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.     

Mechanisms of cannabinoid-receptor-mediated inhibition of synaptic transmission in cultured hippocampal pyramidal neurons.

Cannabinoids, such as marijuana, are known to impair learning and memory perhaps through their actions in the hippocampus where cannabinoid receptors are expressed at high density. Although cannabinoid receptor activation decreases glutamatergic synaptic transmission in cultured hippocampal neurons, the mechanisms of this action are not known. Cannabinoid receptor activation also inhibits calcium channels that support neurotransmitter release in these cells, making modulation of these channels a candidate for cannabinoid-receptor-mediated effects on synaptic transmission. Whole cell patch-clamp recordings of glutamatergic neurons cultured from the CA1 and CA3 regions of the hippocampus were used to identify the mechanisms of the effects of cannabinoids on synaptic transmission. Cannabinoid receptor activation reduced excitatory postsynaptic current (EPSC) size by approximately 50% but had no effect on the amplitude of spontaneous miniature EPSCs (mEPSCs). This reduction in EPSC size was accompanied by an increase in paired-pulse facilitation measured in low (1 mM) extracellular calcium and by a decrease in paired-pulse depression measured in normal (2.5 mM) extracellular calcium. Together, these results strongly support the hypothesis that cannabinoid receptor activation decreases EPSC size by reducing release of neurotransmitter presynaptically while having no effect on postsynaptic sensitivity to glutamate. Further experiments were done to identify the molecular mechanisms underlying this cannabinoid-receptor-mediated decrease in neurotransmitter release. Cannabinoid receptor activation had no effect on the size of the presynaptic pool of readily releasable neurotransmitter-filled vesicles, eliminating reduction in pool size as a mechanism for cannabinoid-receptor-mediated effects. After blockade of Q- and N-type calcium channels with omega-agatoxin TK and omega-conotoxin GVIA; however, activation of cannabinoid receptors reduced EPSC size by only 14%. These results indicate that cannabinoid receptor activation reduces the probability that neurotransmitter will be released in response to an action potential via an inhibition of presynaptic Q- and N-type calcium channels. This molecular mechanism most likely contributes to the impairment of learning and memory produced by cannabinoids and may participate in the analgesic, antiemetic, and anticonvulsive effects of these drugs as well.     

Nociceptin/orphanin FQ (N/OFQ) inhibits excitatory and inhibitory synaptic signaling in the suprachiasmatic nucleus (SCN)

Environmental synchronization of the endogenous mammalian circadian rhythm involves glutamatergic and GABAergic neurotransmission within the hypothalamic suprachiasmatic nucleus (SCN). The neuropeptide nociceptin/orphanin FQ (N/OFQ) inhibits light-induced phase shifts, evokes K(+)-currents and reduces the intracellular Ca(2+) concentration in SCN neurons. Since these effects are consistent with a modulatory role for N/OFQ on synaptic transmission in the SCN, we examined the effects of N/OFQ on evoked and spontaneous excitatory and inhibitory currents in the SCN. N/OFQ produced a consistent concentration-dependent inhibition of glutamate-mediated excitatory postsynaptic currents (EPSC) evoked by optic nerve stimulation. N/OFQ did not alter the amplitude of currents induced by application of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-d-aspartate (NMDA) nor the amplitude of miniature EPSC (mEPSC) consistent with a lack of N/OFQ effect on postsynaptic AMPA or NMDA receptors. N/OFQ significantly reduced the mEPSC frequency. The inhibitory actions of N/OFQ were blocked by omega-conotoxin GVIA, an N-type Ca(2+)channel antagonist and partially blocked by omega-agatoxin TK, a P/Q type Ca(2+) channel blocker. These data indicate that N/OFQ reduces evoked EPSC, in part, by inhibiting the activity of N- and P/Q-type Ca(2+) channels. In addition, N/OFQ produced a consistent reduction in baseline Ca(2+) levels in presynaptic retinohypothalamic tract terminals. N/OFQ also inhibited evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSC) in a concentration dependent manner. However, N/OFQ had no effect on currents activated by muscimol application or on the amplitude of miniature IPSC (mIPSC) and significantly reduced the mIPSC frequency consistent with an inhibition of GABA release downstream from Ca(2+) entry. Finally, N/OFQ inhibited the paired-pulse depression observed in SCN GABAergic synapses consistent with a presynaptic mechanism of action. Together these results suggest a widespread modulatory role for N/OFQ on the synaptic transmission in the SCN.     

Mu-opioid-mediated inhibition of glutamate synaptic transmission in rat central amygdala neurons

The central nucleus of the amygdala (CeA) plays an important role both in stimulus-reward learning for the reinforcing effects of drugs of abuse and in environmental condition-induced analgesia. Both of these two CeA functions involve the opioid system within the CeA. However, the pharmacological profiles of its opioid receptor system have not been fully studied and the synaptic actions of opioid receptors in the CeA are largely unknown. In this study with whole-cell voltage-clamp recordings in brain slices in vitro, we examined actions of opioid agonists on glutamate-mediated excitatory postsynaptic currents (EPSCs) in CeA neurons. Opioid peptide methionine-enkephalin (ME; 10 microM) produced a significant inhibition (38%) in the amplitude of evoked EPSCs, an action mimicked by the mu-opioid receptor agonist [D-Ala(2),N-MePhe(4),Gly-ol(5)]-enkephalin (DAMGO; 1 microM, 44%). Both effects of ME and DAMGO were abolished by the mu receptor antagonist CTAP (1 microM), suggesting a mu receptor-mediated effect. Neither delta-opioid receptor agonist [D-Pen(2),D-Pen(5)]-enkephalin (1 microM) nor kappa-opioid receptor agonist U69593 (300 nM) had any effect on the glutamate EPSC. ME significantly increased the paired-pulse ratio of the evoked EPSCs and decreased the frequency of miniature EPSCs without altering the amplitude of miniature EPSCs. Furthermore, the mu-opioid inhibition of the EPSC was blocked by 4-aminopyridine (4AP; 100 microM), a voltage-dependent potassium channel blocker, and by phospholipase A(2) inhibitors AACOCF(3) (10 microM) and quinacrine (10 microM). These results indicate that only the mu-opioid receptor is functionally present on presynaptic glutamatergic terminals in normal CeA neurons, and its activation reduces the probability of glutamate release through a signaling pathway involving phospholipase A(2) and the presynaptic, 4AP-sensitive potassium channel. This study provides evidence for the presynaptic regulation of glutamate synaptic transmission by mu-opioid receptors in CeA neurons.     

Cannabinoid-induced presynaptic inhibition at the primary afferent trigeminal synapse of juvenile rat brainstem slices

Systemic or intraventricular administration of cannabinoids causes analgesic effects, but relatively little is known for their cellular mechanism. Using brainstem slices with the mandibular nerve attached, we examined the effect of cannabinoids on glutamatergic transmission in superficial trigeminal caudal nucleus of juvenile rats. The exogenous cannabinoid receptor agonist WIN 55,212-2 (WIN), as well as the endogenous agonist anandamide, hyperpolarized trigeminal caudal neurones and depressed the amplitude of excitatory postsynaptic potentials (EPSPs) or currents (EPSCs) monosynaptically evoked by stimulating mandibular nerves in a concentration-dependent manner. The inhibitory action of WIN was blocked or fully reversed by the CB₁ receptor antagonist SR 141716A. WIN had no effect on the amplitude of miniature excitatory postsynaptic currents (mEPSCs) recorded in the presence of tetrodotoxin or cadmium. The inhibitory effect of WIN on EPSCs was greater for those with longer synaptic latency, suggesting that cannabinoids have a stronger effect on C-fibre EPSPs than on Aδ-fibre EPSPs. Ba²⁺ (100 μ m ) blocked the hyperpolarizing effect of cannabinoids, but did not affect their inhibitory effect on EPSPs. The N-type Ca²⁺ channel blocker ω-conotoxin GVIA (ω-CgTX) occluded the WIN-mediated presynaptic inhibition, whereas the P/Q-type Ca²⁺ channel blocker ω-agatoxin TK (ω-Aga) had no effect. These results suggest that cannabinoids preferentially activate CB₁ receptors at the nerve terminal of small-diameter primary afferent fibres. Upon activation, CB₁ receptors may selectively inhibit presynaptic N-type Ca²⁺ channels and exocytotic release machinery, thereby attenuating the transmitter release at the trigeminal nociceptive synapses.     

Kappa opioid receptor activation in the nucleus accumbens inhibits glutamate and GABA release through different mechanisms

Through their actions in the nucleus accumbens (NAc), kappa opioid (KOP) receptors and their endogenous ligand, dynorphin, modify behaviors associated with the administration of drugs of abuse and are regulated by exposure to such drugs. Despite their demonstrated behavioral significance, the synaptic actions of KOP receptor ligands in the NAc are not clearly understood. Using whole-cell voltage-clamp recordings of NAc medium spiny neurons, we have found that, in addition to suppressing glutamate release, the KOP receptor agonist also inhibits GABA release. Interestingly, the mechanism of inhibition of the release of glutamate differs from that controlling GABA. reduces the frequency of Ca(2+)-independent miniature excitatory postsynaptic currents, but not miniature inhibitory postsynaptic currents. Furthermore, while the inhibition of GABAergic transmission is blocked by the N-type Ca(2+) channel blocker omega-CgTx, the inhibition of excitatory glutamatergic transmission by is unaffected by N-type Ca(2+) channel blockade. These results indicate that KOP receptor activation inhibits GABA release by reducing Ca(2+) influx, but inhibits glutamate release at a step downstream of Ca(2+) entry.     

Muscarinic and nicotinic presynaptic modulation of EPSCs in the nucleus accumbens during postnatal development

We have studied the modulatory effects of cholinergic agonists on excitatory postsynaptic currents (EPSCs) in nucleus accumbens (nAcb) neurons during postnatal development. Recordings were obtained in slices from postnatal day 1 (P1) to P27 rats using the whole cell patch-clamp technique. EPSCs were evoked by local electrical stimulation, and all experiments were conducted in the presence of bicuculline methchloride in the bathing medium and with QX-314 in the recording pipette. Under these conditions, postsynaptic currents consisted of glutamatergic EPSCs typically consisting of two components mediated by AMPA/kainate (KA) and N-methyl-D-aspartate (NMDA) receptors. The addition of acetylcholine (ACh) or carbachol (CCh) to the superfusing medium resulted in a decrease of 30-60% of both AMPA/KA- and NMDA-mediated EPSCs. In contrast, ACh produced an increase ( approximately 35%) in both AMPA/KA and NMDA receptor-mediated EPSCs when administered in the presence of the muscarinic antagonist atropine. These excitatory effects were mimicked by the nicotinic receptor agonist 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) and blocked by the nicotinic receptor antagonist mecamylamine, showing the presence of a cholinergic modulation mediated by nicotinic receptors in the nAcb. The antagonistic effects of atropine were mimicked by pirenzepine, suggesting that the muscarinic depression of the EPSCs was mediated by M(1)/M(4) receptors. In addition, the inhibitory effects of ACh on NMDA but not on AMPA/KA receptor-mediated EPSC significantly increased during the first two postnatal weeks. We found that, under our experimental conditions, cholinergic agonists produced no changes on membrane holding currents, on the decay time of the AMPA/KA EPSC, or on responses evoked by exogenous application of glutamate in the presence of tetrodotoxin, but they produced significant changes in paired pulse ratio, suggesting that their action was mediated by presynaptic mechanisms. In contrast, CCh produced consistent changes in the membrane and firing properties of medium spiny (MS) neurons when QX-314 was omitted from the recording pipette solution, suggesting that this substance actually blocked postsynaptic cholinergic modulation. Together, these results suggest that ACh can decrease or increase glutamatergic neurotransmission in the nAcb by, respectively, acting on muscarinic and nicotinic receptors located on excitatory terminals. The cholinergic modulation of AMPA/KA and NMDA receptor-mediated neurotransmission in the nAcb during postnatal development could play an important role in activity-dependent developmental processes in refining the excitatory drive on MS neurons by gating specific inputs.     

Glial glutamate transporter 1 regulates the spatial and temporal coding of glutamatergic synaptic transmission in spinal lamina II neurons

Glutamatergic synaptic transmission is a dynamic process determined by the amount of glutamate released by presynaptic sites, the clearance of glutamate in the synaptic cleft, and the properties of postsynaptic glutamate receptors. Clearance of glutamate in the synaptic cleft depends on passive diffusion and active uptake by glutamate transporters. In this study, we examined the role of glial glutamate transporter 1 (GLT-1) in spinal sensory processing. Excitatory postsynaptic currents (EPSCs) of substantia gelatinosa neurons recorded from spinal slices of young adult rats were analyzed before and after GLT-1 was pharmacologically blocked by dihydrokainic acid. Inhibition of GLT-1 prolonged the EPSC duration and the EPSC decay phase. The EPSC amplitudes were increased in neurons with weak synaptic input but decreased in neurons with strong synaptic input upon inhibition of GLT-1. We suggest that presynaptic inhibition, desensitization of postsynaptic AMPA receptors, and glutamate "spillover" contributed to the kinetic change of EPSCs induced by the blockade of GLT-1. Thus, GLT-1 is a key component in maintaining the spatial and temporal coding in signal transmission at the glutamatergic synapse in substantia gelatinosa neurons.     

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.     

Group II metabotropic glutamate receptor modulation of excitatory transmission in rat subthalamic nucleus

Patch pipettes were used to record currents in whole-cell configuration to study the effects of group II metabotropic glutamate receptor (mGluR) stimulation on synaptic transmission in slices of rat subthalamic nucleus. Evoked glutamatergic excitatory postsynaptic currents (EPSCs) were reversibly reduced by the selective group II mGluR agonist (2' S ,2' R ,3' R )-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) in a concentration-dependent manner, with an IC₅₀ of 0.19 ± 0.05 µ m . DCG IV (1 µ m ) had no effect on inhibitory postsynaptic currents mediated by GABA. DCG IV-induced inhibition of EPSCs was reversed by the selective group II mGluR antagonist LY 341495 (100 n m ) and mimicked by another selective group II agonist (2 S ,1' S ,2' S )-2-(carboxycyclopropyl)glycine ( l -CCG-I). Inhibition of EPSC amplitude by DCG IV and l -CCG-I was associated with an increase in the paired-pulse ratio of EPSCs. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (2 µ m ) reduced the inhibitory effect of DCG IV on EPSCs. However, the response to DCG IV was not affected by the protein kinase A (PKA) activator forskolin (20 µ m ), by the adenylyl cyclase inhibitor MDL 12230A (20 µ m ), or by the phosphodiesterase inhibitor Ro 20–1724 (50 µ m ). DCG IV-induced inhibition of EPSCs was reduced by the non-selective protein kinase inhibitors H-7 (100 µ m ), H-8 (50 µ m ) and HA-1004 (100 µ m ). These results suggest that group II mGluR stimulation acts presynaptically to inhibit glutamate release by a PKC-dependent mechanism in the subthalamic nucleus.     

Selective modulation of excitatory transmission by mu-opioid receptor activation in rat supraoptic neurons

Opioid peptides have profound inhibitory effects on the production of oxytocin and vasopressin, but their direct effects on magnocellular neuroendocrine neurons appear to be relatively weak. We tested whether a presynaptic mechanism is involved in this inhibition. The effects of mu-opioid receptor agonist D-Ala(2), N-CH(3)-Phe(4), Gly(5)-ol-enkephalin (DAGO) on excitatory and inhibitory transmission were studied in supraoptic nucleus (SON) neurons from rat hypothalamic slices using whole cell recording. DAGO reduced the amplitude of evoked glutamatergic excitatory postsynaptic currents (EPSCs) in a dose-dependent manner. In the presence of tetrodotoxin (TTX) to block spike activity, DAGO also reduced the frequency of spontaneous miniature EPSCs without altering their amplitude distribution, rising time, or decaying time constant. The above effects of DAGO were reversed by wash out, or by addition of opioid receptor antagonist naloxone or selective mu-antagonist Cys(2)-Tyr(3)-Orn(5)-Pen(7)-NH(2) (CTOP). In contrast, DAGO had no significant effect on the evoked and spontaneous miniature GABAergic inhibitory postsynaptic currents (IPSCs) in most SON neurons. A direct membrane hyperpolarization of SON neurons was not detected in the presence of DAGO. These results indicate that mu-opioid receptor activation selectively inhibits excitatory activity in SON neurons via a presynaptic mechanism.     

Postnatal development of excitatory postsynaptic currents in nucleus accumbens medium spiny neurons

We have recorded excitatory postsynaptic currents (EPSCs) evoked by local electrical stimulation in 243 nucleus accumbens (nAcb) neurons in vitro during postnatal development from the day of birth (postnatal day 0; P0) to P27 and in young adults rats (P59-P71). An EPSC sensitive to glutamatergic antagonists was found in all neurons. In the majority of cases (189/243), the EPSC had two distinct components: an early one sensitive to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and a late one that was sensitive to d-2-amino-5-phosphonovaleric acid (APV) showing that early and late components of the EPSC were mediated by AMPA/kainate (KA) and N-methyl-d-aspartate (NMDA) receptors respectively. During the first four postnatal days, the amplitudes of both the AMPA/KA and NMDA components of the EPSC were relatively small and then began to increase until the end of the second postnatal week. Whereas the amplitude of the early component appeared to stabilize from that point on, the late component began to decrease and became virtually undetectable in preparations from animals older than 3 weeks unless the AMPA/KA response was blocked with CNQX. In addition, the ratio between the amplitude of the NMDA and AMPA/KA receptor-mediated components of the EPSC followed a developmental pattern parallel to that of the NMDA receptor component showing an increase during the first two postnatal weeks followed by a decrease. Together, these results show that, during postnatal development, there is a period when NMDA receptor-mediated EPSC are preeminent and that time frame might represent a period during which the development of the nAcb might be sensitive to environmental manipulation.     

Subthalamic stimulation evokes complex EPSCs in the rat substantia nigra pars reticulata in vitro

The subthalamic nucleus (STN) plays an important role in movement control by exerting its excitatory influence on the substantia nigra pars reticulata (SNR), a major output structure of the basal ganglia. Moreover, excessive burst firing of SNR neurons seen in Parkinson's disease has been attributed to excessive transmission in the subthalamonigral pathway. Using the 'blind' whole-cell patch clamp recording technique in rat brain slices, we found that focal electrical stimulation of the STN evoked complex, long-duration excitatory postsynaptic currents (EPSCs) in SNR neurons. Complex EPSCs lasted 200–500 ms and consisted of an initial monosynaptic EPSC followed by a series of late EPSCs superimposed on a slow inward shift in holding current. Focal stimulation of regions outside the STN failed to evoke complex EPSCs. The late component of complex EPSCs was markedly reduced by ionotropic glutamate receptor antagonists (2-amino-5-phosphonopentanoic acid and 6-cyano-7-nitro-quinoxalone) and by a GABA_A receptor agonist (isoguvacine) when these agents were applied directly to the STN using a fast-flow microapplicator. Moreover, the complex EPSC was greatly enhanced by bath application of the GABA_A receptor antagonists picrotoxin or bicuculline. These data suggest that recurrent glutamate synapses in the STN generate polysynaptic, complex EPSCs that are under tonic inhibition by GABA. Because complex EPSCs are expected to generate bursts of action potentials in SNR neurons, we suggest that complex EPSCs may contribute to the pathological burst firing that is associated with the symptoms of Parkinson's disease.     

5-HT2 receptors modulate excitatory neurotransmission to cardiac vagal neurons within the nucleus ambiguus evoked during and after hypoxia

To examine the role of 5-HT2 receptors in the central cardiorespiratory network, and in particular the respiratory modulation of parasympathetic activity to the heart, we used an in vitro medullary slice that allowed simultaneous examination of rhythmic inspiratory-related activity recorded from hypoglossal rootlet and excitatory inspiratory-related neurotransmission to cardioinhibitory vagal neurons (CVNs) within the nucleus ambiguus (NA). Focal application of ketanserin, a 5-HT2 receptor antagonist, did not significantly alter the frequency of spontaneous excitatory postsynaptic excitatory currents (EPSCs) in CVNs in control conditions. However, ketanserin diminished spontaneous excitatory neurotransmission to CVNs during hypoxia. The inhibitory action of ketanserin was on 5-HT3 mediated EPSCs during hypoxia since these responses were blocked by the 5-HT3 receptor antagonist ondansetron. In addition, a robust inspiratory-related excitatory neurotransmission was recruited during recovery from hypoxia. Focal application of ketanserin during this posthypoxia period evoked a significant augmentation of the frequency of inspiratory-related, but not spontaneous EPSCs in CVNs. This excitatory effect of ketanserin was prevented by application of the purinergic receptor blocker pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS). These results demonstrate 5-HT2 receptors differentially modulate excitatory neurotransmission to CVNs during and after hypoxia. Activation of 5-HT2 receptors acts to maintain excitatory neurotransmission to CVNs during hypoxia, likely via presynaptic facilitation of 5-HT3 receptor-mediated neurotransmission to CVNs. However, activation of 5HT2 receptors diminishes the subsequent inspiratory-related excitatory neurotransmission to CVNs that is recruited during the recovery from hypoxia likely exerting an inhibitory action on inspiratory-related purinergic signaling.     

Kappa opioid receptor inhibition of glutamatergic transmission in the nucleus accumbens shell

Microinjection of kappa-opioid receptor agonists into the nucleus accumbens produces conditioned place aversion. While attention has focused primarily on the inhibition of dopamine release by kappa-receptor agonists as the synaptic mechanism underlying this effect, recent anatomical studies have raised the possibility that regulation of noncatecholaminergic transmission also contribute. We have investigated the effects of kappa-receptor activation on fast excitatory synaptic transmission in an in vitro slice preparation using whole cell voltage-clamp or extracellular field recordings in the shell region of the nucleus accumbens. The kappa-receptor agonist U69593 produces a pronounced, dose-dependent inhibition of glutamatergic excitatory postsynaptic currents (EPSCs) that can be reversed by 100 nM nor-BNI. Furthermore, U69593 causes an increase in the paired-pulse ratio as well as a decrease in the frequency of spontaneous miniature events, suggesting a presynaptic site of action. Despite anatomical evidence for kappa-receptor localization on dendritic spines of nucleus accumbens neurons, no electrophysiological evidence of a postsynaptic effect was found. This presynaptic inhibition of excitatory synaptic transmission in the nucleus accumbens shell provides a novel mechanism that may contribute to the kappa-receptor-mediated aversion observed in intact animals.     

Spontaneous synaptic activity in chick vestibular nucleus neurons during the perinatal period

The principal cells of the chick tangential nucleus are second-order vestibular neurons involved in the vestibuloocular and vestibulocollic reflexes. The spontaneous synaptic activity of morphologically identified principal cells was characterized in brain slices from 1-day-old hatchlings (H1) using whole-cell voltage-clamp recordings and Cs-gluconate pipet solution. The frequency was 1.45 Hz for spontaneous excitatory postsynaptic currents (sEPSCs) and 1.47 Hz for spontaneous inhibitory postsynaptic currents (sIPSCs). Using specific neurotransmitter receptor antagonists, all of the sEPSCs were identified as AMPA receptor-mediated events, whereas 56% of the sIPSCs were glycine and 44% were GABA(A) receptor-mediated events. On exposure to TTX, the frequency of EPSCs decreased by 68%, while the frequency of IPSCs decreased by 33%, indicating greater EPSC dependency on presynaptic action potentials. These data on spontaneous synaptic activity at H1 were compared with those obtained in previous studies of 16-day old embryos (E16). After birth, the spontaneous synaptic activity exhibited increased EPSC frequency, increased ratio for excitatory to inhibitory events, increased percentage of TTX-dependent EPSCs, and faster kinetics. In addition, the ratio for glycine/GABA receptor-mediated events increased significantly. Altogether, these data indicate that at hatching spontaneous synaptic activity of vestibular nucleus neurons in brain slices of the chick tangential nucleus undergoes appreciable changes, with increased frequency of EPSCs and glycinergic activity playing more important roles compared with the late-term chick embryo when GABAergic activity prevailed. The definition of this developmental pattern of synaptic activity in vestibular nucleus neurons should contribute to understanding how vestibular reflex activity is established in the hatchling chick.     

Effects of corticosteroids on synaptic transmission in rat dorsolateral septal nucleus

The effects of corticosteroids on synaptic transmission in the rat dorsolateral septal nucleus (DLSN) were examined, in vitro, by using intracellular and voltage-clamp recording methods. Prednisolone (100 microM) increased the amplitude of excitatory postsynaptic potential (EPSP) and depressed both fast and slow inhibitory postsynaptic potentials (IPSP). Under voltage-clamp conditions, prednisolone (100 microM) increased the amplitude of excitatory postsynaptic current (EPSC) and depressed the fast and slow inhibitory postsynaptic currents (IPSCs). Corticosterone (100 microM) mimicked the effects of prednisolone on the postsynaptic currents (PSCs). To examine the direct effects of prednisolone on the EPSC and slow IPSC, the fast IPSC was blocked by bicuculline (20 microM). Under these experimental conditions, prednisolone (100 microM) did not alter the isolated EPSC but depressed slow IPSC by 22 +/- 3% (n = 10). The fast IPSC was isolated by pretreatment with kynurenic acid and CGP55845A, where the EPSC and slow IPSC were blocked. Prednisolone (100 microM) depressed the isolated fast IPSC in DLSN neurons. Prednisolone (100 microM) did not change either the inward current produced by glutamate or the outward current produced by gamma-aminobutyric acid (GABA). The results suggest that corticosteroids facilitate excitatory synaptic transmission in the DLSN by reducing the release of GABA from the presynaptic nerve terminals of interneurons.     

Cannabinoid-induced presynaptic inhibition of glutamatergic EPSCs in substantia gelatinosa neurons of the rat spinal cord.

The effect of cannabinoids on excitatory transmission in the substantia gelatinosa was investigated using intracellular recording from visually identified neurons in a transverse slice preparation of the juvenile rat spinal cord. In the presence of strychnine and bicuculline, perfusion of the cannabinoid receptor agonist WIN55,212-2 reduced the frequency and the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs). Furthermore, the frequency of miniature EPSCs (mEPSCs) was also decreased by WIN55,212-2, whereas their amplitude was not affected. Similar effects were reproduced using the endogenous cannabinoid ligand anandamide. The effects of both agonists were blocked by the selective CB(1) receptor antagonist SR141716A. Electrical stimulation of high-threshold fibers in the dorsal root evoked a monosynaptic EPSC in lamina II neurons. In the presence of WIN55,212-2, the amplitude of the evoked EPSC (eEPSCs) was reduced, and the paired-pulse ratio was increased. The reduction of the eEPSC following CB(1) receptor activation was unlikely to have a postsynaptic origin because the response to AMPA, in the presence of 1 microM TTX, was unchanged. To investigate the specificity of this synaptic inhibition, we selectively activated the nociceptive C fibers with capsaicin, which induced a strong increase in the frequency of EPSCs. In the presence of WIN55,212-2, the response to capsaicin was diminished. In conclusion, these results strongly suggest a presynaptic location for CB(1) receptors whose activation results in inhibition of glutamate release in the spinal dorsal horn. The strong inhibitory effect of cannabinoids on C fibers may thereby contribute to the modulation of the spinal excitatory transmission, thus producing analgesia at the spinal level.     

Subthalamic nucleus evokes similar long lasting glutamatergic excitations in pallidal,entopeduncular and nigral neurons in the basal ganglia slice

The subthalamic nucleus (STN) modulates the activity of globus pallidus (GP), entopeduncular nucleus (EP) and substantia nigra pars reticulata (SNr) neurons via its direct glutamatergic projections. To investigate the mechanism by which STN affects activity in these structures and whether STN induced activity is comparable among STN target neurons, we performed patch clamp recordings in a tilted, parasagittal, basal ganglia slice (BGS) that preserves these functional connections. We report that single, brief stimulation of the STN generates a brief monosynaptic AMPA-mediated excitatory postsynaptic current (EPSC) in GP, EP and SNr neurons. A higher intensity, supra-threshold activation evokes a compound EPSC consisting of an early monosynaptic component followed by a slow inward NMDA-mediated current with an overlying barrage of AMPA-mediated EPSCs. These late EPSCs were polysynaptic and gave rise to bursts of spikes that lasted several hundreds of milliseconds. They were eliminated by surgical removal of the STN from the BGS slice, indicating that the STN is required for their generation. Reconstruction of biocytin-filled STN neurons revealed that a third of STN neurons project intra-STN axon collaterals that may underlie polysynaptic activity. We propose that activation of the STN yields comparable long lasting excitations in its target neurons by means of a polysynaptic network.     

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.