GABAergic Presynaptic Terminals Research Articles

5-Hydroxytryptamine 1A/7 and 4α Receptors Differentially Prevent Opioid-Induced Inhibition of Brain Stem Cardiorespiratory Function

Opioids evoke respiratory depression, bradycardia, and reduced respiratory sinus arrhythmia, whereas serotonin (5-HT) agonists stimulate respiration and cardiorespiratory interactions. This study tested whether serotonin agonists can prevent the inhibitory effects of opioids on cardiorespiratory function. Spontaneous and rhythmic inspiratory-related activity and gamma-aminobutyric acid (GABA) neurotransmission to premotor parasympathetic cardioinhibitory neurons in the nucleus ambiguus were recorded simultaneously in an in vitro thick slice preparation. The mu-opioid agonist fentanyl inhibited respiratory frequency. The 5-hydroxytryptamine 1A/7 receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin increased respiratory frequency by itself and also prevented the fentanyl-induced respiratory depression. The 5-hydroxytryptamine 4alpha agonist BIMU-8 did not by itself change inspiratory activity but prevented the mu-opioid-mediated respiratory depression. Both spontaneous and inspiratory-evoked GABAergic neurotransmission to cardiac vagal neurons were inhibited by fentanyl. 8-Hydroxy-2-(di-n-propylamino)tetralin inhibited spontaneous but not inspiratory-evoked GABAergic activity to parasympathetic cardiac neurons. However, 8-hydroxy-2-(di-n-propylamino)tetralin differentially altered the opioid-mediated depression of inspiratory-evoked GABAergic activity but did not change the opioid-induced reduction in spontaneous GABAergic neurotransmission. In contrast, BIMU-8 did not alter GABAergic neurotransmission to cardiac vagal neurons by itself but prevented the fentanyl depression of both spontaneous and inspiratory-elicited GABAergic neurotransmission to cardiac vagal neurons. In the presence of tetrodotoxin, the inhibition of GABAergic inhibitory postsynaptic currents with fentanyl is prevented by coapplication of BIMU-8, indicating that BIMU-8 acts at presynaptic GABAergic terminals to prevent fentanyl-induced depression. These results suggest that activation of 5-hydroxytryptamine receptors, particularly 5-hydroxytryptamine 4alpha agonists, may be a useful therapeutic approach in preventing opioid-evoked cardiorespiratory depression.

Neuregulin-1 Enhances Depolarization-Induced GABA Release

Neuregulin-1 (NRG1), a regulator of neural development, has been shown to regulate neurotransmission at excitatory synapses. Although ErbB4, a key NRG1 receptor, is expressed in glutamic acid decarboxylase (GAD)-positive neurons, little is known about its role in GABAergic transmission. We show that ErbB4 is localized at GABAergic terminals of the prefrontal cortex. Our data indicate a role of NRG1, both endogenous and exogenous, in regulation of GABAergic transmission. This effect was blocked by inhibition or mutation of ErbB4, suggesting the involvement of ErbB4. Together, these results indicate that NRG1 regulates GABAergic transmission via presynaptic ErbB4 receptors, identifying a novel function of NRG1. Because both NRG1 and ErbB4 have emerged as susceptibility genes of schizophrenia, these observations may suggest a mechanism for abnormal GABAergic neurotransmission in this disorder.

Presynaptic AMPA and kainate receptors increase the size of GABAergic terminals and enhance GABA release

In the developing cerebellum, NMDA receptors promote the maturation of axonal terminals of inhibitory interneurons. We compared the effects of AMPA/kainate receptor agonists in cultured cerebellar cells from GAD65-eGFP mice. Both AMPA and kainate augmented granule cell survival without affecting interneurons. The action of kainate was blocked by an AMPA but not by a NMDA receptor antagonist, suggesting AMPA receptor involvement. AMPA and kainate increased the size of the GABAergic terminals and the action of kainate was insensitive to NMDA blockers. Whole cell recordings in granule neurons revealed that chronic treatment for 5 days with kainate as well as NMDA decreased AMPA receptor expression while interneuronal kainate receptors were depressed by kainate treatment. Acute kainate application increased mIPSCs frequency in both granule neurons and interneurons and this effect was only partially blocked by an AMPA receptor antagonist. In contrast to what was reported for NMDA, chronic treatment with kainate induced a significant decrease of the basal mIPSCs frequency but increased the acute action of kainate on mIPSCs. Direct recordings from presynaptic GABAergic terminals suggest that AMPA and kainate receptors are present in developing GABAergic terminals and their activation affects the size of GABAergic terminals and spontaneous GABA release.

Inhibitory synaptic transmission in area postrema neurons of the rat showing robust presynaptic facilitation mediated by nicotinic ACh receptors

Inhibitory synaptic transmission and its modulation in neurons of the area postrema (AP), one of autonomic nuclei in the medulla, were studied using whole-cell patch-electrodes applied to slices from rats on postnatal days 10–24. When glycine (100 μM) or GABA (10 μM) was applied to AP neurons from a “Y tube”, large outward currents that showed reversal potential of − 67 mV (approximate Cl − equilibrium potential estimated) were induced. At a holding potential of − 10 mV, application of high K + to the AP neurons evoked massive inhibitory postsynaptic currents (IPSCs) in the neurons. Most of the evoked synaptic currents were blocked by bicuculline, while the remaining currents were sensitive to strychnine, indicating that the major inhibitory transmission in the area postrema was GABAergic. When nicotine (5–100 μM) was applied to AP neurons, robust IPSCs having GABAergic identity were evoked. Even in the presence of tetrodotoxin, nicotine could induce GABAergic IPSCs, most of which, however, disappeared in the presence of 5 mM Mg 2+. Presynaptic facilitation was also induced by other nicotinic agonists, including cytisine, 1,1-dimethyl-4-phenyl-piperazinium iodide, ACh and choline. The nicotine-induced presynaptic facilitation was inhibited by mecamylamine and slightly inhibited by dihydro-β-erythroidine or α-Bungarotoxin. These results indicate that nicotinic receptors are expressed at GABAergic presynaptic terminals in the area postrema and induce Ca 2+ influx to trigger vesicular release. The major nicotinic receptors involved are thought to be heteromeric subtypes such as α3β4 receptors, which may regulate inhibitory transmission potently responding to endogenous or exogenous nicotinic agents appeared in this area.

Molecular and synaptic organization of GABAA receptors in the cerebellum: Effects of targeted subunit gene deletions

GABAA receptors form heteromeric GABA-gated chloride channels assembled from a large family of subunit genes. In cerebellum, distinct GABAA receptor subtypes, differing in subunit composition, are segregated between cell types and synaptic circuits. The cerebellum therefore represents a useful system to investigate the significance of GABAA receptor heterogeneity. For instance, studies of mice carrying targeted deletion of major GABAA receptor subunit genes revealed the role of alpha subunit variants for receptor assembly, synaptic targeting, and functional properties. In addition, these studies unraveled mandatory association between certain subunits and demonstrated distinct pharmacology of receptors mediating phasic and tonic inhibition. Although some of these mutants have a profound loss of GABAA receptors, they exhibit only minor impairment of motor function, suggesting activation of compensatory mechanisms to preserve inhibitory networks in the cerebellum. These adaptations include an altered balance between phasic and tonic inhibition, activation of voltage-independent K+ conductances, and upregulation of GABAA receptors in interneurons that are not affected directly by the mutation. Deletion of the alpha1 subunit gene leads to complete loss of GABAA receptors in Purkinje cells. A striking alteration occurs in these mice, whereby presynaptic GABAergic terminals are preserved in the molecular layer but make heterologous synapses with spines, characterized by a glutamatergic-like postsynaptic density. During development of alpha1(0/0) mice, GABAergic synapses are initially formed but are replaced upon spine maturation. These findings suggest that functional GABAA receptors are required for long-term maintenance of GABAergic synapses in Purkinje cells.

The Na+/H+ exchanger is a major pH regulator in GABAergic presynaptic nerve terminals synapsing onto rat CA3 pyramidal neurons

The effects of pH(i) on GABAergic miniature inhibitory postsynaptic currents (mIPSCs) were studied in mechanically dissociated CA3 pyramidal neurons, by use of ammonium prepulse and whole-cell patch-clamp techniques, under the voltage-clamp condition. NH(4)Cl itself, which is expected to alkalinize pH(i), increased GABAergic mIPSC frequency in a concentration-dependent manner. In contrast, NH(4)Cl decreased mIPSC frequency, either in the presence of 200 microm Cd(2+) or in Ca(2+)-free external solution, suggesting that intraterminal alkalosis decreased GABAergic mIPSC frequency while [NH4(+)] itself may activate Ca(2+) channels by depolarizing the terminal. On the other hand, GABAergic mIPSC frequency was greatly increased immediately after NH(4)Cl removal, a condition expected to acidify pH(i), and recovered to the control level within 2 min after NH(4)Cl removal. This explosive increase in mIPSC frequency observed after NH(4)Cl removal was completely eliminated after depletion of Ca(2+) stores with 1 microm thapsigargin in the Ca(2+)-free external solution, suggesting that acidification increases in intraterminal Ca(2+) concentration via both extracellular Ca(2+) influx and Ca(2+) release from the stores. However, the acidification-induced increase in mIPSC frequency had not recovered by 10 min after NH(4)Cl removal either in the Na(+)-free external solution or in the presence of 10 microm 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), a specific Na(+)/H(+) exchanger (NHE) blocker. The present results suggest that NHEs are major intraterminal pH regulators on GABAergic presynaptic nerve terminals, and that the NHE-mediated regulation of pH(i) under normal physiological or pathological conditions might play an important role in the neuronal excitability by increasing inhibitory tones.

Identification and characterization of two novel splice forms of GRIP1 in the rat brain

We cloned two novel alternatively-spliced mRNA isoforms of glutamate receptor interacting protein 1 (GRIP1) which we named GRIP1d and GRIP1e 4-7. GRIP1d is a 135 kDa, 7-PDZ-domain variant of GRIP1, containing the 12 amino acid C-terminus originally described for the 4-PDZ-domain GRIP1c 4-7. GRIP1e 4-7 is a 75 kDa 4-PDZ-domain variant of GRIP1, containing the 12 amino acid C-terminus originally described for the 7-PDZ-domain GRIP1a/b. Northern blots indicated that GRIP1d mRNA is 5.1 kb long and abundant in brain. An antibody to the C-terminus of the 75 kDa GRIP1c 4-7 also recognized an abundant 135 kDa protein, consistent with the predicted size of GRIP1d. Similarly, an antibody to the C-terminus of the 135 kDa GRIP1a/b also recognized a low abundance 75 kDa protein, consistent with the predicted size of GRIP1e 4-7. Immunocytochemistry of hippocampal cultures and intact brain using these antibodies showed that (i) these isoforms are present in both GABAergic and glutamatergic synapses, and (ii) the isoforms co-localize in individual synapses. While GRIP1a/b isoforms are abundant in interneurons and highly concentrated in GABAergic presynaptic terminals, the isoforms recognized by the antibody to the C-terminus common to GRIP1c 4-7 and GRIP1d are much less abundant in interneurons and preferentially concentrate at the postsynaptic complex.

Subthalamic high frequency stimulation resets subthalamic firing and reduces abnormal oscillations

High frequency stimulation (HFS) of the subthalamic nucleus (STN) is a well-established therapeutic approach for the treatment of late-stage Parkinson's disease. Although the underlying cause of this illness remains a mystery, changes in firing rate and synchronized activity in different basal ganglia nuclei have been related to its symptoms. Here we investigated the impact of STN-HFS on firing rate as well as correlated and oscillatory activity in the STN network in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned non-human primates by using simultaneous extracellular single-unit recordings. STN-HFS reduced (i) the firing rate of STN neurons, (ii) the oscillatory activity at an individual STN neuron level as well as (iii) the correlated and oscillatory activity between pairs of STN neurons, while contralateral rigidity was improved. A detailed analysis showed that the decrease of mean firing rate resulted from the resetting of firing probability to virtually zero by the stimulus pulse. Subsequently, STN neurons resumed their activity after a mean duration of 2.9 +/- 0.1 ms and their firing probability returned to baseline values approximately 7 ms after the onset of the stimulus pulse, the recovery of the firing probability being represented by a sigmoid function. Thus, the overall decrease of the mean firing rate resulted from the repetition of this dynamical process with a frequency of 130 Hz (interstimulus interval approximately 7.7 ms), allowing the neuron to fire with its baseline firing rate only for a very short period. Although the mechanisms underlying the desynchronization of neuronal activity in the STN network remain unclear, the resetting of STN neuron firing probability by the electrical stimulus would rather be expected to increase oscillatory activity at an individual neuron level as well as correlated and oscillatory activity between pairs of STN neurons. However, assuming the resetting of firing rate to be the consequence of a transient GABAergic inhibition through excitation of presynaptic GABAergic axon terminals, different recovery periods of STN neurons might delay the appearance of synchronized oscillations, particularly if they are not generated locally. In conclusion, our study provides new evidence that STN-HFS decreases oscillatory activity in the STN network. Although the exact relation between oscillatory activity and Parkinson's disease symptoms remains to be determined, the present results suggest that STN-HFS might at least partially exert its beneficial effects through the reduction of oscillatory activity in the STN network and consequently in the entire cortex-basal ganglia-cortex network.

GRIP1 in GABAergic synapses

The glutamate receptor-interacting protein GRIP1 is present in glutamatergic synapses and interacts with the GluR2/3/4c subunits of the AMPA receptors. This interaction plays important roles in trafficking, synaptic targeting, and recycling of AMPA receptors as well as in the plasticity of glutamatergic synapses. Although GRIP1 has been shown to be present at GABAergic synapses in cultured neurons, the use of EM (electron microscopy) immunocytochemistry in the intact brain has failed to convincingly reveal the presence of GRIP1 in GABAergic synapses. Therefore, most studies on GRIP1 have focused on glutamatergic synapses. By using mild tissue fixation and embedding in EM, we show that in the intact brain the 7-PDZ domain GRIP1a/b is present not only in glutamatergic synapses but also in GABAergic synapses. In GABAergic synapses GRIP1a/b localizes both at the presynaptic terminals and postsynaptically, being frequently localized on the synaptic membranes or the synaptic junctional complex. Considerably higher density of GRIP1a/b is found in the presynaptic GABAergic terminals than in the glutamatergic terminals, while the density of GRIP1a/b in the postsynaptic complex is similar in both types of synapses. The results also show that the 7-PDZ and the shorter 4-PDZ domain splice forms of GRIP1 (GRIP1c 4-7) frequently colocalize with each other in individual GABAergic and glutamatergic synapses. The results suggest that GRIP1 splice forms might play important roles in brain GABAergic synapses.

A Conceptualization of Integrated Actions of Ethanol Contributing to its GABAmimetic Profile: A Commentary

Early behavioral investigations supported the contention that systemic ethanol displays a GABAmimetic profile. Microinjection of GABA agonists into brain and in vivo electrophysiological studies implicated a regionally specific action of ethanol on GABA function. While selectivity of ethanol to enhance the effect of GABA was initially attributed an effect on type-I-benzodiazepine (BZD)-GABA(A) receptors, a lack of ethanol's effect on GABA responsiveness from isolated neurons with this receptor subtype discounted this contention. Nonetheless, subsequent work identified GABA(A) receptor subtypes, with limited distribution in brain, sensitive to enhancement of GABA at relevant ethanol concentrations. In view of these data, it is hypothesized that the GABAmimetic profile for ethanol is due to activation of mechanisms associated with GABA function, distinct from a direct action on the majority of postsynaptic GABA(A) receptors. The primary action proposed to account for ethanol's regional specificity on GABA transmission is its ability to release GABA from some, but not all, presynaptic GABAergic terminals. As systemic administration of ethanol increases neuroactive steroids, which can enhance GABA responsiveness, this elevated level of neurosteroids is proposed to magnify the effect of GABA released by ethanol. Additional factors contributing to the degree to which ethanol interacts with GABA function include an involvement of GABA(B) and other receptors that influence ethanol-induced GABA release, an effect of phosphorylation on GABA responsiveness, and a regional reduction of glutamatergic tone. Thus, an integration of these consequences induced by ethanol is proposed to provide a logical basis for its in vivo GABAmimetic profile.

Capsaicin augments synaptic transmission in the rat medial preoptic nucleus

The medial preoptic nucleus (MPN) is the major nucleus of the preoptic area (POA), a hypothalamic area involved in the regulation of body-temperature. Injection of capsaicin into this area causes hypothermia in vivo. Capsaicin also causes glutamate release from hypothalamic slices. However, no data are available on the effect of capsaicin on synaptic transmission within the MPN. Here, we have studied the effect of exogenously applied capsaicin on spontaneous synaptic activity in hypothalamic slices of the rat. Whole-cell patch-clamp recordings were made from visually identified neurons located in the MPN. In a subset of the studied neurons, capsaicin enhanced the frequency of spontaneous glutamatergic EPSCs. Remarkably, capsaicin also increased the frequency of GABAergic IPSCs, an effect that was sensitive to removal of extracellular calcium, but insensitive to tetrodotoxin. This suggests an action of capsaicin at presynaptic GABAergic terminals. In contrast to capsaicin, the TRPV4 agonist 4α-PDD did not affect GABAergic IPSCs. Our results show that capsaicin directly affects synaptic transmission in the MPN, likely through actions at presynaptic terminals as well as on projecting neurons. Our data add to the growing evidence that capsaicin receptors are not only expressed in primary afferent neurons, but also contribute to synaptic processing in some CNS regions.

Maturation of glutamatergic and GABAergic synapse composition in hippocampal neurons

It is commonly accepted that glutamatergic and GABAergic presynaptic terminals form perfectly matched appositions opposite their appropriate receptors and associated binding proteins. However, recent reports indicate that certain synaptic proteins that are commonly used to identify excitatory or inhibitory synapses can be mismatched, particularly during development. In order to construct a more comprehensive scheme of synapse composition during development, we co-immunolabeled for several principle excitatory and inhibitory proteins over the course of synaptogenesis in cultured hippocampal neurons. We find that although the majority of synaptic appositions are composed of matched clusters of pre- and postsynaptic proteins appropriate for a particular neurotransmitter, many are initially mismatched, even in dendrites receiving both glutamatergic and GABAergic innervation. Over time, the fidelity of GABAergic synapse composition increases such that, despite the persistence of some mismatched components at glutamatergic sites, the incidence of mismatch diminishes at both inhibitory and excitatory synapses. Activation of either GABA-A or NMDA receptors promotes fidelity at GABAergic sites, but NMDA receptor activation promotes mismatching among glutamatergic synapses. Thus, apposition of pre- and postsynaptic elements can occur independent of neurotransmitter specificity and synaptic activity modifies these associations. Our findings support the idea that synapse maturation occurs in several distinct stages, and that these stages are regulated by a combination of activity-dependent and -independent factors.

GABAergic and glutamatergic axons innervate the axon initial segment and organize GABA(A) receptor clusters of cultured hippocampal pyramidal cells.

We have studied gamma-aminobutyric acid (GABA)(A) receptor (GABA(A)R) clustering within the axon initial segment (AIS) in low-density cultures of hippocampal pyramidal cells following GABAergic and glutamatergic innervation of the AIS. Large, intensely fluorescent, and postsynaptic GABA(A)R clusters were present in the AIS. More than 95% of these clusters colocalized with presynaptic GABAergic or glutamatergic terminals, forming matched or mismatched synapses, respectively. Less than 5% of the GABA(A)R clusters of the AIS did not colocalize with GABAergic or glutamatergic terminals, suggesting that GABA(A)Rs normally do not form clusters unless the AIS received GABAergic or glutamatergic innervation. Few or no clusters of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors or the postsynaptic density-95 protein (PSD-95) were found in the AIS, even when the AIS was innervated by glutamatergic axons. Glutamatergic innervation of the AIS that formed mismatched synapses with postsynaptic GABA(A)R clusters mainly occurred when the AIS did not receive GABAergic innervation. However, when the AIS was innervated by GABAergic axons, the formation of matched GABAergic synapses predominated and coincided with large reductions in both the density of glutamatergic terminals from the AIS and the mismatching of GABA(A)R clusters. A similar effect was observed at axo-dendritic synapses, where GABAergic innervation also led to a large decrease in mismatched GABA(A)R clusters and a smaller, but significant, decrease in glutamatergic terminal density in dendrites that received GABAergic innervation. We hypothesize that competition between GABAergic and glutamatergic innervation of the AIS in the intact hippocampus leads to the exclusive presence of GABAergic inhibitory synapses in the AIS of pyramidal cells.

GABAergic and glutamatergic terminals differentially influence the organization of GABAergic synapses in rat cerebellar granule cells in vitro

Synapse formation in CNS neurons requires appropriate sorting and clustering of neurotransmitter receptors and associated proteins at postsynaptic sites. In GABAergic synapses, clustering of GABA A receptors requires gephyrin, but it is not known whether presynaptic signals are also involved in this process. To investigate this issue, we analyzed the subcellular distribution of GABA A receptors and gephyrin in primary cultures of cerebellar granule cells, by comparing cells receiving GABAergic input with cells devoid of such afferents. Using immunofluorescence staining, we show that the GABA A receptor α1 and γ2 subunit, but not α6 or δ subunit, form clusters co-localized with gephyrin in granule cell neurites, irrespective of the presence of GABAergic axons. GABAergic terminals typically were surrounded by groups of gephyrin clusters, pointing to the presence of multiple synaptic sites. In contrast, in neurites devoid of GABAergic input, gephyrin clusters were distributed at random and apposed to glutamatergic terminals, suggesting the formation of mismatched synapses. Both populations of gephyrin clusters were co-localized with GABA A receptor subunits, indicating that these proteins are associated also in non-GABAergic synapses. To determine whether signaling mediated by GABA A receptors is required for the formation of appropriately matched gephyrin clusters, cultures were treated chronically with bicuculline, or with either muscimol or 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol. All these treatments failed to influence the distribution of gephyrin clusters. We conclude that although GABAergic presynaptic terminals have a preponderant influence on the distribution of gephyrin clusters in dendrites of cerebellar granule cells, GABA transmission is dispensable for postsynaptic clustering of gephyrin and GABA A receptors and for the formation of appropriately matched GABAergic synapses.

GABAergic Terminals Are Required for Postsynaptic Clustering of Dystrophin But Not of GABAA Receptors and Gephyrin

In rat hippocampal cultures, we show by multilabeling immunocytochemistry that pyramidal cells, which receive little or no GABAergic input, mistarget alpha2-GABA(A) receptors and gephyrin to glutamatergic terminals. This mismatch does not occur in neurons innervated by numerous GABAergic terminals. A similar phenomenon has been reported for isolated autaptic hippocampal neurons (Rao et al., 2000). GABAergic synapses typically form multiple release sites apposed to GABA(A) receptor and gephyrin clusters. Remarkably, dystrophin, a protein highly abundant in skeletal muscle membranes, is extensively colocalized with alpha2-GABA(A) receptors exclusively opposite GABAergic terminals. In addition, selective apposition of syntrophin and beta-dystroglycan to GABAergic presynaptic terminals suggests that the entire dystrophin-associated protein complex (DPC) clusters at GABAergic synapses. In contrast to gephyrin and GABA(A) receptors, DPC proteins are not mistargeted to glutamatergic synapses, indicating independent clustering mechanisms. This was confirmed in hippocampal neurons cultured from GABA(A) receptor gamma2 subunit-deficient mice. Clustering of GABA(A) receptor and gephyrin in these neurons was strongly impaired, whereas clustering of dystrophin and associated proteins was unaffected by the absence of the gamma2 subunit. Our results indicate that accumulation of dystrophin and DPC proteins at GABAergic synapses occurs independently of postsynaptic GABA(A) receptors and gephyrin. We suggest that selective signaling from GABAergic terminals contributes to postsynaptic clustering of dystrophin.

Inhibitory effects of 1,4-DHP antagonists on synaptic GABA release modulated by BAY-K 8644 in mechanically dissociated rat substantia innominata

The effects of dihydropyridine (1,4-DHP) agonist and antagonists on miniature inhibitory postsynaptic currents (mIPSCs) were investigated in mechanically dissociated rat substantia innominata neurons attached to native GABAergic presynaptic nerve terminals, namely ‘synaptic bouton preparation’, using nystatin perforated patch recording mode under voltage-clamp conditions. BAY-K 8644 (BAY-K), an L-type Ca 2+ channel agonist, reversibly and concentration dependently facilitated the GABAergic mIPSC frequency without altering the distribution of current amplitudes. Removal of extracellular Ca 2+ completely suppressed the facilitatory effect of BAY-K on mIPSC frequency. The facilitatory effect of BAY-K on mIPSC frequency was maintained even in the presence of selective N-, P- and Q-type Ca 2+ channel antagonists, such as 3 × 10 −6 M ω-conotoxin-GVIA (ω-CgTX-GVIA), 3 × 10 −8 M ω-agatoxin-IVA (ω-AgTX-IVA) and 3 × 10 −6M ω-conotoxin-MVIIC (ω-CmTX-MVIIC). However, nicardipine (3 × 10 −6 M) and nimodipine (3 × 10 −6 M), 1,4-DHP antagonists, significantly inhibited the mIPSC frequency enhanced by BAY-K by 37 ± 5 and 42 ± 6%, respectively. These results suggest the possible existence of L-type Ca 2+ channels in GABAergic presynaptic nerve terminals.

Presynaptic inhibition of GABAergic miniature currents by metabotropic glutamate receptor in the rat CNS

The modulation of spontaneous miniature GABAergic inhibitory postsynaptic currents (mIPSC) by the metabotropic glutamate receptors was investigated in the mechanically dissociated rat nucleus basalis of Meynert neurons using the conventional whole-cell patch recording configuration. An application of (±)-1-aminocyclopentane- trans-1,3-dicarboxylic acid (tACPD) reversibly reduced the frequency of mIPSC without affecting the current amplitude distribution. The application of K + channel blockers such as 4-aminopyridine, Cs +, Ba 2+ or tetraethylammonium increased the mIPSC frequency, but failed to inhibit the tACPD action on mIPSC. Although the removal of Ca 2+ from the extracellular solution reduced the mIPSC frequency, the inhibitory effect of tACPD on mIPSC was unaltered. These results suggested that neither voltage-dependent K + or Ca 2+ channels are involved in the inhibitory effect of tACPD on mIPSC frequency. Forskolin, an activator of adenylate cyclase, facilitated the mIPSC frequency in a concentration-dependent manner and inhibited the tACPD-induced suppression of mIPSC frequency. 8-Br-cAMP, a membrane permeable analog of cAMP, also prevented the inhibitory action of tACPD. However, Sp-cAMP, an activator of protein kinase A, could not prevent the inhibitory action of tACPD. L-CCG-I and (2 R,4 R)-APDC, group II mGluR agonists, mimicked the tACPD action on mIPSC frequency, but L-AP4, a group III mGluR agonist, had no such effect. MCCG, a group II mGluR antagonist, fully blocked the tACPD action. It was concluded that the activation of group II mGluR on the GABAergic presynaptic nerve terminals projecting to the rat nucleus basalis of Meynert neurons therefore inhibits the GABA release by reducing the activity of the cAMP-dependent pathway.

Agonist-Induced Internalization and Trafficking of Cannabinoid CB1Receptors in Hippocampal Neurons

Agonist-induced internalization of G-protein-coupled receptors is an important mechanism for regulating receptor abundance and availability at the plasma membrane. In this study we have used immunolabeling techniques and confocal microscopy to investigate agonist-induced internalization and trafficking of CB(1) receptors in rat cultured hippocampal neurons. The levels of cell surface CB(1) receptor immunoreactivity associated with presynaptic GABAergic terminals decreased markedly (by up to 84%) after exposure to the cannabinoid agonist (+)-WIN55212, in a concentration-dependent (0.1-1 microm) and stereoselective manner. Inhibition was maximal at 16 hr and abolished in the presence of SR141716A, a selective CB(1) receptor antagonist. Methanandamide (an analog of an endogenous cannabinoid, anandamide) also reduced cell surface labeling (by 43% at 1 microm). Differential labeling of cell surface and intracellular pools of receptor demonstrated that the reduction in cell surface immunoreactivity reflects agonist-induced internalization and suggests that the internalized CB(1) receptors are translocated toward the soma. The internalization process did not require activated G-protein alpha(i) or alpha(o) subunits. A different pattern of cell surface CB(1) receptor expression was observed using an undifferentiated F-11 cell line, which had pronounced somatic labeling. In these cells substantial CB(1) receptor internalization was also observed after exposure to (+)-WIN55212 (1 microm) for relatively short periods (30 min) of agonist exposure. In summary, this dynamic modulation of CB(1) receptor expression may play an important role in the development of cannabinoid tolerance in the CNS. Agonist-induced internalization at presynaptic terminals has important implications for the modulatory effects of G-protein-coupled receptors on neurotransmitter release.

Presynaptic Kainate Receptors that Enhance the Release of GABA on CA1 Hippocampal Interneurons

We report that kainate receptors are present on presynaptic GABAergic terminals contacting interneurons and that their activation increases GABA release. Application of kainate increased the frequency of miniature inhibitory postsynaptic currents recorded in CA1 interneurons. Local applications of glutamate but not of AMPA or NMDA also increased GABA quantal release. Application of kainate as well as synaptically released glutamate reduced the number of failures of GABAergic neurotransmission between interneurons. Thus, activation of presynaptic kainate receptors increases the probability of GABA release at interneuron–interneuron synapses. Glutamate may selectively control the communication between interneurons by increasing their mutual inhibition.

Myelinated dendrites in the mormyrid electrosensory lobe.

This is the third paper in a series on the morphology, immunohistochemistry, and synaptology of the mormyrid electrosensory lateral line lobe (ELL). The ELL is a highly laminated, cerebellum-like structure in the rhombencephalon that subserves an active electric sense: Objects in the nearby environment are detected on the basis of changes in the reafferent electrosensory signals that are generated by the animal's own electric organ discharge. This paper concentrates on the intermediate (cell and fiber) layer of the medial zone of the ELL and pays particular attention to the large multipolar neurons of this layer (LMI cells). LMI cells are gamma-aminobutyric acid (GABA)ergic and have one axon and three to seven proximal dendrites that all become myelinated after their last proximal branching point. The axon projects to the contralateral homotopic region and has ipsilateral collaterals. Both ipsilaterally and contralaterally, it terminates in the deep and superficial granular layers. The myelinated dendrites end in the deep granular layer, where they most likely do not make postsynaptic specializations, but do make presynaptic specializations, similar to those of the LMI axons. Because it is not possible to distinguish between axonal and dendritic LMI terminals in the granular layer, the authors refer to both as LMI terminals. These are densely filled with small, flattened vesicles and form large appositions with ELL granular cell somata and dendrites with symmetric synaptic membrane specializations. LMI cells do not receive direct electrosensory input on their somata, but electrophysiological recordings suggest that they nevertheless respond strongly to electrosensory signals (Bell [1990] J. Neurophysiol. 63:303-318). Consequently, the authors speculate that the myelinated dendrites of LMI cells are excited ephaptically (i.e., by electric field effects) by granular cells, which, in turn, are excited via mixed synapses by mormyromast primary afferents. The authors suggest that this ephaptic activation of the GABAergic presynaptic terminals of the myelinated dendrites may trigger immediate synaptic release of GABA and, thus, may provide a very fast local feedback inhibition of the excited granular cells in the center of the electrosensory receptive field. Subsequent propagation of the dendritic excitation down the myelinated dendrites to the somata and axon hillocks of LMI cells probably generates somatic action potentials, resulting in the spread of inhibition through axonal terminals to a wide region around the receptive field center and in the contralateral ELL. Similar presynaptic myelinated dendrites that subserve feedback inhibition, until now, have not been described elsewhere in the brain of vertebrates.