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PubMed2026

Induced pluripotent stem cell reprogramming: methodological evolution and challenges in clinical translation.

Cell reprogramming can transform somatic cells into induced pluripotent stem cells providing a platform for patient-specific disease modeling, drug screening and regenerative medicine research. Since the advent of OKSM-mediated reprogramming, the system of technical approaches has evolved continuously - from integrated viral vectors to non-integrated episomal systems and, more recently, chemical reprogramming and CRISPR approaches. The simultaneous advances in single-cell multi-omics, biomaterials engineering, and artificial intelligence have further refined the controllability and precision of the reprogramming process. Despite these innovations, problems persist that hinder clinical translation: incomplete epigenetic resetting, ongoing clonal heterogeneity, genomic instability in long-term culture, and the lack of standardized Good Manufacturing Practice protocols for large-scale manufacturing. This review summarizes the trajectory of iPSC reprogramming technologies, with special emphasis on the translational applicability of each modality. We evaluated viral and nonviral delivery systems, chemical reprogramming, strategies that aid gene editing, and emerging engineering platforms, including microfluidics, smart biomaterials, and artificial-intelligence-driven process optimization. We further identify the core "translational triltrilas", namely, the inherent tradeoffs between security, homogeneity, and scalability, and propose a comprehensive strategy to overcome these bottlenecks. By linking basic mechanistic understandings with industrial and regulatory considerations, this review aims to provide a route for transitioning iPSC technology from a laboratory tool to a clinically viable manufacturing platform.

clinical translation↗
PubMed2002

Opioid peptides inhibit excitatory but not inhibitory synaptic transmission in the rat dorsal motor nucleus of the vagus.

Opioid peptides produce gastrointestinal inhibition and increase feeding when applied to the brainstem. The present studies were designed to determine the actions of opioid peptides on synaptic transmission within the dorsal motor nucleus of the vagus (DMV) and the localization of mu-opioid receptors. Whole-cell recordings were made from identified gastrointestinal-projecting DMV neurons in thin brainstem slices of the rat. Electrical stimulation of the nucleus of the tractus solitarius evoked EPSCs and IPSCs. In all neurons tested, methionine (Met)-enkephalin (0.003-30 microm) inhibited the peak amplitude of the EPSCs. The effect was prevented by naloxone (1 microm) as well as by naloxonazine (0.2 microm). An increase in the ratio of the evoked paired pulses indicated that the inhibition was attributable to actions at presynaptic receptors. This presynaptic inhibitory action was mimicked by [d-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (0.1 microm) and the analgesic dipeptide kyotorphin (10 microm) but not by cyclic[d-Pen(2), d-Pen(5)]-enkephalin (1 microm) and trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate (1 microm). In contrast, the amplitude of evoked IPSCs was not altered either by Met-enkephalin or by any of the opioid receptor-selective agonists. Immunohistochemical studies revealed that nerve terminals apposing DMV neurons showed immunoreactivity to mu-opioid receptors colocalized with glutamate immunoreactivity but not glutamic acid decarboxylase immunoreactivity. These results suggest that within the DMV, mu-opioid receptors are present on the nerve terminals of excitatory but not inhibitory inputs to GI motoneurons. Such specificity may imply that the central inhibitory action of opioid peptides on gastrointestinal function targets selected pathways.

Animals↗
PubMed2002

Presynaptic cannabinoid sensitivity is a major determinant of depolarization-induced retrograde suppression at hippocampal synapses.

Recent studies have clarified that endogenous cannabinoids (endocannabinoids) are released from depolarized postsynaptic neurons in a Ca(2+)-dependent manner and act retrogradely on presynaptic cannabinoid receptors to suppress inhibitory or excitatory neurotransmitter release. This type of modulation has been found in the hippocampus and cerebellum and was called depolarization-induced suppression of inhibition (DSI) or excitation (DSE). In this study, we quantitatively examined the effects of postsynaptic depolarization and a cannabinoid agonist on excitatory and inhibitory synapses in rat hippocampal slices and cultures. We found that both DSE and DSI can be induced, but DSE was much less prominent than DSI. For the induction of DSE, the necessary duration of depolarization was longer than for DSI. The magnitude of DSE was much smaller than that of DSI. To explore the reasons for these differences, we tested the sensitivity of EPSCs and IPSCs to a cannabinoid agonist, WIN55,212-2, in hippocampal cultures. IPSCs were dichotomized into two distinct populations, one with a high sensitivity to WIN55,212-2 (50% block at 2 nm) and the other with no sensitivity. In contrast, EPSCs were homogeneous and exhibited a low sensitivity to WIN55,212-2 (50% block at 60 nm). We estimated that the 5 sec depolarization elevated the local endocannabinoid concentration to a level equivalent to several nanomoles of WIN55,212-2. Using CB1 knock-out mice, we verified that both DSI and DSE were mediated by the cannabinoid CB1 receptor. These results indicate that presynaptic cannabinoid sensitivity is a major factor that determines the extent of DSI and DSE.

Animals↗
PubMed2002

In vivo imaging of functional inhibitory networks on the mauthner cell of larval zebrafish.

Noninvasive in vivo calcium imaging was used to observe and characterize inhibitory circuitry in intact larval zebrafish. In the teleost hindbrain, the inhibitory network onto the major pair of reticulospinal neurons known as Mauthner cells (M-cells) has been described in detail. There are three sources of inhibition onto M-cells: recurrent inhibition mediated by an ipsilateral collateral of the M-cell axon, feedforward inhibition driven by sensory afferents, and reciprocal inhibition between bilaterally opposed M-cells. To visualize these inhibitions, M-cells were retrogradely loaded with the calcium indicator calcium green dextran. Recurrent inhibition attenuated the Ca(2+) response associated with an action potential in M-cells. Whole-cell recording revealed recurrent IPSCs, the conductance of which may underlie the shunting effect on action potentials and the attenuation of the Ca(2+) signal in M-cells. Blocking synaptic transmission within the recurrent network abolished both the Ca(2+) signal attenuation and the IPSCs. Electrical stimulation of the otic vesicle to activate VIII nerve afferents resulted in feedforward suppression of antidromically evoked test Ca(2+) responses in the contralateral M-cell. Orthodromic activation of M-cells produced a reciprocal reduction of the test Ca(2+) response in the contralateral M-cell. Thus, in the present study, we visualized the three types of inhibition and demonstrated that they are functional at 4 d after fertilization. The use of noninvasive techniques to image inhibition in vivo suggest the plausibility of studying the hypothesis previously tested in adult goldfish that use-dependent changes in inhibitions underlie sound conditioning in escape behavior.

Action Potentials↗
PubMed2002

Two different mechanisms of disinhibition produced by GABAA receptor mutations linked to epilepsy in humans.

The first mutations of the GABA(A) receptor channel linked to familial epilepsy in humans were reported recently (Baulac et al., 2001; Wallace et al., 2001). Preliminary functional analysis of alpha1beta2gamma2 GABA(A) receptors expressed in Xenopus oocytes suggested that the gamma2 subunit R43Q mutation abolished current enhancement by the benzodiazepine, diazepam, and that the gamma2 subunit K289M mutation decreased current amplitudes. We used single-channel recording and concentration jump techniques applied to outside out patches to evaluate the impact of these mutations on GABA(A) receptor channel function of the highly conserved rat ortholog subunits expressed in human embryonic kidney cells. When coexpressed with alpha1 and beta3 subunits, no differences were observed between wild-type and mutant GABA(A) receptor current activation rates or rates or extent of desensitization during prolonged (400 msec) GABA application (1 mm). Although deactivation after brief (5 msec) or prolonged (400 msec) GABA application was unaltered by the R43Q mutation, deactivation (a correlate of IPSC duration) was accelerated for the K289M mutation. Faster deactivation was likely a consequence of altered gating, because single-channel openings had shorter mean duration. Interestingly, the R43Q mutation did not alter diazepam potentiation. It did, however, substantially decrease current amplitude, which was not caused by decreased single-channel conductance or open time, suggesting reduced surface expression of functional receptors. The two gamma2 subunit mutations likely produce disinhibition and familial epilepsy by distinct mechanisms, suggesting that maintenance of neuronal inhibition depends not only on the peak amplitude of IPSCs, but also on their time course.

Cells, Cultured↗
PubMed1999

Chlorpromazine inhibits miniature GABAergic currents by reducing the binding and by increasing the unbinding rate of GABAA receptors.

Recent studies have emphasized that nonequilibrium conditions of postsynaptic GABAA receptor (GABAAR) activation is a key factor in shaping the time course of IPSCs (Puia et al., 1994; Jones and Westbrook, 1995). Such nonequilibrium, resulting from extremely fast agonist time course, may affect the interaction between pharmacological agents and postsynaptic GABAARs. In the present study we found that chlorpromazine (CPZ), a widely used antipsychotic drug known to interfere with several ligand and voltage-gated channels, reduces the amplitude and accelerates the decay of miniature IPSCs (mIPSCs). A good qualitative reproduction of the effects of CPZ on mIPSCs was obtained when mIPSCs were mimicked by responses to ultrafast GABA applications to excised patches. Our experimental data and model simulations indicate that CPZ affects mIPSCs by decreasing the binding (kon) and by increasing the unbinding (koff) rates of GABAARs. Because of reduction of kon by CPZ, the binding reaction becomes rate-limiting, and agonist exposure of GABAARs during mIPSC is too short to activate the receptors to the same extent as in control conditions. The increase in unbinding rate is implicated as the mechanism underlying the acceleration of mIPSC decaying phase. The effect of CPZ on GABAAR binding rate, resulting in slower onset of GABA-evoked currents, provides a tool to estimate the speed of synaptic clearance of GABA. Moreover, the onset kinetics of recorded responses allowed the estimate the peak synaptic GABA concentration.

Animals↗
PubMed2000

Effects of halothane on GABA(A) receptor kinetics: evidence for slowed agonist unbinding.

Many anesthetics, including the volatile agent halothane, prolong the decay of GABA(A) receptor-mediated IPSCs at central synapses. This effect is thought to be a major factor in the production of anesthesia. A variety of different kinetic mechanisms have been proposed for several intravenous agents, but for volatile agents the kinetic mechanisms underlying this change remain unknown. To address this question, we used rapid solution exchange techniques to apply GABA to recombinant GABA(A) receptors (alpha(1)beta(2)gamma(2s)) expressed in HEK 293 cells, in the absence and presence of halothane. To differentiate between different microscopic kinetic steps that may be altered by the anesthetic, we studied a variety of measures, including peak concentration-response characteristics, macroscopic desensitization, recovery from desensitization, maximal current activation rates, and responses to the low-affinity agonist taurine. Experimentally observed alterations were compared with predictions based on a kinetic scheme that incorporated two agonist binding steps, and open and desensitized states. We found that, in addition to slowing deactivation after a brief pulse of GABA, halothane increased agonist sensitivity and slowed recovery from desensitization but did not alter macroscopic desensitization or maximal activation rate and only slightly slowed rapid deactivation after taurine application. This pattern of responses was found to be consistent with a reduction in the microscopic agonist unbinding rate (k(off)) but not with changes in channel gating steps, such as the channel opening rate (beta), closing rate (alpha), or microscopic desensitization. We conclude that halothane slows IPSC decay by slowing dissociation of agonist from the receptor.

Anesthetics, Inhalation↗
PubMed2000

Functional GluR6 kainate receptors in the striatum: indirect downregulation of synaptic transmission.

Kainate receptors (KARs) are abundantly expressed in the basal ganglia, but their function in synaptic transmission has not been established. In the present study, we show that the GluR6 subunit of KARs is expressed in both substance P- and enkephalin-containing GABAergic projection neurons of the mouse striatum. Using whole-cell voltage-clamp recordings in brain slices, we demonstrate the presence of functional KARs in the dorsal striatum activated by low concentrations of the AMPA/KAR agonist domoate in wild-type but not GluR6-deficient mice. Despite the abundance of KARs, we found no evidence for synaptic activation of these receptors after single or repetitive stimulation of glutamatergic afferents. Domoate induces a transient increase in the frequency of spontaneous IPSCs of small amplitude and a sustained depression of large IPSCs evoked by minimal electrical stimulation within the striatum in wild-type mice but not in GluR6-deficient mice. This depressant effect is inhibited in presence of adenosine A(2A) receptor antagonists, ZM-241385 and SCH-58261. These data strongly suggest that, in striatal neurons, KARs depress GABAergic synaptic transmission indirectly via release of adenosine acting on A(2A) receptors.

Animals↗
PubMed2000

Analysis of presynaptic Ca2+ influx and transmitter release kinetics during facilitation at the inhibitor of the crayfish neuromuscular junction.

The inhibitory synapse of the crayfish neuromuscular junction was used to examine mechanisms underlying the F2 component of synaptic facilitation. Because previous studies have shown accelerated transmitter release during facilitation, we examined whether an activity-dependent plasticity in I(Ca) could underlie this acceleration. We established that fluorescent transients generated by Magnesium Green can resolve small differences in presynaptic Ca(2+) influx that correlate with changes in IPSC waveform. However, there was no change in Ca(2+) transients associated with the accelerated release. Analyzing the initial rise of IPSC and the duration of the presynaptic spike yielded a depolarization-release coupling plot that captures the impact of spike waveform on the initial rate of release. We conclude that accelerated release during F2 facilitation cannot be attributed to plasticity of I(Ca) or modulation of spike waveform. Kinetic analysis showed a reduction in synaptic delay during facilitation only when broad action potentials were used. In unfacilitated release, synaptic delay increased as spike duration lengthened. We propose that small single Ca(2+) channel currents during the plateau phase of broad action potentials raise local Ca(2+) concentration only enough to fill a high-affinity site. Occupation of this site in itself, or events downstream, would convert a vesicle from control to facilitated state. If the conversion were a slow process, it could explain the changes in synaptic delay reported here. This hypothesis can also account for a number of observations related to Ca(2+) cooperativity and synaptic facilitation.

Action Potentials↗
PubMed2001

Disinhibition in rat superior colliculus mediated by GABAc receptors.

The stratum griseum superficiale (SGS) of the superior colliculus contains a high concentration of the recently described GABA(C) receptor. In a previous study, it was postulated that activation of these receptors on inhibitory interneurons functions to disinhibit projection cells that relay visual information to the thalamus and brainstem. To test this model, we used in vitro whole-cell patch-clamp methods to measure effects of GABA and muscimol on EPSCs and IPSCs evoked in rat SGS by electrical optic layer stimulation. The neurons were filled with biocytin for later morphological characterization. As expected, bath applications of GABA and muscimol always strongly depressed evoked PSCs at concentrations of >100 and >1 micrometer, respectively. However, at lower agonist concentrations, which most likely activate GABA(C) but not GABA(A) receptors, effects were not uniform. Evoked responses were suppressed by both agonists in 48% of the neurons, whereas the remaining cells exhibited enhanced responses with increased evoked EPSCs, decreased evoked IPSCs, or both types of change. Most morphologically identified cells with suppressed responses (14 of 17 cells) had morphological characteristics of putative GABAergic interneurons, whereas almost all cells with enhanced responses (8 of 10 cells) had morphological characteristics of projection cells. Finally, all effects of GABA and muscimol at low concentrations were blocked by (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid, a specific GABA(C) receptor antagonist, but not by the specific GABA(A) receptor antagonist bicuculline. Taken together, these results indicate that in SGS, GABA(C) receptors are predominantly expressed by GABAergic neurons and that activation of these receptors leads to disinhibition of SGS projection cells.

Animals↗
PubMed2001

Rapid signaling at inhibitory synapses in a dentate gyrus interneuron network.

Mutual synaptic interactions between GABAergic interneurons are thought to be of critical importance for the generation of network oscillations and for temporal encoding of information in the hippocampus. However, the functional properties of synaptic transmission between hippocampal interneurons are largely unknown. We have made paired recordings from basket cells (BCs) in the dentate gyrus of rat hippocampal slices, followed by correlated light and electron microscopical analysis. Unitary GABA(A) receptor-mediated IPSCs at BC-BC synapses recorded at the soma showed a fast rise and decay, with a mean decay time constant of 2.5 +/- 0.2 msec (32 degrees C). Synaptic transmission at BC-BC synapses showed paired-pulse depression (PPD) (32 +/- 5% for 10 msec interpulse intervals) and multiple-pulse depression during repetitive stimulation. Detailed passive cable model simulations based on somatodendritic morphology and localization of synaptic contacts further indicated that the conductance change at the postsynaptic site was even faster, decaying with a mean time constant of 1.8 +/- 0.6 msec. Sequential triple recordings revealed that the decay time course of IPSCs at BC-BC synapses was approximately twofold faster than that at BC-granule cell synapses, whereas the extent of PPD was comparable. To examine the consequences of the fast postsynaptic conductance change for the generation of oscillatory activity, we developed a computational model of an interneuron network. The model showed robust oscillations at frequencies >60 Hz if the excitatory drive was sufficiently large. Thus the fast conductance change at interneuron-interneuron synapses may promote the generation of high-frequency oscillations observed in the dentate gyrus in vivo.

Action Potentials↗
PubMed2001

ATP-sensitive K+ channels and cellular actions of morphine in periaqueductal gray slices of neonatal and adult rats.

ATP-sensitive K+ (K(ATP)) channels were reported to be involved in morphine analgesia in vivo. The present study, using patch-clamp technique in brain slices of neonatal (P12-P16) and adult rats, investigated cellular actions of K(ATP) channel ligands and their interactions with morphine in the ventrolateral periaqueductal gray (PAG), a crucial site for morphine analgesia. In neonatal PAG neurons, morphine depressed evoked inhibitory postsynaptic currents (IPSCs) in almost all tested neurons and elicited an inwardly rectifying K+ current in one-third of tested neurons. Glibenclamide (1-10 microM), a K(ATP) channel blocker, did not affect the membrane current or synaptic current per se but also failed to affect the effects of morphine. No outward current was elicited upon using microelectrodes containing ATP-free internal solution. In adult neurons, morphine, at the concentration up to 300 microM, failed to activate K+ current in all 25 neurons tested but depressed IPSCs to a comparable extent as that in neonatal neurons. Glibenclamide also failed to alter the effect of morphine in adult neurons. The openers of K(ATP) channels, lemakalim (10-30 microM) and diazoxide (10-500 microM), unlike morphine, did not increase membrane currents in both neonatal and adult neurons. However, diazoxide induced a glibenclamide-sensitive outward current in hippocampal CA1 neurons. It is concluded that K(ATP) channels display little functional role per se and might not be involved in effects of morphine in the ventrolateral PAG. The correlation between the insensitivity in K+ channel activation and the less antinociceptive response to morphine in adults was discussed.

ATP-Binding Cassette Transporters↗
PubMed2001

Metabotropic glutamate receptors 1 and 5 differentially regulate CA1 pyramidal cell function.

The activation of group I metabotropic glutamate receptors (mGluRs) produces a variety of actions that lead to alterations in excitability and synaptic transmission in the CA1 region of the hippocampus. The group I mGluRs, mGluR1 and mGluR5, are activated selectively by (S)-3,5-dihydroxyphenylglycine (DHPG). To identify which of these mGluR subtypes are responsible for the various actions of DHPG in area CA1, we took advantage of two novel subtype-selective antagonists. (S)-(+)-alpha-amino-a-methylbenzeneacetic acid (LY367385) is a potent competitive antagonist that is selective for mGluR1, whereas 2-methyl-6-(phenylethynyl)-pyridine (MPEP) is a potent noncompetitive antagonist that is selective for mGluR5. The use of these compounds in experiments with whole-cell patch-clamp recording and Ca(2+)-imaging techniques revealed that each group I mGluR subtype plays distinct roles in regulating the function of CA1 pyramidal neurons. The block of mGluR1 by LY367385 suppressed the DHPG-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the direct depolarization of CA1 hippocampal neurons. In addition, the increase in the frequency of spontaneous IPSCs (sIPSCs) caused by the DHPG-induced depolarization of inhibitory interneurons also was blocked by LY367385, as was the DHPG-induced inhibition of transmission at the Schaffer collateral-->CA1 synapse. On the other hand, the block of mGluR5 by MPEP antagonized the DHPG-induced suppression of the Ca(2+)-activated potassium current (I(AHP)) and potentiation of the NMDA receptor. Finally, antagonism of the DHPG-induced suppression of evoked IPSCs required the blockade of both mGluR1 and mGluR5. These data suggest that mGluR1 and mGluR5 play distinct roles in the regulation of the excitability of hippocampal CA1 pyramidal neurons.

Animals↗
PubMed2002

Antioscillatory effects of nociceptin/orphanin FQ in synaptic networks of the rat thalamus.

Postsynaptic and presynaptic effects of nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the opioid-like orphan receptor, were investigated in an in vitro slice preparation of the rat thalamic reticular nucleus (NRT) and ventrobasal complex (VB). In NRT as well as VB, all tested neurons developed an outward current on application of 1 micrometer N/OFQ. Basic properties of the N/OFQ-induced current included inward rectification, dependence on extracellular K(+), reduction by 100 micrometer Ba(+), antagonistic effect of [Nphe(1)]nociceptin(1-13)NH(2), and sensitivity to internal GDP-beta-S. Miniature IPSCs (mIPSCs) mediated by GABA(A) receptors in VB neurons were not affected by 1 micrometer N/OFQ. In addition, paired-pulse depression of evoked IPSCs was unchanged, indicating a lack of presynaptic effects. By comparison, N/OFQ application resulted in a reduction in frequency of miniature EPSCs (mEPSCs) in a subpopulation of NRT neurons, whereas paired-pulse facilitation of evoked EPSCs was not altered. In either nucleus, current-clamp experiments revealed a hyperpolarization and associated decrease in input resistance in response to N/OFQ. Although N/OFQ had no measurable effect on calcium-mediated burst activity evoked by depolarizing steps from hyperpolarized values of the membrane potential, rebound bursts on relief of hyperpolarizing current steps were decreased. Slow thalamic oscillations induced in vitro by extracellular stimulation were dampened by N/OFQ in VB and NRT, as seen by delayed onset of rhythmic multiple-unit activity and reduction in amplitude and duration. We conclude that N/OFQ reduces the excitability of NRT and VB neurons predominantly through an increase of a G-protein-coupled inwardly rectifying K(+) conductance.

Animals↗
PubMed2002

The cannabinoid CB1 receptor mediates retrograde signals for depolarization-induced suppression of inhibition in cerebellar Purkinje cells.

Action potential firing or depolarization of the postsynaptic neuron can induce a transient suppression of inhibitory synaptic inputs to the depolarized neuron in the cerebellum and hippocampus. This phenomenon, termed depolarization-induced suppression of inhibition (DSI), is initiated postsynaptically by an elevation of intracellular Ca2+ concentration ([Ca2+]i) and is expressed presynaptically as a suppression of the transmitter release. It is, therefore, thought that some retrograde signal must exist from the depolarized postsynaptic neurons to the presynaptic terminals. Recent studies on hippocampal neurons have revealed that endogenous cannabinoids (endocannabinoids) play a key role as a retrograde messenger. There are, however, conflicting reports that glutamate may be a candidate retrograde messenger for cerebellar DSI that acts on presynaptic group II metabotropic glutamate receptors (mGluRs). In this study, we examined whether endocannabinoids mediate retrograde signal for cerebellar DSI. We recorded IPSCs from Purkinje cells by stimulating putative basket cell axons in mouse cerebellar slices. DSI was readily induced in evoked IPSCs by a depolarizing pulse train. We found that DSI was completely occluded by a cannabinoid agonist, WIN55,212-2, was totally eliminated by a specific antagonist of the type 1 cannabinoid (CB1) receptor, SR141716A, and was deficient in the CB1 knock-out mouse. In contrast, a group II mGluR-specific agonist, (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine, did not completely occlude DSI, and an mGluR antagonist, (RS)-alpha-methyl-4-carboxyphenylglycine, had no depressant effect on DSI. These results clearly indicate that the CB1 receptor mediates retrograde signal for DSI in cerebellar Purkinje cells.

Animals↗
PubMed2002

New intrathalamic pathways allowing modality-related and cross-modality switching in the dorsal thalamus.

Transmission through the dorsal thalamus involves nuclei that convey different aspects of sensory or motor information. Cells in the dorsal thalamus are strongly inhibited by the GABAergic cells of the thalamic reticular nucleus (TRN). Here we show that stimulation of cells in specific dorsal thalamic nuclei evokes robust IPSCs or IPSPs in other specific dorsal thalamic nuclei and vice versa. These IPSCs are GABA(A) receptor-mediated currents and are consistent with the activation of disynaptic intrathalamic pathways mediated by TRN. Thus, cells engaged in sensory analyses in the ventrobasal complex or the medial division of the posterior complex can interact with cells responsive to sensory events in the caudal intralaminar nuclei, whereas cells engaged in motor analyses in the ventrolateral nucleus can interact with cells responsive to motor events in the rostral intralaminar nuclei. Furthermore, sensory event-related cells in the caudal intralaminar nuclei can interact with motor event-related cells in the rostral intralaminar nuclei. In addition, single cells in one dorsal thalamic nucleus can receive convergent inhibitory inputs after stimulation of cells in two or more other dorsal thalamic nuclei, and TRN-mediated inhibitory inputs can momentarily switch off tonic firing of action potentials in dorsal thalamic cells. Our findings provide the first direct evidence for a rich network of intrathalamic pathways that allows modality-related and cross-modality inhibitory modulation between dorsal thalamic nuclei. Moreover, TRN-mediated switching between dorsal thalamic nuclei could provide a mechanism for the selection of competing transmissions of sensory and/or motor information through the dorsal thalamus.

Action Potentials↗
PubMed2003

GABA(B) receptors inhibit dendrodendritic transmission in the rat olfactory bulb.

In the mammalian olfactory bulb, mitral cell dendrites release glutamate onto the dendritic spines of granule cells, which in turn release GABA back onto mitral dendrites. This local synaptic circuit forms the basis for reciprocal dendrodendritic inhibition mediated by ionotropic GABA(A) receptors in mitral cells. Surprisingly little is known about neurotransmitter modulation of dendrodendritic signaling in the olfactory bulb. In this study, we examine whether metabotropic GABA(B) receptors modulate dendrodendritic signaling between mitral and granule cells. We find that the selective GABA(B) agonist baclofen reduces mitral cell recurrent inhibition mediated by dendrodendritic synapses. GABA(B) receptor activation causes only a weak inhibition of field EPSCs in the external plexiform layer and only slightly reduces glutamate-mediated mitral cell self-excitation. Although GABA(B) receptors depress mitral cell glutamate release only weakly, baclofen causes a marked reduction in the amplitude of granule-cell-evoked, GABA(A)-mediated IPSCs in mitral cells. In addition to reducing the amplitude of granule-cell-evoked IPSCs, baclofen causes a change from paired-pulse depression to paired-pulse facilitation, suggesting that GABA(B) receptors modulate GABA release from granule cells. To explore the mechanism of action of GABA(B) receptors further, we show that baclofen inhibits high-voltage-activated calcium currents in granule cells. Together, these findings suggest that GABA(B) receptors modulate dendrodendritic inhibition primarily by inhibiting granule cell calcium channels and reducing the release of GABA. Furthermore, we show that endogenous GABA regulates the strength of dendrodendritic inhibition via the activation of GABA(B) autoreceptors.

Animals↗
PubMed2003

Neuronal glutamate uptake Contributes to GABA synthesis and inhibitory synaptic strength.

Neurons must maintain a supply of neurotransmitter in their presynaptic terminals to fill synaptic vesicles. GABA is taken up into inhibitory terminals by transporters or is synthesized from glutamate by glutamic acid decarboxylase. Here we report that glutamate transporters supply GABAergic terminals in the hippocampus with glutamate, which is then used to synthesize GABA for filling synaptic vesicles. Glutamate transporter antagonists reduced IPSC and miniature IPSC (mIPSC) amplitudes, consistent with a reduction in the amount of GABA packaged into each synaptic vesicle. This reduction occurred rapidly and independently of synaptic activity, suggesting that modulation of vesicular GABA content does not require vesicle release and refilling. Raising extracellular glutamate levels increased mIPSC amplitudes by enhancing glutamate uptake and, consequently, GABA synthesis. These results indicate that neuronal glutamate transporters strengthen inhibitory synapses in response to extracellular glutamate. This modulation appears to occur under normal conditions and may constitute a negative feedback mechanism to combat hyperexcitability.

Amino Acid Transport System X-AG↗