Galphimine B modulates synaptic transmission on dopaminergic ventral tegmental area neurons

Galphimine B (GB) is a bioactive compound isolated from the plant Galphimia glauca Cav. (Malpighiaceae) and has been shown to have central nervous system depressant properties. In an earlier study, it was reported that both systemic and local administration of GB modified the extracellular spontaneous spiking activity in the ventral tegmental area (VTA) neurons. In the present study we analyzed the synaptic effects of this compound on dopaminergic neurons. Recordings were made in brain slices using intracellular and patch-clamp techniques, in dopaminergic (DA) VTA neurons. Spontaneous miniature excitatory postsynaptic currents (mEPSCs), excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic potentials (IPSPs) were recorded in control situation and after bath infusions of several concentrations of GB (1 microM-5 mM), GABA (1 nM-100 microM) and the GABA A blockers, picrotoxin (100 microM) and bicuculline (10 microM), and GABA B blocker saclofen (200 microM). GB administration reduced the frequency (p < 0.05) but not the amplitude of mESPCs. However, GABA (IC 50 = 645 nM) and GB (IC 50 = 174.5 microM) infusion significantly reduced the amplitude of stimuli induced EPSCs, Bicuculline (10 microM) co-administration only reduced GABA effects and did not modify the GB depressant action. Finally, isolated GABAergic IPSPs were modified by the addition of picrotoxin, but GB had no effect on these evoked synaptic responses. The present results indicate that GB modifies synaptic activity on dopaminergic VTA neurons by a non-GABAergic mechanism.     

GABA(B) receptor-mediated presynaptic inhibition of glutamatergic and GABAergic transmission in the basolateral amygdala

The information processing at central synapses is mediated not only by homosynaptic transmission with direct synaptic connections but also by heterosynaptic interactions between distinct synaptic inputs. Using rat brain slices and whole-cell recordings this study aimed to examine the roles of GABA(B) receptors in synaptic interactions in the basolateral amygdala (BLA), a critical brain structure related to fear and anxiety. Stimulation in the BLA produced non-NMDA type glutamate receptor antagonist-sensitive excitatory postsynaptic currents (EPSCs) and bicuculline-sensitive inhibitory postsynaptic currents (IPSCs) in the BLA neurons. The GABA(B) receptor agonist baclofen markedly inhibited both EPSCs and IPSCs in a concentration-dependent manner, and the baclofen-induced inhibition was selectively abolished by the GABA(B) receptor antagonist CGP55845A. The paired-pulse ratio of EPSC and IPSC amplitude was increased by baclofen. The effect of baclofen was mimicked by lowering the external Ca2+ concentration but not by glutamate- and GABA(A)-receptor antagonists. The frequency but not the mean amplitude of miniature EPSCs and IPSCs was decreased by baclofen. The findings suggest that activation of GABA(B) receptors by baclofen reduces the strength of excitatory and inhibitory transmission in the BLA by a presynaptic mechanism. Repetitive conditioning stimulation applied to GABAergic synaptic inputs exerted an inhibitory action on glutamatergic excitatory transmission, and the stimulation-induced inhibition was abolished by CGP55845A. Furthermore, the paired-pulse ratio of EPSCs was increased during the stimulation-induced inhibition. The results in this study provide evidence that synaptic activation of GABA(B) heteroreceptors elicits presynaptic inhibition of glutamatergic excitatory transmission in the BLA.     

Tonically activated GABAA receptors in hippocampal neurons are high-affinity,low-conductance sensors for extracellular GABA

In the hippocampus, two distinct forms of GABAergic inhibition have been identified, phasic inhibitory postsynaptic currents that are the consequence of the vesicular release of GABA and a tonic conductance that is activated by low ambient concentrations of extracellular GABA. It is not known what accounts for the distinct properties of receptors that mediate the phasic and tonic inhibitory conductances. Moreover, the physiological role of the tonic inhibitory conductance remains uncertain because pharmacological tools that clearly distinguish tonic and phasic receptors are lacking. Here, we demonstrate that GABAA receptors that generate a tonic conductance in cultured hippocampal neurons from embryonic mice have different pharmacological properties than those in cerebellar granule neurons or pyramidal neurons in the dentate gyrus. The tonic conductance in cultured hippocampal neurons is enhanced by the benzodiazepine, midazolam, and is insensitive to the inhibitory effects of the competitive antagonist, gabazine (< or =10 microM). We also identify penicillin as an uncompetitive antagonist that selectively inhibits the synaptic but not tonic conductance. GABA was applied to hippocampal neurons to investigate the properties of synaptic and extrasynaptic receptors. GABA-evoked current was composed of two components: a rapidly desensitizing current that was blocked by penicillin and a nondesensitizing current that was insensitive to penicillin blockade. The potency of GABA was greater for the penicillin-insensitive nondesensitizing current. Single-channel studies show that the gabazine-insensitive GABAA receptors have a lower unitary conductance (12 pS) than that estimated for synaptic receptors. Thus, specialized GABAA receptors with an apparent higher affinity for GABA that do not readily desensitize mediate the persistent tonic conductance in hippocampal neurons. The receptors underlying tonic and phasic inhibitory conductances in hippocampal neurons are pharmacologically and biophysically distinct, suggesting that they serve different physiological roles.     

Distinct functional and pharmacological properties of tonic and quantal inhibitory postsynaptic currents mediated by gamma-aminobutyric acid(A) receptors in hippocampal neurons

gamma-Aminobutyric acid (GABA), the principal inhibitory neurotransmitter, activates a persistent low amplitude tonic current in several brain regions in addition to conventional synaptic currents. Here we demonstrate that GABA(A) receptors mediating the tonic current in hippocampal neurons exhibit functional and pharmacological properties different from those of quantal synaptic currents. Patch-clamp techniques were used to characterize miniature inhibitory postsynaptic currents (mIPSCs) and the tonic GABAergic current recorded in CA1 pyramidal neurons in rat hippocampal slices and in dissociated neurons grown in culture. The competitive GABA(A) receptor antagonists, bicuculline and picrotoxin, blocked both the mIPSCs and the tonic current. In contrast, mIPSCs but not the tonic current were inhibited by gabazine (SR-95531). Coapplication experiments and computer simulations revealed that gabazine bound to the receptors responsible for the tonic current but did not prevent channel activation. However, gabazine competitively inhibited bicuculline blockade. The unitary conductance of the GABA(A) receptors underlying the tonic current (approximately 6 pS) was less than the main conductance of channels activated during quantal synaptic transmission (approximately 15--30 pS). Furthermore, compounds that potentiate GABA(A) receptor function including the benzodiazepine, midazolam, and anesthetic, propofol, prolonged the duration of mIPSCs and increased tonic current amplitude in cultured neurons to different extents. Clinically-relevant concentrations of midazolam and propofol caused a greater increase in tonic current compared with mIPSCs, as measured by total charge transfer. In summary, the receptors underlying the tonic current are functionally and pharmacologically distinct from quantally activated synaptic receptors and these receptors represent a novel target for neurodepressive drugs.     

Alpha 2-adrenergic receptor-mediated presynaptic inhibition of GABAergic IPSPs in rat histaminergic neurons

Nuclei of the brainstem involved in behavioral state control are mutually interconnected. Histaminergic neurons of the posterior hypothalamus receive inputs from brainstem noradrenergic cell groups as well as from the locus coeruleus. The role of adrenergic inputs in histaminergic function is unclear. We examined the actions of adrenergic agonists on histaminergic neurons of the tuberomamillary nucleus (TM) using electrophysiological methods in a brain slice preparation. Evoked GABAergic inhibitory postsynaptic potentials (IPSPs) in histaminergic neurons were reduced in amplitude following the application of norepinephrine (NE) (2-20 microM) or clonidine (10 microM) but were not affected by isoproterenol (10 microM). Norepinephrine application caused no changes in membrane properties of TM neurons. Responses to exogenously applied GABA were unaffected by adrenergic agonists. Clonidine reduced the frequency of spontaneous IPSPs, an action that was blocked by yohimbine. Norepinephrine did not alter the amplitude distribution of bicuculline-sensitive miniature inhibitory postsynaptic currents (mIPSCs). Thus, GABA release onto TM neurons is modulated presynaptically by adrenergic alpha(2)-receptors. Inputs from noradrenergic neurons of the brainstem will reduce the inhibitory actions of GABAergic inputs resulting in disinhibition of histaminergic neurons.     

Presynaptic modulation of synaptic gamma-aminobutyric acid transmission by tandospirone in rat basolateral amygdala

Nystatin-perforated patch recordings were made from mechanically dissociated neurons (in which functional native presynaptic nerve terminals are preserved), isolated from the basolateral amygdala regions to investigate the effects of tandospirone on gamma-aminobutyric acidergic (GABAergic) inhibition. Two types of neurons, ovoid-shaped and pyramidal-shaped neurons, were obtained from the basolateral amygdala nuclei and the electrophysiological characteristics of these two types of neurons supported the morphological classification of these isolated neurons. From the ovoid-shaped neurons, bicuculline-sensitive GABA(A)ergic miniature inhibitory postsynaptic currents (miniature IPSC) were recorded in the presence of tetrodotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and DL-2-amino-5-phosphovaleric acid (DL-AP5). Tandospirone (10 microM) reversibly and continuously inhibited the GABAergic miniature synaptic events to 66.3+/-2.1% of control (P<0.01, n=17) without affecting the miniature IPSC amplitude (104.0+/-3.1% of control, n=17). The similar inhibition of miniature IPSC frequency was mimicked by a specific 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT, 1 microM), and the effects of tandospirone were prevented in the presence of a specific 5-HT1A receptor antagonist 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl] piperazine hydrobromide (NAN-190, 1 microM). Activation of 5-HT1A receptors by 8-OH-DPAT (1 microM) evoked no direct postsynaptic effects in enzyme-treated isolated basolateral amygdala neurons, suggesting that tandospirone acts at presynaptic 5-HT1A receptors. Furthermore, this presynaptic inhibition by tandospirone was prevented after treatment with a pertussis toxin-sensitive GTP-binding protein (G-protein) inhibitor, N-ethylmaleimide (at 3 microM for 5 min). In conclusion, in the basolateral amygdala nuclei, tandospirone activated presynaptic 5-HT1A receptors on the GABAergic nerve terminals projecting to ovoid-shaped neurons and inhibited synaptic GABA transmission via G-proteins.     

Presynaptic inhibition preferentially reduces in NMDA receptor-mediated component of transmission in rat midbrain dopamine neurons.

We used patch pipettes to record whole-cell currents from single dopamine neurons in slices of rat midbrain. Pharmacological methods were used to isolate EPSCs evoked by focal electrical stimulation. Baclofen was significantly more potent for inhibiting NMDA receptor-mediated EPSCs (IC50=0.24 microM) compared with inhibition of EPSCs mediated by AMPA receptors (IC50=1.72 microM). The increased potency of baclofen for inhibiting the NMDA component persisted in superfusate that contained zero Mg2+ and when postsynaptic K+ conductances were reduced by Cs+ and QX-314. Effects of baclofen on EPSCs were blocked by the GABA(B) receptor antagonist CGP-35348. Adenosine was 20 fold more potent for reducing the NMDA component of transmission (IC50=31 microM) compared with inhibition of AMPA receptor-mediated EPSCs (IC50=654 microM). Effects of adenosine on EPSCs were blocked by the A1 receptor antagonist DPCPX. Both baclofen and adenosine significantly increased the ratio of EPSCs in paired-pulse studies, suggesting presynaptic sites of action. Although adenosine (1 mM) did not reduce currents evoked by exogenous NMDA (10 microM), baclofen (1 microM) reduced NMDA currents by 29%. Neither baclofen nor adenosine altered currents evoked by exogenous AMPA (1 microM). We conclude that adenosine acts at presynaptic A1 receptors to cause a preferential reduction in the NMDA component of synaptic transmission. In contrast, baclofen preferentially reduces NMDA EPSCs by acting at both pre- and postsynaptic GABA(B) receptors. By regulating NMDA receptor function, A1 and GABA(B) receptors may play important roles in regulating the excitability of dopamine neurons.     

辣椒素受体参与骶髓后联合核神经元突触传递

目的研究辣椒素受体对大鼠骶髓后联合核(SDCN)神经元突触传递的影响。方法在脊髓骶段横切薄片上,利用全细胞膜片钳法记录骶髓后联合核神经元谷氨酸能兴奋性突触后电流(EPSCs)和γ-氨基丁酸(GABA)能抑制性突触后电流(IPSCs),比较激动辣椒素受体后上述突触电流的变化;观察激动辣椒素受体对SDCN神经元动作电位发放的影响。结果辣椒素受体被其特异性激动剂辣椒素(1μmol.L-1)激动后,自发EPSCs(sEPSCs)的频率和振幅均有明显增加(P<0.05,n=17)。在河豚毒素(0.5μmol.L-1)存在的条件下,辣椒素明显增加微小EPSCs(mEPSCs)的频率(P<0.01,n=13),但对mEPSCs的振幅无影响(P>0.05,n=13),提示辣椒素的作用在突触前。辣椒素也明显增加动作电位发放(P<0.05,n=19)。上述作用均可被辣椒素受体特异性拮抗剂capsazepine(10μmol.L-1)阻断。辣椒素也增加GABA能的自发IPSCs(sIPSCs)的频率(P<0.05,n=20),但对其不依赖动作电位的微小IPSCs(mIPSCs)的频率或振幅均无作用(P>0.05,n=9)。结论在SDCN,辣椒素受体主要表达于兴奋性突触终末;激动辣椒素受体影响兴奋性和抑制性突触活动,并可能参与痛觉信息在脊髓水平的传递和调制。     

Role of GABA receptors in nitric oxide inhibition of dorsolateral periaqueductal gray neurons

Nitric oxide (NO) affects neuronal activity of the midbrain periaqueductal gray (PAG). The purpose of this report was to investigate the role of GABA receptors in NO modulation of neuronal activity through inhibitory and excitatory synaptic inputs within the dorsolateral PAG (dl-PAG). First, spontaneous miniature inhibitory postsynaptic currents (mIPSCs) and excitatory postsynaptic currents (mEPSCs) were recorded using whole cell voltage-clamp methods. Increased NO by either S-nitroso-N-acetyl-penicillamine (SNAP, 100 microM) or L-arginine (50 microM) significantly augmented the frequency of mIPSCs of the dl-PAG neurons without altering their amplitudes or decay time constants. The effects were eliminated after bath application of carboxy-PTIO (NO scavenger), and 1-(2-trifluorom-ethylphenyl) imidazole (NO synthase inhibitor). In contrast, SNAP and L-arginine did not alter mEPSCs in dl-PAG neurons. However the frequency of mEPSCs was significantly increased with prior application of the GABA(B) receptors antagonist, CGP55845. In addition, NO significantly decreased the discharge rate of spontaneous action potentials in the dl-PAG neurons and the effect was reduced in the presence of the GABA(A) receptor antagonist, bicuculline. Our data show that within the dl-PAG NO potentiates the synaptic release of GABA, while NO-induced GABA presynaptically inhibits glutamate release through GABA(B) receptors. Overall, NO suppresses neuronal activity of the dl-PAG via a potentiation of GABAergic synaptic inputs and via GABA(A) receptors.     

Tonically active GABAA receptors in hippocampal pyramidal neurons exhibit constitutive GABA-independent gating

Phasic and tonic inhibitory currents of hippocampal pyramidal neurons exhibit distinct pharmacological properties. Picrotoxin and bicuculline methiodide inhibited both components, consistent with a role for GABAA receptors; however, gabazine, at a concentration that abolished miniature GABAergic inhibitory postsynaptic currents and responses to exogenous GABA, had no effect on tonic currents. Because all GABA-activated GABAA receptors in pyramidal neurons are gabazine-sensitive, it follows that tonic currents are not GABA-activated. Furthermore, picrotoxin-sensitive spontaneous single-channel events recorded from outside-out patches had the same chord conductance as GABA-activated channels and were gabazine-resistant. Therefore, we hypothesize that GABAA receptors, constitutively active in the absence of GABA, mediate tonic current; the failure of gabazine to block tonic current reflects a lack of negative intrinsic efficacy of the antagonist. We compared the negative efficacies of bicuculline and gabazine using the general anesthetic propofol to directly activate GABAA receptors native to pyramidal neurons or alpha1beta3gamma2 receptors recombinantly expressed in human embryonic kidney 293 cells. Propofol activated gabazine-resistant, bicuculline-sensitive currents when applied to either preparation. Although gabazine had negligible efficacy as an inhibitor of propofol-activated currents, it prevented inhibition by bicuculline, which acts as an inverse agonist inhibiting GABA-independent gating. Recombinant alpha1beta1/3gamma2 receptors also mediated agonist-independent tonic currents that were resistant to gabazine and inhibited by bicuculline. Thus, gabazine is a competitive antagonist with negligible negative efficacy and is therefore unable to inhibit GABAA receptors that are active in the absence of GABA because of either anesthetic or spontaneous gating. Moreover, spontaneously active GABAA receptors mediate gabazine-resistant tonic currents in pyramidal neurons.     

Effects of endomorphin on substantia gelatinosa neurons in rat spinal cord slices

1. Whole-cell patch recordings were made from substantia gelatinosa (SG) neurons in transverse lumbar spinal cord slices of 15- to 30-day-old rats. 2. Endomorphin 1 (EM-1) or EM-2 (相似文献   

Secondhand tobacco smoke exposure differentially alters nucleus tractus solitarius neurons at two different ages in developing non-human primates

Exposing children to secondhand tobacco smoke (SHS) is associated with increased risk for asthma, bronchiolitis and SIDS. The role for changes in the developing CNS contributing to these problems has not been fully explored. We used rhesus macaques to test the hypothesis that SHS exposure during development triggers neuroplastic changes in the nucleus tractus solitarius (NTS), where lung sensory information related to changes in airway and lung function is first integrated. Pregnant monkeys were exposed to filtered air (FA) or SHS for 6 h/day, 5 days/week starting at 50-day gestational age. Mother/infant pairs continued the exposures postnatally to age 3 or 13 months, which may be equivalent to approximately 1 or 4 years of human age, respectively. Whole-cell recordings were made of second-order NTS neurons in transverse brainstem slices. To target the consequences of SHS exposure based on neuronal subgroups, we classified NTS neurons into two phenotypes, rapid-onset spiking (RS) and delayed-onset spiking (DS), and then evaluated intrinsic and synaptic excitabilities in FA-exposed animals. RS neurons showed greater cell excitability especially at age of 3 months while DS neurons received greater amplitudes of excitatory postsynaptic currents (EPSCs). Developmental neuroplasticity such as increases in intrinsic and synaptic excitabilities were detected especially in DS neurons. In 3 month olds, SHS exposure effects were limited to excitatory changes in RS neurons, specifically increases in evoked EPSC amplitudes and increased spiking responses accompanied by shortened action potential width. By 13 months, the continued SHS exposure inhibited DS neuronal activity; decreases in evoked EPSC amplitudes and blunted spiking responses accompanied by prolonged action potential width. The influence of SHS exposure on age-related and phenotype specific changes may be associated with age-specific respiratory problems, for which SHS exposure can increase the risk, such as SIDS and bronchiolitis in infants and asthma in older children.     

Distinct modulatory effects of 5-HT on excitatory synaptic transmissions in the nucleus tractus solitarius of the rat

The second-order relay neurons in the nucleus tractus solitarius (NTS) receive numerous peripheral afferent inputs mainly from the vagus nerve. Their activity is modified by several neuromodulators and hence autonomic responses are properly regulated. Serotonin (5-HT) is an important candidate for such neuromodulators, since serotonergic inputs and distribution of 5-HT receptors are provided in the NTS. However, its mechanism of action remains unclear. To evaluate the serotonergic modulation of synaptic transmission, we examined the effects of 5-HT (1.0-10.0 μM) on the solitary tract-evoked excitatory postsynaptic currents (eEPSCs) and spontaneously occurring EPSCs (sEPSCs) in the preselected second-order neurons of the rat NTS. 5-HT concentration-dependently decreased the amplitude of eEPSCs, which was accompanied by an increase in paired-pulse ratio. The inhibitory effect of 5-HT was mimicked by α-methylserotonin and blocked by ketanserin. 5-HT had no effect on the inward current induced in the NTS neurons by topically applied α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). On the other hand, 5-HT increased the frequency of sEPCSs without effect on their amplitude. This excitatory effect of 5-HT was mimicked by 2-methylserotonin and antagonized by ondansetron. The results suggest a dual modulation of the excitatory synaptic transmission by 5-HT in the NTS; presynaptic inhibition of the peripheral inputs synapsing to the relay neurons via 5-HT(2) receptors and presynaptic excitation of inputs from the intrinsic local network via 5-HT(3) receptors. These effects of 5-HT may provide important means of optimizing the autonomic responses mediated by the NTS network.     

Kavalactones and dihydrokavain modulate GABAergic activity in a rat gastric-brainstem preparation

Using an in vitro neonatal rat gastric-brainstem preparation, the activity of majority neurons recorded in the nucleus tractus solitarius (NTS) of the brainstem were significantly inhibited by GABA A receptor agonist, muscimol (30 microM), and this inhibition was reversed by selective GABA A receptor antagonist, bicuculline (10 microM). Application of kavalactones (300 microg/ml) and dihydrokavain (300 microM) into the brainstem compartment of the preparation also significantly reduced the discharge rate of these NTS neurons (39 % and 32 %, respectively, compared to the control level), and this reduction was partially reversed by bicuculline (10 microM). Kavalactones or dihydrokavain induced inhibitory effects were not reduced after co-application of saclofen (10 microM; a selective GABA B receptor antagonist) or naloxone (100 nM; an opioid receptor antagonist). Pretreatment with kavalactones (300 microg/ml) or dihydrokavain (300 microM) significantly decreased the NTS inhibitory effects induced by muscimol (30 microM), approximately from 51 % to 36 %. Our results demonstrated modulation of brainstem GABAergic mechanism by kavalactones and dihydrokavain, and suggested that these compounds may play an important role in regulation of GABAergic neurotransmission.     

Glutamatergic neural transmission in the nucleus tractus solitarius: N-methyl-D-aspartate receptors

1. The nucleus tractus solitarius (NTS) is the first central site where the reflex control of autonomic, including baroreceptor, reflex function is coordinated. Autonomic signals are transmitted from the first-order visceral afferent fibres to second-order NTS neurons by L-glutamate. It is well established that activation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptors, which mediate the fast component of L-glutamate signalling, is required for generating changes in membrane potentials of the second-order NTS neurons. The contribution of the slower-developing, longer-lasting N-methyl-D-aspartate (NMDA) receptor-mediated component of glutamate signalling to synaptic transmission at these synapses is less well understood. 2. The aim of this work is to highlight evidence that functional NMDA receptors exist on second-order NTS neurons in autonomic, including baroreceptor, afferent pathways by determining whether NMDA receptors can be activated by: (i) exogenous application of NMDA; and (ii) endogenous release of L-glutamate from autonomic afferent fibres. Studies were performed on second-order neurons in transverse and horizontal brainstem slices containing the intermediate NTS and the tractus solitarius. Second-order NTS neurons were identified by electrophysiological criteria or by attached fluorescent-labelled aortic depressor nerve (ADN) boutons. 3. N-Methyl-D-aspartate (50 nmol(-2) micromol) dose-dependently evoked excitatory post-synaptic currents in second-order NTS neurons (P = 0.004; n = 4). The NMDA receptor-mediated currents were also synaptically evoked by low-frequency stimulation of the autonomic afferent fibres in the tractus solitarius. The NMDA receptor-mediated currents were blocked by the NMDA receptor antagonist AP5 (n = 7; P = 0.027). 4. The findings suggest that functional NMDA receptors exist on second-order NTS neurons. While the NMDA receptor- mediated currents may not be required for signal transmission when the second-order neurons are at resting membrane potential, their activation may help to modulate autonomic signal transmission in the NTS under conditions in which the membrane is depolarized by high frequency or convergent inputs.     

Differential modulation of glutamatergic transmission by 3,5-dibromo-L-phenylalanine

An increasing body of evidence supports the hypothesis that diminished function of N-methyl-D-aspartate (NMDA) receptors and the associated increase in glutamate release and overstimulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors are critical elements of the pathophysiology of schizophrenia. Here, we describe a halogenated derivative of the aromatic amino acid L-phenylalanine that 1) activates NMDA receptors, 2) depresses presynaptic glutamate release, and 3) blocks AMPA/kainate receptors. The experiments were conducted in rat cerebrocortical cultured neurons by using the patch-clamp technique. 3,5-Dibromo-L-phenylalanine (3,5-DBr-L-Phe) augmented NMDA miniature excitatory postsynaptic currents (mEPSCs) and activated the steady-state current, effects that were eliminated by NMDA receptor antagonists DL-2-amino-5-phosphonopentanoic acid and MK-801 (dizocilpine maleate; 5H-dibenzo[a,d]cyclohepten-5,10-imine). 3,5-DBr-L-Phe was a partial agonist at the glutamate-binding site of NMDA receptors with an EC50 of 331.6 +/- 78.6 microM and with an efficacy of 30.5 +/- 4.7% compared with NMDA. 3,5-DBr-L-Phe depressed both amplitude and frequency of AMPA/kainate mEPSCs. The IC50 of 3,5-DBr-L-Phe to inhibit AMPA/kainate mEPSC frequency was 29.4 +/- 4.3 microM. 3,5-DBr-L-Phe significantly decreased paired pulse depression of AMPA/kainate EPSCs and attenuated current activated by AMPA with higher efficacy at lower concentration of AMPA. 3,5-DBr-L-Phe neither affected GABA miniature inhibitory postsynaptic currents nor elicited action potentials. By enhancing NMDA receptor function, reducing glutamate release and blocking AMPA/kainate receptors 3,5-DBr-L-Phe represents a new type of polyvalent modulator of glutamatergic synaptic transmission with potential therapeutic applications.     

Neuroprotective agent riluzole potentiates postsynaptic GABA(A) receptor function.

The antiepileptic drug riluzole is a use-dependent blocker of voltage-gated Na(+) channels and selectively depresses action potential-driven glutamate over gamma-aminobutyric acid (GABA) release. Here we report that in addition to its presynaptic effect, riluzole at higher concentrations also strongly potentiates postsynaptic GABA(A) responses both in cultured hippocampal neurons and in Xenopus oocytes expressing recombinant receptors. Although peak inhibitory postsynaptic currents (IPSCs) of autaptic hippocampal neurons were inhibited, 20-100 microM riluzole significantly prolonged the decay of IPSCs, resulting in little change in total charge transfer. The effect was dose-dependent and reversible. Riluzole selectively increased miniature IPSC fast and slow decay time constants, without affecting their relative proportions. Miniature IPSC peak amplitude, rise time and frequency were unaffected, indicating a postsynaptic mechanism. In the Xenopus oocyte expression system, riluzole potentiated GABA responses by lowering the EC(50) for GABA activation. Riluzole directly gated a GABA(A) current that was partially blocked by bicuculline and gabazine. Pharmacological experiments suggest that the action of riluzole did not involve a benzodiazepine, barbiturate, or neurosteroid site. Instead, riluzole-induced potentiation was inhibited by the lactone antagonist alpha-isopropyl-alpha-methyl-gamma-butyrolatone (alpha-IMGBL). While most anticonvulsants either block voltage-gated Na(+) channels or potentiate GABA(A) receptors, our results suggest that riluzole may define an advantageous class of anticonvulsants with both effects.     

Ethanol inhibition of synaptically evoked kainate responses in rat hippocampal CA3 pyramidal neurons.

Many studies have demonstrated that intoxicating concentrations of ethanol (10-100 mM) can selectively inhibit the component of glutamatergic synaptic transmission mediated by N-methyl-D-aspartate (NMDA) receptors while having little or no effect on excitatory synaptic transmission mediated by non-NMDA receptors [i.e., alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and/or kainate (KA) receptors]. However, until the recent development of highly selective AMPA receptor antagonists, it was not possible to assess the relative contribution of AMPA and KA receptors to non-NMDA receptor-mediated synaptic transmission or to determine whether these glutamate receptor subtypes differed in their sensitivity to ethanol. In the present experiments, we used the highly selective AMPA receptor antagonist LY 303070 to pharmacologically isolate KA receptor-mediated excitatory postsynaptic currents (EPSCs) in rat hippocampal CA3 pyramidal neurons and tested their sensitivity to ethanol. Concentrations of ethanol as low as 20 mM significantly and reversibly depressed KA EPSCs. Ethanol also inhibited KA currents evoked by direct pressure application of KA in the presence of LY 303070, suggesting that this inhibition was mediated by a postsynaptic action. In contrast, ethanol had no effect on AMPA EPSCs in these cells, even at the highest concentration tested (80 mM). Ethanol significantly inhibited NMDA EPSCs in these neurons, but these responses were less sensitive to ethanol than KA EPSCs. These results suggest that in addition to its well-described depressant effect on NMDA receptor-mediated synaptic transmission, ethanol has an even greater inhibitory effect on glutamatergic synaptic transmission mediated by KA receptors in rat hippocampal CA3 pyramidal neurons.     

Serotonergic modulation of GABAergic and glutamatergic synaptic transmission in mechanically isolated rat medial preoptic area neurons.

The medial preoptic area (MPOA) of the hypothalamus is critically involved in the regulation of male sexual behavior and has been implicated in several homeostatic processes. Serotonin (5-hydroxytryptamine, 5-HT) inhibits sexual behavior via effects in the MPOA, where there are high densities of 5-HT(1A) and 5-HT(1B) receptor subtypes. We used whole-cell recordings under voltage-clamp conditions to investigate the serotonergic modulation of gamma-aminobutyric acid (GABA)ergic and glutamatergic synaptic transmission in mechanically dissociated rat MPOA neurons with native presynaptic nerve endings. Spontaneous GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in the MPOA were completely blocked by bicuculline. Serotonin reversibly reduced the GABAergic mIPSC frequency without affecting the mean current amplitude. Serotonergic inhibition of mIPSC frequency was mimicked by (+/-)-8-hydroxy-2-dipropylaminotetralin hydrobromide, a specific 5-HT(1A) receptor agonist, and blocked by 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl] piperazine hydrobromide, a specific 5-HT(1A) receptor antagonist. 6-Cyano-7-nitroquinoxaline-2,3-dione completely blocked spontaneous glutamatergic miniature excitatory postsynaptic currents (mEPSCs) in the MPOA. Serotonin reversibly decreased the glutamatergic mEPSC frequency without affecting the mean current amplitude. Serotonergic inhibition of mEPSC frequency was mimicked by CGS 12066B, a specific 5-HT(1B) receptor agonist, and blocked by SB 216641, a specific 5-HT(1B) receptor antagonist. Stimulation of adenylyl cyclase with forskolin increased the frequencies of GABAergic mIPSCs and glutamatergic mEPSCs, and blocked the inhibitory effects of 5-HT. H-89, a selective protein kinase A (PKA) inhibitor, decreased the frequencies of GABAergic mIPSCs and glutamatergic mEPSCs, and blocked their reduction by 5-HT. These findings suggest that 5-HT reduces the frequency of GABAergic mIPSCs and glutamatergic mEPSCs through 5-HT(1A) and 5-HT(1B) receptor-mediated inhibition, respectively, of the PKA-dependent pathway in the presynaptic nerve terminals of MPOA neurons.     

High-affinity, slowly desensitizing GABAA receptors mediate tonic inhibition in hippocampal dentate granule cells

The tonic form of GABA-mediated inhibition requires the presence of slowly desensitizing GABA(A) receptors with high affinity, which has not yet been directly demonstrated in hippocampal neurons. Low concentration of GABA (1 microM) persistently increased baseline noise, increased membrane slope conductance, but did not affect spontaneous inhibitory postsynaptic currents (sIPSCs) in dentate granule cells (DGCs). Higher concentrations of GABA (10-100 microM) desensitized synaptic currents quickly, and there was a large residual current. Saturating concentration of GABA (1 mM) completely desensitized synaptic currents and revealed a slowly desensitizing, persistent current. Penicillin (300 microM) inhibited baseline noise without affecting mean current and inhibited decay time of sIPSCs. GABA(A) receptors mediating baseline noise in DGCs were sensitive to allopregnanolone, furosemide, and loreclezole and insensitive to diazepam and zolpidem. These studies demonstrate persistently open GABA(A) receptors on DGCs with high affinity for GABA, slow desensitization rate, and pharmacological properties similar to those of recombinant receptors containing alpha(4), beta(1), and the delta subunits.