(2+) concentration with no effect on their amplitude distribution. Pharmacological analyses using SS-receptor subtype-specific drugs suggest that SS-induced action of the IPSCs is mediated mostly by the sst(2) subtype, whereas sst subtypes mediating SS-induced inhibition of EPSCs are mainly sst(1) or sst(4). These findings suggest that SS presynaptically inhibits both GABA and glutamate release onto BF cholinergic neurons in a Ca(2+)-dependent way, and that SS-induced effect on IPSCs and EPSCs are mediated by different sst subtypes.     

In vivo patch-clamp analysis of IPSCs evoked in rat substantia gelatinosa neurons by cutaneous mechanical stimulation

To know a functional role of inhibitory synaptic responses in transmitting noxious and innoxious information from the periphery to the rat spinal dorsal horn, we examined inhibitory postsynaptic currents (IPSCs) elicited in substantia gelatinosa (SG) neurons by mechanical stimuli applied to the skin using the newly developed in vivo patch-clamp technique. In the majority (80%) of SG neurons examined, a brush stimulus applied to the ipsilateral hind limb produced a barrage of IPSCs that persisted during the stimulus, while a pinch stimulus evoked IPSCs only at its beginning and end. The pinch-evoked IPSCs may have been caused by a touch that occurs at the on/off time of the pinch. The evoked IPSCs were blocked by either a glycine-receptor antagonist, strychnine (4 microM), or a GABA(A)-receptor antagonist, bicuculline (20 microM). All SG neurons examined received inhibitory inputs from a wide area throughout the thigh and lower leg. When IPSCs were examined together with excitatory postsynaptic currents (EPSCs) in the same neurons, a brush evoked a persistent activity of both IPSCs and EPSCs during the stimulus while a pinch evoked such an activity of EPSCs but not IPSCs. It is suggested that innoxious mechanical stimuli activate a GABAergic or glycinergic circuitry in the spinal dorsal horn. This inhibitory transmission may play an important role in the modulation of noxious information in the SG.     

Intrinsic gamma-aminobutyric acid neurons in the nucleus of the solitary tract and the rostral ventrolateral medulla of the rat: an immunocytochemical and biochemical study

Sympathoexcitatory neurons in the C1 adrenergic area of the rostral ventrolateral medulla (RVL) are tonically inhibited by gamma-aminobutyric acid (GABA). To identify the source of this GABAergic input, the distribution of neurons containing glutamate decarboxylase (GAD) was determined immunocytochemically in rats treated with colchicine. Numerous GAD-stained neurons were located in the nucleus of the solitary tract (NTS) and in RVL. Unilateral lesions in NTS did not alter GABA content or GAD activity in RVL, indicating that the afferent projection from NTS to RVL is not GABAergic. Intrinsic GABAergic neurons in RVL may provide tonic inhibition of vasomotor neurons in the C1 area.     

5-HT inhibits synaptic transmission in rat subthalamic nucleus neurons in vitro

The subthalamic nucleus (STN) plays a pivotal role in normal and abnormal motor function. We used patch pipettes to study effects of 5-HT on synaptic currents evoked in STN neurons by focal electrical stimulation of rat brain slices. 5-HT (10 microM) reduced glutamate-mediated excitatory postsynaptic currents (EPSCs) by 35+/-4%. However, a much higher concentration of 5-HT (100 microM) was required to inhibit GABA-mediated inhibitory postsynaptic currents (IPSCs) to a comparable extent. Concentration-response curves showed that the 5-HT inhibitory concentration 50% (IC50) for inhibition of IPSCs (20.2 microM) was more than fivefold greater than the IC50 for inhibition of EPSCs (3.4 microM). The 5-HT-induced reductions in EPSCs and IPSCs were accompanied by increases in paired-pulse ratios, indicating that 5-HT acts presynaptically to inhibit synaptic transmission. The 5-HT1B receptor antagonist NAS-181 significantly antagonized 5-HT-induced inhibitions of EPSCs and IPSCs. These studies show that 5-HT inhibits synaptic transmission in the STN by activating presynaptic 5-HT1B receptors.     

Differential modulation of nucleus accumbens synapses

The nucleus accumbens (NAcc) is a brain region involved in functions ranging from motivation and reward to feeding and drug addiction. The NAcc is typically divided into two major subdivisions, the shell and the core. The primary output neurons of both of these areas are medium spiny neurons (MSNs), which are quiescent at rest and depend on the relative input of excitatory and inhibitory synapses to determine when they fire action potentials. These synaptic inputs are, in turn, regulated by a number of neurochemical signaling agents that can ultimately influence information processing in the NAcc. The present study characterized the ability of three major signaling pathways to modulate synaptic transmission in NAcc MSNs and compared this modulation across different synapses within the NAcc. The opioid [Met](5)enkephalin (ME) inhibited excitatory postsynaptic currents (EPSCs) in shell MSNs, an effect mediated primarily by micro-opioid receptors. Forskolin, an activator of adenylyl cyclase, potentiated shell EPSCs. An analysis of miniature EPSCs indicated a primarily presynaptic site of action, although a smaller postsynaptic effect may have also contributed to the potentiation. Adenosine and an adenosine A(1)-receptor agonist inhibited shell EPSCs, although no significant tonic inhibition by endogenous adenosine was detected. The effects of these signaling agents were then compared across four different synapses in the NAcc: glutamatergic EPSCs and GABAergic inhibitory postsynaptic currents (IPSCs) in both the core and shell subregions. ME inhibited all four of these synapses but produced a significantly greater inhibition of shell IPSCs than the other synapses. Forskolin produced an increase in transmission at each of the synapses tested. However, analysis of miniature IPSCs in the shell showed no sign of a postsynaptic contribution to this potentiation, in contrast to the shell miniature EPSCs. Tonic inhibition of synaptic currents by endogenous adenosine, which was not observed in shell EPSCs, was clearly present at the other three synapses tested. These results indicate that neuromodulation can vary between the different subregions of the NAcc and between the different synapses within each subregion. This may reflect differences in neuronal interconnections and functional roles between subregions and may contribute to the effects of drugs acting on these systems.     

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.     

Effects of activation of group III metabotropic glutamate receptors on spinal synaptic transmission in a rat model of neuropathic pain

Chronic neuropathic pain remains an unmet clinical problem because it is often resistant to conventional analgesics. Metabotropic glutamate receptors (mGluRs) are involved in nociceptive processing at the spinal level, but their functions in neuropathic pain are not fully known. In this study, we investigated the role of group III mGluRs in the control of spinal excitatory and inhibitory synaptic transmission in a rat model of neuropathic pain induced by L5/L6 spinal nerve ligation. Whole-cell recording of lamina II neurons was performed in spinal cord slices from control and nerve-ligated rats. The baseline amplitude of glutamatergic EPSCs evoked from primary afferents was significantly larger in nerve-injured rats than in control rats. However, the baseline frequency of GABAergic and glycinergic inhibitory postsynaptic currents (IPSCs) was much lower in nerve-injured rats than in control rats. The group III mGluR agonist l(+)-2-amino-4-phosphonbutyric acid (l-AP4) produced a greater inhibition of the amplitude of monosynaptic and polysynaptic evoked EPSCs in nerve-injured rats than in control rats. l-AP4 inhibited the frequency of miniature EPSCs in 66.7% of neurons in control rats but its inhibitory effect was observed in all neurons tested in nerve-injured rats. Furthermore, l-AP4 similarly inhibited the frequency of GABAergic and glycinergic IPSCs in control and nerve-injured rats. Our study suggests that spinal nerve injury augments glutamatergic input from primary afferents but decreases GABAergic and glycinergic input to spinal dorsal horn neurons. Activation of group III mGluRs attenuates glutamatergic input from primary afferents in nerve-injured rats, which could explain the antinociceptive effect of group III mGluR agonists on neuropathic pain.     

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.     

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.     

GABAB receptors in the medial septum/diagonal band slice from 16-25 day rat

GABA(B) receptors are believed to play a role in rhythmic activity in the mammalian brain. The aim of our study was to examine the presynaptic and postsynaptic locations of these receptors in the medial septal diagonal band area (MS/DB), an area known to pace the hippocampus theta rhythm. Whole-cell patch recordings were made from parasagittal MS/DB slices obtained from the 16-25 day rat. Neurons were classified into GABAergic and cholinergic subtypes according to previous electrophysiological criteria. Bath application of the GABA(B) receptor agonist baclofen in the presence of tetrodotoxin, and brief tetanic fiber stimulation in the presence of ionotropic receptor antagonists, provided evidence for the presence of postsynaptic GABA(B) receptor transmission to GABAergic but not cholinergic neurons. Bath application of baclofen, at concentrations too low to elicit postsynaptic activity in MS/DB neurons, significantly reduced the amplitudes of stimulus-evoked ionotropic receptor inhibitory postsynaptic potentials (IPSPs) and excitatory postsynaptic potentials (EPSPs) and the paired pulse depression of these evoked potentials. Baclofen also significantly reduced the frequencies but not the amplitudes of miniature inhibitory postsynaptic currents (IPSCs) and excitatory postsynaptic currents (EPSCs), indicating the presence of presynaptic GABA(B) receptors on GABAergic and glutamatergic terminals in the MS/DB. Baclofen, also at a concentration too low to elicit postsynaptic activity, reduced the frequencies and amplitudes of spontaneous IPSCs and EPSCs recorded in the presence of 200-400 nM kainate. Rhythmic compound IPSCs at theta frequencies were recorded under these conditions in some neurons, and these rhythmic compound IPSCs were disrupted by the activation but not by the inhibition of GABA(B) receptors. These results suggest that GABA(B) receptors modulate rather than generate rhythmic activity in the MS/DB, and that this modulatory effect occurs via receptors located on presynaptic terminals.     

Brain-derived neurotrophic factor increases inhibitory synapses, revealed in solitary neurons cultured from rat visual cortex

To elucidate chronic actions of brain-derived neurotrophic factor (BDNF) on GABAergic synapses, we examined effects of a long-term application of BDNF for 10-15 days on autapses (synapses) of solitary GABAergic neurons cultured from rat visual cortex. Solitary neuron preparations were used to exclude a possible contamination of BDNF actions on excitatory neurons in dissociated neuron culture or slice preparations. Neurons were confirmed to be GABAergic pharmacologically with bicuculline, a selective antagonist for GABAA receptors and immunocytochemically with antibody against glutamic acid decarboxylase 65, a GABA synthesizing enzyme. To evaluate GABAergic synaptic function, evoked and/or miniature inhibitory postsynaptic currents (IPSCs) were recorded in the whole-cell voltage-clamp mode. The treatment with BDNF at a concentration of 100 ng/ml enhanced the amplitude of evoked IPSCs and the frequency of miniature IPSCs. In contrast, BDNF did not have a detectable effect on the amplitude of miniature IPSCs and the paired pulse ratio of IPSCs evoked by two, successive activations. To evaluate morphological changes, neurons were immunocytochemically stained with antibodies against microtubule-associated protein 2, to visualize somatodendritic region and synapsin I, to visualize presynaptic sites. The quantitative analysis indicated that BDNF increased the area of soma, the numbers of primary dendrites and dendritic branching points, the total length of dendrites and the number of synaptic sites. Such an action of BDNF was seen in both subgroups of GABAergic neurons, parvalbumin-positive and -negative neurons. To visualize functionally active presynaptic sites, neurons were stained with a styryl dye, FM1-43. BDNF increased the number of stained sites that was correlated with the frequency of miniature IPSCs. These results suggest that the chronic treatment with BDNF promotes dendritic and synaptic development of GABAergic neurons in visual cortex.     

Neurotensin inhibition of GABAergic transmission via mGluR-induced endocannabinoid signalling in rat periaqueductal grey

Neurotensin modulates pain via its actions within descending analgesic pathways which include brain regions such as the midbrain periaqueductal grey (PAG). The aim of this study was to examine the cellular actions of neurotensin on PAG neurons. Whole cell patch clamp recordings were made from rat midbrain PAG slices in vitro to examine the postsynaptic effects of neurotensin and its effects on GABA_A mediated inhibitory postsynaptic currents (IPSCs). Neurotensin (100–300 n m ) produced an inward current in subpopulations of opioid sensitive and insensitive PAG neurons which did not reverse over membrane potentials between –50 and –130 mV. The neurotensin induced current was abolished by the NTS1 and NTS1/2 antagonists SR48692 (300 n m ) and SR142948A (300 n m ). Neurotensin also produced a reduction in the amplitude of evoked IPSCs, but had no effect on the rate and amplitude of TTX-resistant miniature IPSCs. The neurotensin induced inhibition of evoked IPSCs was reduced by the mGluR5 antagonist MPEP (5μ m ) and abolished by the cannabinoid CB₁ receptor antagonist AM251 (3μ m ). These results suggest that neurotensin produces direct neuronal depolarisation via NTS1 receptors and inhibits GABAergic synaptic transmission within the PAG. The inhibition of synaptic transmission is mediated by neuronal excitation and action potential dependent release of glutamate, leading to mGluR5 mediated production of endocannabinoids which activate presynaptic CB₁ receptors. Thus, neurotensin has cellular actions within the PAG which are consistent with both algesic and analgesic activity, some of which are mediated via the endocannabinoid system.     

Inhibitory-excitatory synaptic balance is shifted toward increased excitation in magnocellular neurosecretory cells of heart failure rats

Despite the well-established contribution of neurohumoral activation to morbidity and mortality in heart failure (HF) patients, relatively little is known about the underlying central nervous system mechanisms. In this study, we aimed to determine whether changes in GABAergic inhibitory and glutamatergic excitatory synaptic function contribute to altered hypothalamic magnocellular neurosecretory cell (MNC) activity in HF rats. Patch-clamp recordings were obtained from MNCs in brain slices from sham and HF rats. Glutamate excitatory (EPSCs) and GABAergic inhibitory postsynaptic currents (IPSCs) were simultaneously recorded, and changes in their strengths, as well as their interactions, were evaluated. We found a diminished GABAergic synaptic strength in MNCs of HF rats, reflected as faster decaying IPSCs and diminished mean IPSC charge transfer. Opposite changes were observed in glutamate EPSC synaptic strength, resulting in a shift in the GABA-glutamate balance toward a relatively stronger glutamate influence in HF rats. The prolongation of glutamate EPSCs during HF was mediated, at least in part, by an enhanced contribution of AMPA receptor desensitization to the EPSC decay time course. EPSC prolongation, and consequently increased unitary strength, resulted in a stronger AMPA receptor-mediated excitatory drive to firing discharge in MNCs of HF rats. Blockade of GABA(A) synaptic activity diminished the EPSC waveform variability observed among events in sham rats, an effect that was blunted in HF rats. Together, our results suggest that opposing changes in postsynaptic properties of GABAergic and glutamatergic synaptic function contribute to enhanced magnocellular neurosecretory activity in HF rats.     

Altered synaptic input and GABAB receptor function in spinal superficial dorsal horn neurons in rats with diabetic neuropathy

Hyperactivity of spinal dorsal horn neurons plays an important role in the development of diabetic neuropathic pain. However, little is known as to whether synaptic input to spinal dorsal horn neurons is altered in diabetic neuropathy. Also, the function of GABA_B receptors in the control of synaptic input to dorsal horn neurons in diabetes remains poorly understood. To determine the changes in synaptic input to dorsal horn neurons and the GABA_B receptor function in streptozotocin-induced diabetes, we performed whole-cell recording (GDP-β-S included in the internal solution) on lamina II neurons in rat spinal cord slices. The frequency of glutamatergic mEPSCs and the amplitude of monosynaptic EPSCs evoked from the dorsal root were significantly higher in diabetic than in control rats. On the other hand, the basal frequency and amplitude of GABAergic spontaneous IPSCs and mIPSCs and those of glycinergic spontaneous IPSCs and mIPSCs did not differ significantly between control and diabetic rats. The GABA_B agonist baclofen produced a significantly greater reduction in dorsal root-evoked EPSCs and the frequency of mEPSCs in control than in diabetic rats. However, the inhibitory effect of baclofen on GABAergic and glycinergic spontaneous IPSCs and mIPSCs was not significantly different in the two groups. These findings suggest that increased glutamatergic input from primary afferents to dorsal horn neurons may contribute to synaptic plasticity and central sensitization in diabetic neuropathic pain. Furthermore, the function of presynaptic GABA_B receptors at primary afferent terminals, but not that on GABAergic and glycinergic interneurons, in the spinal cord is reduced in diabetic neuropathy.     

Codeine presynaptically inhibits the glutamatergic synaptic transmission in the nucleus tractus solitarius of the guinea pig

Although codeine is the most prominent and centrally acting antitussive agent, the precise sites and mode of its action have not been fully understood yet. In the present study, we examined the effects of codeine on synaptic transmission in second-order neurons of the nucleus tractus solitarius (NTS), which is the first central relay site receiving tussigenic afferent fibers, by using whole-cell patch-clamp recordings in guinea-pig brainstem slices. Codeine (0.3-3 mM) significantly decreased the amplitude of excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation of the tractus solitarius in a naloxone-reversible and concentration-dependent manner, but it had no effect on the decay time of evoked EPSCs (eEPSCs). The inhibition of eEPSCs was accompanied by an increased paired-pulse ratio of two consecutive eEPSCs. The inward current induced by application of AMPA remained unchanged after codeine application. A voltage-sensitive K+ channel blocker, 4-aminopyridine (4-AP) attenuated the inhibitory effect of codeine on eEPSCs. These results suggest that codeine inhibits excitatory transmission from the primary afferent fibers to the second-order NTS neurons through the opioid receptors that activate the 4-AP sensitive K+ channels located at presynaptic terminals.     

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.