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.     

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.     

Presynaptic angiotensin II AT1 receptors enhance inhibitory and excitatory synaptic neurotransmission to motoneurons and other ventral horn neurons in neonatal rat spinal cord

In neonatal spinal cord, we previously reported that exogenous angiotensin II (ANG II) acts at postsynaptic AT1 receptors to depolarize neonatal rat spinal ventral horn neurons in vitro. This study evaluated an associated increase in synaptic activity. Patch clamp recordings revealed that 38/81 thoracolumbar (T7-L5) motoneurons responded to bath applied ANG II (0.3-1 microM; 30 s) with a prolonged (5-10 min) and reversible increase in spontaneous postsynaptic activity, selectively blockable with Losartan (n = 5) but not PD123319 (n = 5). ANG-II-induced events included both spontaneous inhibitory (IPSCs; n = 6) and excitatory postsynaptic currents (EPSCs; n = 5). While most ANG induced events were tetrodotoxin-sensitive, ANG induced a significant tetrodotoxin-resistant increase in frequency but not amplitude of miniature IPSCs (n = 7/13 cells) and EPSCs (n = 2/7 cells). In 35/77 unidentified neurons, ANG II also induced a tetrodotoxin-sensitive and prolonged increase in their spontaneous synaptic activity that featured both IPSCs (n = 5) and EPSCs (n = 4) when tested in the presence of selective amino acid receptor antagonists. When tested in the presence of tetrodotoxin, ANG II was noted to induce a significant increase in the frequency but not the amplitude of mIPSCs (n = 9) and mEPSCs (n = 8). ANG also increased spontaneous motor activity from isolated mouse lumbar ventral rootlets. Collectively, these observations support the existence of a wide pre- and postsynaptic distribution of ANG II AT1 receptors in neonatal ventral spinal cord that are capable of influencing both inhibitory and excitatory neurotransmission.     

Synaptic activity in chronically injured,epileptogenic sensory-motor neocortex

We recorded spontaneous and evoked synaptic currents in pyramidal neurons of layer V in chronically injured, epileptogenic neocortex to assess changes in the efficacy of excitatory and inhibitory neurotransmission that might promote cortical hyperexcitability. Partial sensory-motor neocortical isolations with intact blood supply ("undercuts") were made in 20 rats on postnatal day 21-25 and examined 2-6 wk later in standard brain slice preparations using whole cell patch-clamp techniques. Age-matched, uninjured naive rats (n = 20) were used as controls. Spontaneous and miniature excitatory and inhibitory postsynaptic currents (s- and mEPSCs; s- and mIPSCs) were recorded using patch-clamp techniques. The average frequency of s- and mEPSCs was significantly higher, while that of s- and mIPSCs was significantly lower in neurons of undercuts versus controls. The increased frequency of excitatory events was due to an increase in both s- and mEPSC frequency, suggesting an increased number of excitatory contacts and/or increased release probability at excitatory terminals. No significant difference was observed in 10-90% rise time of these events. The input-output slopes of fast, short-latency, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate (AMPA/KA) receptor-mediated components of evoked EPSCs were steeper in undercuts than in controls. The peak amplitude of the AMPA/KA component of EPSCs evoked by supra-threshold stimuli was significantly greater in the partially isolated neocortex. In contrast, the N-methyl-D-aspartate receptor-mediated component of evoked EPSCs was not significantly different in neurons of injured versus control cortex, suggesting that the increased AMPA/KA component was due to postsynaptic alterations. Results support the conclusion that layer V pyramidal neurons receive increased AMPA/KA receptor-mediated excitatory synaptic drive and decreased GABA(A) receptor-mediated inhibition in this chronically injured, epileptogenic cortex. This shift in the balance of excitatory and inhibitory synaptic activation of layer V pyramidal cells toward excitation might be maladaptive and play a critical role in epileptogenesis.     

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.     

Activation of group III metabotropic glutamate receptors presynaptically reduces both GABAergic and glutamatergic transmission in the rat globus pallidus

To investigate the role of group III metabotropic glutamate receptors (mGluRs) in the globus pallidus (GP), whole-cell recordings were performed using rat brain slice preparations. Application of the group III mGluRs specific agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) suppressed the amplitude of striatal stimulation-induced IPSCs and internal capsule stimulation-induced EPSCs in most of the GP neurons that were capable of generating repetitive firing without spike accommodation. The suppression of IPSCs and EPSCs was accompanied by an increase in the paired-pulse ratio. The L-AP4 effects were antagonized by (R,S)-alpha-cyclopropyl-4-phosphophenylglycine, a blocker for group II/III mGluRs. L-AP4 reduced the frequency of mIPSCs and mEPSCs without changing their amplitude distribution. L-AP4 failed to change iontophoretic glutamate induced responses. These results suggest that the subthalamo-pallidal glutamatergic input might homo- and hetero-synaptically control GABAergic and glutamatergic transmission in the GP.     

Reduction of GABAA receptor-mediated inhibition by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione in cultured neurons of rat brain.

The action of the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on gamma-aminobutyric acid-A (GABAA) receptor-mediated currents was studied in dissociated rat midbrain and hypothalamic cultures using whole-cell recording. Spontaneous synaptic activity consisted of excitatory (EPSCs) and inhibitory postsynaptic currents (IPSCs). Bicuculline (20 microM) blocked IPSCs and increased the frequency of EPSCs. CNQX (1 microM) reduced both EPSCs and IPSCs. In the presence of 0.3 microM tetrodotoxin (TTX), CNQX (1-20 microM) blocked miniature EPSCs and reduced IPSCs. In TTX, increasing K+ (20 mM) evoked EPSCs and IPSCs in a Ca-dependent manner. CNQX (10 microM) blocked evoked EPSCs and diminished evoked IPSCs similarly as miniature IPSCs. Muscimol-(0.2-5 microM) induced currents were dose-dependently reduced by CNQX (10-50 microM). It is concluded that CNQX reduces GABAA receptor-mediated inhibition primarily by reducing the excitatory drive in the evolving network, but, in addition, has a significant blocking effect on the GABAA receptor-channel complex.     

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.     

Action-potential-independent GABAergic tone mediated by nicotinic stimulation of immature striatal miniature synaptic transmission

Stimulation of presynaptic nicotinic acetylcholine receptors (nAChRs) increases the frequency of miniature excitatory synaptic activity (mEPSCs) to a point where they can promote cell firing in hippocampal CA3 neurons. We have evaluated whether nicotine regulation of miniature synaptic activity can be extended to inhibitory transmission onto striatal medium spiny projection neurons (MSNs) in acute brain slices. Bath application of micromolar nicotine typically induced 12-fold increases in the frequency of miniature inhibitory synaptic currents (mIPSCs). Little effect was observed on the amplitude of mIPSCs or mEPSCs under these conditions. Nicotine stimulation of mIPSCs was dependent on entry of extracellular calcium because removal of calcium from perfusate was able to block its action. To assess the potential physiological significance of the nicotine-stimulated increase in mIPSC frequency, we also examined the nicotine effect on evoked IPSCs (eIPSCs). eIPSCs were markedly attenuated by nicotine. This effect could be attributed to two potential mechanisms: transmitter depletion due to extremely high mIPSC rates and/or a reduction in presynaptic excitability associated with nicotinic depolarization. Treatment with low concentrations of K(+) was able to in part mimic nicotine's stimulatory effect on mIPSCs and inhibitory effect on eIPSCs. Current-clamp recordings confirmed a direct depolarizing action of nicotine that could dampen eIPSC activity leading to a switch to striatal inhibitory synaptic transmission mediated by tonic mIPSCs.     

Excitatory and inhibitory postsynaptic currents in a rat model of epileptogenic microgyria

Developmental cortical malformations are common in patients with intractable epilepsy; however, mechanisms contributing to this epileptogenesis are currently poorly understood. We previously characterized hyperexcitability in a rat model that mimics the histopathology of human 4-layered microgyria. Here we examined inhibitory and excitatory postsynaptic currents in this model to identify functional alterations that might contribute to epileptogenesis associated with microgyria. We recorded isolated whole cell excitatory postsynaptic currents and GABA(A) receptor-mediated inhibitory currents (EPSCs and IPSCs) from layer V pyramidal neurons in the region previously shown to be epileptogenic (paramicrogyral area) and in homotopic control cortex. Epileptiform-like activity could be evoked in 60% of paramicrogyral (PMG) cells by local stimulation. The peak conductance of both spontaneous and evoked IPSCs was significantly larger in all PMG cells compared with controls. This difference in amplitude was not present after blockade of ionotropic glutamatergic currents or for miniature (m)IPSCs, suggesting that it was due to the excitatory afferent activity driving inhibitory neurons. This conclusion was supported by the finding that glutamate receptor antagonist application resulted in a significantly greater reduction in spontaneous IPSC frequency in one PMG cell group (PMG(E)) compared with control cells. The frequency of both spontaneous and miniature EPSCs was significantly greater in all PMG cells, suggesting that pyramidal neurons adjacent to a microgyrus receive more excitatory input than do those in control cortex. These findings suggest that there is an increase in numbers of functional excitatory synapses on both interneurons and pyramidal cells in the PMG cortex perhaps due to hyperinnervation by cortical afferents originally destined for the microgyrus proper.     

Transient enhancement of inhibitory synaptic transmission in hippocampal CA1 pyramidal neurons after cerebral ischemia

Pyramidal neurons in hippocampal CA1 regions are highly sensitive to cerebral ischemia. Alterations of excitatory and inhibitory synaptic transmission may contribute to the ischemia-induced neuronal degeneration. However, little is known about the changes of GABAergic synaptic transmission in the hippocampus following reperfusion. We examined the GABA_A receptor-mediated inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal neurons 12 and 24 h after transient forebrain ischemia in rats. The amplitudes of evoked inhibitory postsynaptic currents (eIPSCs) were increased significantly 12 h after ischemia and returned to control levels 24 h following reperfusion. The potentiation of eIPSCs was accompanied by an increase of miniature inhibitory postsynaptic current (mIPSC) amplitude, and an enhanced response to exogenous application of GABA, indicating the involvement of postsynaptic mechanisms. Furthermore, there was no obvious change of the paired-pulse ratio (PPR) of eIPSCs and the frequency of mIPSCs, suggesting that the potentiation of eIPSCs might not be due to the increased presynaptic release. Blockade of adenosine A1 receptors led to a decrease of eIPSCs amplitude in post-ischemic neurons but not in control neurons, without affecting the frequency of mIPSCs and the PPR of eIPSCs. Thus, tonic activation of adenosine A1 receptors might, at least in part, contribute to the enhancement of inhibitory synaptic transmission in CA1 neurons after forebrain ischemia. The transient enhancement of inhibitory neurotransmission might temporarily protect CA1 pyramidal neurons, and delay the process of neuronal death after cerebral ischemia.     

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.     

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.     

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.     

Astrocyte-mediated potentiation of inhibitory synaptic transmission

We investigated the role of astrocytes in activity-dependent modulation of inhibitory synaptic transmission in hippocampal slices. Repetitive firing of an interneuron decreased the probability of synaptic failures in spike-evoked inhibitory postsynaptic currents (unitary IPSCs) in CA1 pyramidal neurons. The GABAB-receptor antagonist CGP55845A abolished this effect. Direct stimulation of astrocytes, or application of the GABAB-receptor agonist baclofen, potentiated miniature inhibitory postsynaptic currents (mIPSCs) in pyramidal neurons. These effects were blocked by inhibition of astrocytic calcium signaling with the calcium chelator BAPTA or by antagonists of the ionotropic glutamate receptors. These observations suggest that interneuronal firing elicits a GABAB-receptor-mediated elevation of calcium in surrounding astrocytes, which in turn potentiates inhibitory transmission. Astrocytes may therefore be a necessary intermediary in activity-dependent modulation of inhibitory synapses in the hippocampus.     

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.     

Reduction of spontaneous inhibitory synaptic activity in experimental heterotopic gray matter

Neuronal heterotopia has a strong association with epilepsy, but the mechanisms that underlie this relationship are largely unknown. We have utilized the in utero irradiated rat model to study circuit abnormalities in experimentally induced subcortical heterotopic gray matter. Spontaneous and miniature inhibitory (IPSCs) and excitatory (EPSCs) postsynaptic currents were recorded from visualized heterotopic pyramidal neurons in in vitro hemispheric slices and compared with control neocortical pyramidal neurons using the whole cell patch-clamp technique. The frequency of spontaneous and miniature IPSCs was significantly reduced in pyramidal neurons from heterotopic cortex. Amplitude and kinetics of IPSCs were not different between the two groups. Spontaneous and miniature EPSCs were not different between the two groups. Short-term synaptic plasticity of stimulus-evoked EPSCs showed depression in heterotopic neurons and facilitation in control pyramidal neurons. This study shows a selective impairment of the GABAergic circuitry in experimental heterotopic gray matter. We have reported similar findings in normotopic dysplastic cortex from this model. Taken together, these studies demonstrate a pervasive defect in inhibition throughout the cortex of irradiated rats with cortical dysplasia and neuronal heterotopia. This may have important implications regarding cortical development and function following in utero injuries.     

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.     

Orexin neurons are directly and indirectly regulated by catecholamines in a complex manner

We reported elsewhere that orexin neurons are directly hyperpolarized by noradrenaline (NA) and dopamine. In the present study, we show that NA, dopamine, and adrenaline all directly hyperpolarized orexin neurons. This response was inhibited by the alpha2 adrenergic receptor (alpha2-AR) antagonist, idazoxan or BRL44408, and was mimicked by the alpha2-AR-selective agonist, UK14304. A low concentration of Ba2+ inhibited NA-induced hyperpolarization, which suggests that activation of G protein coupled inward rectifier potassium channels is involved in the response. In the presence of a high concentration of idazoxan, NA induced depolarization or inward current. This response was inhibited by alpha1-AR antagonist, prazosin, which suggests the existence of alpha1-ARs on the orexin neurons along with alpha2-AR. We also examined the effects of NA on glutamatergic and GABAergic synaptic transmission. NA application dramatically increased the frequency and amplitude of spontaneous inhibitory synaptic currents (sIPSCs) and inhibited excitatory synaptic currents (sEPSCs) in orexin neurons; however, NA decreased the frequency of miniature EPSCs (mEPSCs) and IPSCs and the amplitude of evoked EPSCs and IPSCs through the alpha2-AR, because the NA response on mPSCs was inhibited by idazoxan. These results suggest that the NA-induced increase in sIPSC frequency and amplitude is mediated via alpha1-ARs on the somata of GABAergic neurons that innervate the orexin neurons. Calcium imaging using orexin/YC2.1 transgenic mouse brain revealed that NA-induced inhibition of orexin neurons is not altered by sleep deprivation or circadian time in mice. The evidence presented here revealed that orexin neurons are regulated by catecholamines in a complex manner.     

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.