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We tested the hypothesis that nicotine exposure in utero and after birth [developmental nicotine exposure (DNE)] disrupts development of glycinergic synaptic transmission to hypoglossal motoneurons (XIIMNs). Glycinergic spontaneous and miniature inhibitory postsynaptic currents (sIPSC/mIPSC) were recorded from XIIMNs in brain stem slices from 1- to 5-day-old rat pups of either sex, under baseline conditions and following stimulation of nicotinic acetylcholine (ACh) receptors with nicotine (i.e., an acute nicotine challenge). Under baseline conditions, there were no significant effects of DNE on the amplitude or frequency of either sIPSCs or mIPSCs. In addition, DNE did not alter the magnitude of the whole cell current evoked by bath application of glycine, consistent with an absence of change in postsynaptic glycine-mediated conductance. An acute nicotine challenge (bath application of 0.5 μM nicotine) increased sIPSC frequency in the DNE cells, but not control cells. In contrast, nicotine challenge did not change mIPSC frequency in either control or DNE cells. In addition, there were no significant changes in the amplitude of either sIPSCs or mIPSCs in response to nicotine challenge. The increased frequency of sIPSCs in response to an acute nicotine challenge in DNE cells reflects an enhancement of action potential-mediated input from glycinergic interneurons to hypoglossal motoneurons. This could lead to more intense inhibition of hypoglossal motoneurons in response to exogenous nicotine or endogenous ACh. The former would occur with smoking or e-cigarette use while the latter occurs with changes in sleep state and with hypercapnia.NEW & NOTEWORTHY Here we show that perinatal nicotine exposure does not impact baseline glycinergic neurotransmission to hypoglossal motoneurons but enhances glycinergic inputs to hypoglossal motoneurons in response to activation of nicotinic acetylcholine (ACh) receptors with acute nicotine. Given that ACh is the endogenous ligand for nicotinic ACh receptors, the latter reveals a potential mechanism whereby perinatal nicotine exposure alters motor function under conditions where ACh release increases, such as the transition from non-rapid-eye movement to rapid-eye movement sleep, and during hypercapnia.

Motor neuron (MN) development in early onset spasticity is poorly understood. For example, spastic cerebral palsy (sCP), the most common motor disability of childhood, is poorly predicted by brain imaging, yet research remains focused on the brain. By contrast, MNs, via the motor unit and neurotransmitter signaling, are the target of most therapeutic spasticity treatments and are the final common output of motor control. MN development in sCP is a critical knowledge gap, because the late embryonic and postnatal periods are not only when the supposed brain injury occurs but also are critical times for spinal cord neuromotor development. Using an animal model of early onset spasticity [ spa mouse (B6.Cg- Glrbspa/J) with a glycine (Gly) receptor mutation], we hypothesized that removal of effective glycinergic neurotransmitter inputs to MNs during development will influence MN pruning (including primary dendrites) and MN size. Spa (Glrb−/−) and wild-type (Glrb+/+) mice, ages 4–9 wk, underwent unilateral retrograde labeling of the tibialis anterior muscle MNs via peroneal nerve dip in tetramethylrhodamine. After 3 days, mice were euthanized and perfused with 4% paraformaldehyde, and the spinal cord was excised and processed for confocal imaging. Spa mice had ~61% fewer lumbar tibialis anterior MNs ( P < 0.01), disproportionately affecting larger MNs. Additionally, a ~23% reduction in tibialis anterior MN somal surface area ( P < 0.01) and a 12% increase in primary dendrites ( P = 0.046) were observed. Thus MN pruning and MN somal surface area are abnormal in early onset spasticity. Fewer and smaller MNs may contribute to the spastic phenotype.NEW & NOTEWORTHY Motor neuron (MN) development in early onset spasticity is poorly understood. In an animal model of early onset spasticity, spa mice, we found ~61% fewer lumbar tibialis anterior MNs compared with controls. This MN loss disproportionately affected larger MNs. Thus number and heterogeneity of the MN pool are decreased in spa mice, likely contributing to the spastic phenotype.

The effect of capsaicin on glycinergic synaptic transmission to juvenile rat hypoglossal motor neurons in acute brainstem slices was evaluated in the presence of TTX. Capsaicin caused a robust decrease in miniature IPSC frequency, amplitude, and half-width, showing that this effect is independent of action potential generation. In the presence of capsazepine, a classic TRPV1 antagonist, capsaicin was still able to reduce spontaneous inhibitory postsynaptic current (IPSC) amplitude and frequency. We further investigated whether the effect of capsaicin on glycinergic transmission to hypoglossal motor neurons is pre- or postsynaptic in nature by recording pairs of evoked IPSCs. Interestingly, capsaicin also reduced evoked IPSC amplitude without affecting paired-pulse ratio, indicating a postsynaptic mechanism of action. Significant reduction was also observed in evoked IPSC half-width, rise time, and decay tau. We also show that capsaicin does not have any effect on either transient (It) or sustained (Is) potassium currents. Finally, we also show that the hyperpolarization-activated cationic current (Ih) also remains unchanged after capsaicin application. NEW & NOTEWORTHY Capsaicin reduces the amplitude of quantal and evoked glycinergic inhibitory neurotransmission to brainstem motor neurons without altering activity-dependent transmitter release. This effect of capsaicin is not due to activation of TRPV1 receptors, as it is not blocked by capsazepine, a TRPV1 receptor antagonist. Capsaicin does not alter voltage-dependent potassium current or the hyperpolarization-activated cationic current in brainstem motor neurons.

During development GABA and glycine synapses are initially excitatory before they gradually become inhibitory. This transition is due to a developmental increase in the activity of neuronal potassium-chloride cotransporter 2 (KCC2), which shifts the chloride equilibrium potential ( ECl) to values more negative than the resting membrane potential. While the role of early GABA and glycine depolarizations in neuronal development has become increasingly clear, the role of the transition to hyperpolarization in synapse maturation and circuit refinement has remained an open question. Here we investigated this question by examining the maturation and developmental refinement of GABA/glycinergic and glutamatergic synapses in the lateral superior olive (LSO), a binaural auditory brain stem nucleus, in KCC2-knockdown mice, in which GABA and glycine remain depolarizing. We found that many key events in the development of synaptic inputs to the LSO, such as changes in neurotransmitter phenotype, strengthening and elimination of GABA/glycinergic connection, and maturation of glutamatergic synapses, occur undisturbed in KCC2-knockdown mice compared with wild-type mice. These results indicate that maturation of inhibitory and excitatory synapses in the LSO is independent of the GABA and glycine depolarization-to-hyperpolarization transition.

In the central nervous system, inhibition shapes neuronal excitation. In spinal cord glycinergic inhibition predominates, whereas GABAergic inhibition predominates in the brain. The retina uses GABA and glycine in approximately equal proportions. Glycinergic crossover inhibition, initiated in the On retinal pathway, controls glutamate release from presynaptic OFF cone bipolar cells (CBCs) and directly shapes temporal response properties of OFF retinal ganglion cells (RGCs). In the retina, four glycine receptor (GlyR) α-subunit isoforms are expressed in different sublaminae and their synaptic currents differ in decay kinetics. GlyRα1, expressed in both On and Off sublaminae of the inner plexiform layer, could be the glycinergic isoform that mediates On-to-Off crossover inhibition. However, subunit-selective glycine contributions remain unknown because we lack selective antagonists or cell class-specific subunit knockouts. To examine the role of GlyRα1 in direct inhibition in mature RGCs, we used retrogradely transported adeno-associated virus (AAV) that performed RNAi and eliminated almost all glycinergic spontaneous and visually evoked responses in PV5 (OFFαTransient) RGCs. Comparisons of responses in PV5 RGCs infected with AAV-scrambled-short hairpin RNA (shRNA) or AAV- Glra1-shRNA confirm a role for GlyRα1 in crossover inhibition in cone-driven circuits. Our results also define a role for direct GlyRα1 inhibition in setting the resting membrane potential of PV5 RGCs. The absence of GlyRα1 input unmasked a serial and a direct feedforward GABAAergic modulation in PV5 RGCs, reflecting a complex interaction between glycinergic and GABAAergic inhibition.

Neurons in the medial nucleus of the trapezoid body (MNTB) are well known for their prominent excitatory inputs, mediated by the calyx of Held. Less attention has been paid to the prominent inhibitory inputs that MNTB neurons also receive. Because of their auditory nature, both excitatory and inhibitory synapses are highly active in vivo. These high levels of activity are known to reduce excitatory synaptic currents considerably, such that in vivo synaptic currents produced by the calyx are smaller than typically measured in standard brain slice experiments. The goal of this study was to investigate the properties of the inhibitory inputs in the Mongolian gerbil ( Meriones unguiculatus) under activity levels that correspond to those in the intact brain to facilitate a direct comparison between the two inputs. Our results suggest that inhibitory inputs to MNTB are largely mediated by a fast and phasic glycinergic component, and to a lesser degree by a GABAergic component. The glycinergic component can sustain prolonged high levels of activity. Even when challenged with stimulus patterns consisting of thousands of stimuli over tens of minutes, glycinergic inputs to MNTB maintain large conductances and fast decays and even facilitate substantially when the stimulation frequency is increased. The inhibition is mediated by a relatively small number of independent input fibers. The data presented here suggest that inhibitory inputs to MNTB sustain high levels of activity and need to be considered for a full understanding of mechanisms underlying processing of auditory information in MNTB.

Processing of signals within the cerebral cortex requires integration of synaptic inputs and a coordination between excitatory and inhibitory neurotransmission. In addition to the classic form of synaptic inhibition, another important mechanism that can regulate neuronal excitability is tonic inhibition via sustained activation of receptors by ambient levels of inhibitory neurotransmitter, usually GABA. The purpose of this study was to determine whether this occurs in layer II/III pyramidal neurons (PNs) in the prelimbic region of the mouse medial prefrontal cortex (mPFC). We found that these neurons respond to exogenous GABA and to the α4δ-containing GABAA receptor (GABAAR)-selective agonist gaboxadol, consistent with the presence of extrasynaptic GABAAR populations. Spontaneous and miniature synaptic currents were blocked by the GABAAR antagonist gabazine and had fast decay kinetics, consistent with typical synaptic GABAARs. Very few layer II/III neurons showed a baseline current shift in response to gabazine, but almost all showed a current shift (15–25 pA) in response to picrotoxin. In addition to being a noncompetitive antagonist at GABAARs, picrotoxin also blocks homomeric glycine receptors (GlyRs). Application of the GlyR antagonist strychnine caused a modest but consistent shift (∼15 pA) in membrane current, without affecting spontaneous synaptic events, consistent with the tonic activation of GlyRs. Further investigation showed that these neurons respond in a concentration-dependent manner to glycine and taurine. Inhibition of glycine transporter 1 (GlyT1) with sarcosine resulted in an inward current and an increase of the strychnine-sensitive current. Our data demonstrate the existence of functional GlyRs in layer II/III of the mPFC and a role for these receptors in tonic inhibition that can have an important influence on mPFC excitability and signal processing.

Glutamate release at bipolar to ganglion cell synapses activates NMDA and AMPA/kainic acid (KA) ionotropic glutamate receptors. Their relative strength determines the output signals of the retina. We found that this balance is tightly regulated by presynaptic inhibition that preferentially suppresses NMDA receptor (NMDAR) activation. In transient ON-OFF neurons, block of GABA and glycine feedback enhanced total NMDAR charge by 35-fold in the ON response and 9-fold in the OFF compared with a 1.7-fold enhancement of AMPA/KA receptors. Blocking only glycine receptors enhanced the NMDAR excitatory postsynaptic current 10-fold in the ON and 2-fold in the OFF pathway. Blocking GABAA or GABAC receptors (GABACRs or GABAARs) produced small changes in total NMDAR charge. When both GABAARs and GABACRs were blocked, the total NMDAR charge increased ninefold in the ON and fivefold in the OFF pathway. This exposed a strong GABACR feedback to bipolar cells that was suppressed by serial amacrine cell synapses mediated by GABAARs. The results indicate that NMDAR currents are large but latent, held in check by dual GABA and glycine presynaptic inhibition. One example of this controlled NMDAR activation is the cross talk between ON and OFF pathways. Blocking the ON pathway increased NMDAR relative strength in the OFF pathway. Stimulus prolongation similarly increased the NMDAR relative strength in the OFF response. This NMDAR enhancement was produced by a diminution in GABA and glycine feedback. Thus the retinal network recruits NMDAR pathways through presynaptic disinhibition.

Localization of low-frequency acoustic stimuli is processed in dedicated neural pathways where coincidence-detecting neurons compare the arrival time of sound stimuli at the two ears, or interaural time disparity (ITD). ITDs occur in the submillisecond range, and vertebrates have evolved specialized excitatory and inhibitory circuitry to compute these differences. Glycinergic inhibition is a computationally significant and prominent component of the mammalian ITD pathway. However, evidence for glycinergic transmission is limited in birds, where GABAergic inhibition has been thought to be the dominant or exclusive inhibitory transmitter. Indeed, previous work showed that GABA antagonists completely eliminate inhibition in avian nuclei specialized for processing temporal features of sound, nucleus magnocellularis (NM) and nucleus laminaris (NL). However, more recent work shows that glycine is coexpressed with GABA in synaptic terminals apposed to neurons in both nuclei (Coleman WL, Fischl MJ, Weimann SR, Burger RM. J Neurophysiol 105: 2405–2420, 2011; Kuo SP, Bradley LA, Trussell LO. J Neurosci 29: 9625–9634, 2009). Here we show complementary evidence of functional glycine receptor (GlyR) expression in NM and NL. Additionally, we show that glycinergic input can be evoked under particular stimulus conditions. Stimulation at high but physiologically relevant rates evokes a slowly emerging glycinergic response in NM and NL that builds over the course of the stimulus. Glycinergic response magnitude was stimulus rate dependent, representing 18% and 7% of the total inhibitory current in NM and NL, respectively, at the end of the 50-pulse, 200-Hz stimulus. Finally, we show that the glycinergic component is functionally relevant, as its elimination reduced inhibition of discharges evoked by current injection into NM neurons.

Interaural intensity differences are analyzed in neurons of the lateral superior olive (LSO) by integration of an inhibitory input from the medial nucleus of the trapezoid body (MNTB), activated by sound from the contralateral ear, with an excitatory input from the ipsilateral cochlear nucleus. The early postnatal refinement of this inhibitory MNTB-LSO projection along the tonotopic axis of the LSO has been extensively studied. However, little is known to what extent physiological changes at these inputs also occur after the onset of sound-evoked activity. Using whole-cell patch-clamp recordings of LSO neurons in acute brain stem slices, we analyzed the developmental changes of inhibitory synaptic currents evoked by MNTB fiber stimulation occurring after hearing onset. We compared these results in gerbils and mice, two species frequently used in auditory research. Our data show that neither the number of presumed input fibers nor the conductance of single fibers significantly changed after hearing onset. Also the amplitude of miniature inhibitory currents remained constant during this developmental period. In contrast, the kinetics of inhibitory synaptic currents greatly accelerated after hearing onset. We conclude that tonotopic refinement of inhibitory projections to the LSO is largely completed before the onset of hearing, whereas acceleration of synaptic kinetics occurs to a large part after hearing onset and might thus be dependent on proper auditory experience. Surprisingly, inhibitory input characteristics, as well as basic membrane properties of LSO neurons, were rather similar in gerbils and mice.

One of the characteristics of respiratory motor output is the presence of fast synchronous oscillations, at rates far exceeding the basic breathing rhythm, within a given functional population. However, the mechanisms responsible for organizing phrenic output into two dominant bands in vivo, medium (MFO)- and high (HFO)-frequency oscillations, have yet to be elucidated. We hypothesize that GABAAergic and glycinergic inhibition within the phrenic motor nucleus underlies the specific organization of these oscillations. To test this, the phrenic nuclei (C4) of 14 unanesthetized, decerebrate adult male Sprague-Dawley rats were microinjected unilaterally with either 4 mM strychnine ( n = 7) or GABAzine ( n = 7) to block glycine or GABAA receptors, respectively. Application of GABAzine caused an increase in overall phrenic amplitude during all three phases of respiration (inspiration, postinspiration, and expiration), while the increases caused by strychnine were most pronounced during postinspiration. Neither antagonist produced changes in inspiratory duration or respiratory rate. Power spectral analysis of inspiratory phrenic bursts showed that blockade of inhibition caused significant reduction in the relative power of MFO (GABAA and glycine receptors) and HFO (GABAA receptors only). In addition, analysis of the coherence between the firing of the ipsi- and contralateral phrenic nerves revealed that HFO coupling was significantly reduced by both antagonists and that of MFO was significantly reduced only by strychnine. We conclude that both GABAA and glycine receptors play critical roles in the organization of fast oscillations into MFO and HFO bands in the phrenic nerve, as well as in their bilateral coupling.

The effects of Ca2+, Sr2+, and Ba2+on spontaneous and evoked glycinergic inhibitory postsynaptic currents (mIPSCs and eIPSCs) were studied using the “synaptic bouton” preparation of rat spinal neurons and conventional whole cell recording under voltage-clamp conditions. In response to application of Ca2+-free solution, the frequency of mIPSC initially rapidly decreased to 40∼50% of control followed by a gradual further decline in mIPSC frequency to ∼30% of control. Once mIPSC frequency had significantly decreased in Ca2+-free solution, application of Ca2+, Sr2+, or Ba2+increased mIPSC frequency. The rank order of effect in restoring mIPSCs was Ba2+≫ Ca2+> Sr2+. Moreover, the application of excess external [K+]osolution (30 mM) containing Sr2+or Ba2+after 2 h in Ca2+-free solution also increased mIPSC frequency in the order Sr2+≧ Ba2+> Ca2+. The mean mIPSC amplitude was not affected at all. In contrast, eIPSCs produced by focal stimulation of single boutons were completely abolished in Ca2+-free solution or when Ca2+was replaced by Sr2+or Ba2+(2 mM each). However, eIPSCs were restored in increased concentrations of Sr2+or Ba2+(5 mM each). The results show that these divalent cations affect mIPSC and eIPSCs differently and indicate that the mechanisms underlying transmitter release that generates eIPSCs and mIPSC in presynaptic nerve terminals are different. The different mechanisms might be explained by the different sensitivity of synaptotagmin isoforms to Ca2+, Sr2+, and Ba2+.

We studied the interactions between excitation and inhibition in morphologically identified amacrine cells in the light-adapted rabbit retinal slice under patch clamp. The majority of on amacrine cells received glycinergic off inhibition. About half of the off amacrine cells received glycinergic on inhibition. Neither class received any GABAergic inhibition. A minority of on, off, and on–off amacrine cells received both glycinergic on and GABAergic off inhibition. These interactions were found in cells with diverse morphologies having both wide and narrow processes that stratify in single or multiple layers of the inner plexiform layer (IPL). Most on–off amacrine cells received no inhibition and have monostratified processes confined to the middle of the IPL. The most common interaction between amacrine cells that we measured was “crossover inhibition,” where off inhibits on and on inhibits off. Although the morphology of amacrine cells is diverse, the interactions between excitation and inhibition appear to be relatively limited and specific.

The development of the cerebral cortex depends on genetic factors and early electrical activity patterns that form immature neuronal networks. Subplate neurons (SPn) are involved in the construction of thalamocortical innervation, generation of oscillatory network activity, and in the proper formation of the cortical columnar architecture. Because glycine receptors play an important role during early corticogenesis, we analyzed the functional consequences of glycine receptor activation in visually identified SPn in neocortical slices from postnatal day 0 (P0) to P4 rats using whole cell and perforated patch-clamp recordings. In all SPn the glycinergic agonists glycine, β-alanine, and taurine induced dose-dependent inward currents with the affinity for glycine being higher than that for β-alanine and taurine. Glycine-induced responses were blocked by the glycinergic antagonist strychnine, but were unaffected by either the GABAergic antagonist gabazine, the N-methyl-d-aspartate–receptor antagonist d-2-amino-5-phosphonopentanoic acid, or picrotoxin and cyanotriphenylborate, antagonists of α-homomeric and α1-subunit–containing glycine receptors, respectively. Under perforated-patch conditions, glycine induced membrane depolarizations that were sufficient to trigger action potentials (APs) in most cells. Furthermore, glycine and taurine decreased the injection currents as well as the synaptic stimulation strength required to elicit APs, indicating that glycine receptors have a consistent excitatory effect on SPn. Inhibition of taurine transport and application of hypoosmolar solutions induced strychnine-sensitive inward currents, suggesting that taurine can act as a possible endogenous agonist on SPn. In summary, these results demonstrate that SPn express glycine receptors that mediate robust excitatory membrane responses during early postnatal development.

The zebrafish is an attractive model system for studying the function of the spinal locomotor network by combining electrophysiological, imaging, and genetic approaches. Thus far, most studies have been focusing on embryonic and larval stages. In this study we have developed an in vitro preparation of the isolated spinal cord from adult zebrafish in which locomotor activity can be induced while the activity of single neurons can be monitored using whole cell recording techniques. Application of NMDA elicited rhythmic locomotor activity that was monitored by recording from muscles or ventral roots in semi-intact or isolated spinal cord preparations, respectively. This rhythmic activity displayed a left–right alternation and a rostrocaudal delay. Blockade of glycinergic synaptic transmission by strychnine switched the alternating activity into synchronous bursting in the left and right sides as well as along the rostrocaudal axis. Whole cell recordings from motoneurons showed that they receive phasic synaptic inputs that were correlated with the locomotor activity recorded in ventral roots. This newly developed in vitro preparation of the adult zebrafish spinal cord will allow examination of the organization of the spinal locomotor network in an adult system to complement studies in zebrafish larvae and new born rodents.

Although extracellular Zn2+ is an endogenous biphasic modulator of strychnine-sensitive glycine receptors (GlyRs), the physiological significance of this modulation remains poorly understood. Zn2+ modulation of GlyR may be especially important in the hippocampus where presynaptic Zn2+ is abundant. Using cultured embryonic mouse hippocampal neurons, we examined whether 1 μM Zn2+, a potentiating concentration, enhances the inhibitory effects of GlyRs activated by sustained glycine applications. Sustained 20 μM glycine (EC25) applications alone did not decrease the number of action potentials evoked by depolarizing steps, but they did in 1 μM Zn2+. At least part of this effect resulted from Zn2+ enhancing the GlyR-induced decrease in input resistance. Sustained 20 μM glycine applications alone did not alter neuronal bursting, a form of hyperexcitability induced by omitting extracellular Mg2+. However, sustained 20 μM glycine applications depressed neuronal bursting in 1 μM Zn2+. Zn2+ did not enhance the inhibitory effects of sustained 60 μM glycine (EC70) applications in these paradigms. These results suggest that tonic GlyR activation could decrease neuronal excitability. To test this possibility, we examined the effect of the GlyR antagonist strychnine and the Zn2+ chelator tricine on action potential firing by CA1 pyramidal neurons in mouse hippocampal slices. Co-applying strychnine and tricine slightly but significantly increased the number of action potentials fired during a depolarizing current step and decreased the rheobase for action potential firing. Thus Zn2+ may modulate neuronal excitability normally and in pathological conditions such as seizures by potentiating GlyRs tonically activated by low agonist concentrations.

Cartwheel cells are glycinergic interneurons that modify somatosensory input to the dorsal cochlear nucleus. They are characterized by firing of mixtures of both simple and complex action potentials. To understand what ion channels determine the generation of these two types of spike waveforms, we recorded from cartwheel cells using the gramicidin perforated-patch technique in brain slices of mouse dorsal cochlear nucleus and applied channel-selective blockers. Complex spikes were distinguished by whether they arose directly from a negative membrane potential or later during a long depolarization. Ca2+ channels and Ca2+-dependent K+ channels were major determinants of complex spikes. Onset complex spikes required T-type and possibly R-type Ca2+ channels and were shaped by BK and SK K+ channels. Complex spikes arising later in a depolarization were dependent on P/Q- and L-type Ca2+ channels as well as BK and SK channels. BK channels also contributed to fast repolarization of simple spikes. Simple spikes featured an afterdepolarization that is probably the trigger for complex spiking and is shaped by T/R-type Ca2+ and SK channels. Fast spikes were dependent on Na+ channels; a large persistent Na+ current may provide a depolarizing drive for spontaneous activity in cartwheel cells. Thus the diverse electrical behavior of cartwheel cells is determined by the interaction of a wide variety of ion channels with a prominent role played by Ca2+.

Activation of spinal muscarinic acetylcholine receptors (mAChRs) produces analgesia and inhibits dorsal horn neurons through potentiation of GABAergic/glycinergic tone and inhibition of glutamatergic input. To investigate the mAChR subtypes involved in the inhibitory effect of mAChR agonists on glutamate release, evoked excitatory postsynaptic currents (eEPSCs) were recorded in lamina II neurons using whole cell recordings in rat spinal cord slices. The nonselective mAChR agonist oxotremorine-M concentration-dependently inhibited the monosynaptic and polysynaptic EPSCs elicited by dorsal root stimulation. Interestingly, oxotromorine-M caused a greater inhibition of polysynaptic EPSCs (64.7%) than that of monosynaptic EPSCs (27.9%). In rats pretreated with intrathecal pertussis toxin, oxotremorine-M failed to decrease monosynaptic EPSCs but still partially inhibited the polysynaptic EPSCs in some neurons. This remaining effect was blocked by a relatively selective M3 antagonist 4-DAMP. Himbacine, an M2/M4 antagonist, or AFDX-116, a selective M2 antagonist, completely blocked the inhibitory effect of oxotremorine-M on monosynaptic EPSCs. However, the specific M4 antagonist MT-3 did not alter the effect of oxotremorine-M on monosynaptic EPSCs. Himbacine also partially attenuated the effect of oxotremorine-M on polysynaptic EPSCs in some cells and this effect was abolished by 4-DAMP. Furthermore, oxotremorine-M significantly decreased spontaneous EPSCs in seven of 22 (31.8%) neurons, an effect that was blocked by 4-DAMP. This study provides new information that the M2 mAChRs play a critical role in the control of glutamatergic input from primary afferents to dorsal horn neurons. The M3 and M2/M4 subtypes on a subpopulation of interneurons are important for regulation of glutamate release from interneurons in the spinal dorsal horn.

Fast inhibitory synaptic transmission in the medial vestibular nucleus (MVN) is mediated by GABAA receptors (GABAARs) and glycine receptors (GlyRs). To assess their relative contribution to inhibition in the MVN, we recorded miniature inhibitory postsynaptic currents (mIPSCs) in physiologically characterized type A and type B MVN neurons. Transverse brain stem slices were prepared from mice (3–8 wk old), and whole cell patch-clamp recordings were obtained from visualized MVN neurons (CsCl internal; Vm = –70 mV; 23°C). In 81 MVN neurons, 69% received exclusively GABAAergic inputs, 6% exclusively glycinergic inputs, and 25% received both types of mIPSCs. The mean amplitude of GABAAR-mediated mIPSCs was smaller than those mediated by GlyRs (22.6 ± 1.8 vs. 35.3 ± 5.3 pA). The rise time and decay time constants of GABAAR- versus GlyR-mediated mIPSCs were slower (1.3 ± 0.1 vs. 0.9 ± 0.1 ms and 10.5 ± 0.3 vs. 4.7 ± 0.3 ms, respectively). Comparison of type A ( n = 20) and type B ( n = 32) neurons showed that type A neurons received almost exclusively GABAAergic inhibitory inputs, whereas type B neurons received GABAAergic inputs, glycinergic inputs, or both. Intracellular labeling in a subset of MVN neurons showed that morphology was not related to a MVN neuron's inhibitory profile ( n = 15), or whether it was classified as type A or B ( n = 29). Together, these findings indicate that both GABA and glycine contribute to inhibitory synaptic processing in MVN neurons, although GABA dominates and there is a difference in the distribution of GABAA and Gly receptors between type A and type B MVN neurons.

An inhibitory role for strychnine-sensitive glycine-gated chloride channels (GlyRs) in mature hippocampus is beginning to be appreciated. We have reported previously that CA1 pyramidal cells and GABAergic interneurons recorded in 3- to 4-wk-old rat hippocampal slices express functional GlyRs, dispelling previous misconceptions that GlyR expression ceases in early development. However, the effect of GlyR activation on cell excitability and synaptic circuits in hippocampus has not been fully explored. Using whole cell current-clamp recordings, we show that activation of strychnine-sensitive GlyRs through exogenous glycine application causes a significant decrease in input resistance and prevents somatically generated action potentials in both CA1 pyramidal cells and interneurons. Furthermore, GlyR activation depresses the synaptic network by reducing suprathreshold excitatory postsynaptic potentials (EPSPs) to subthreshold events in both cell types. Blockade of postsynaptic GlyRs with the chloride channel blocker 4, 4′-diisothiocyanatostilbene-2-2′-disulfonic acid (DIDS) or altering the chloride ion driving force in recorded cells attenuates the synaptic depression, strongly indicating that a postsynaptic mechanism is responsible. Increasing the local glycine concentration by blocking reuptake causes a strychnine-sensitive synaptic depression in interneuron recordings, suggesting that alterations in extracellular glycine will impact excitability in hippocampal circuits. Finally, using immunohistochemical methods, we show that glycine and the glycine transporter GlyT2 are co-localized selectively in GABAergic interneurons, indicating that interneurons contain both inhibitory neurotransmitters. Thus we report a novel mechanism whereby activation of postsynaptic GlyRs can function to depress activity in the synaptic network in hippocampus. Moreover, the co-localization of glycine and GABA in hippocampal interneurons, similar to spinal cord, brain stem, and cerebellum, suggests that this property is likely to be a general characteristic of inhibitory interneurons throughout the CNS.

Neurotransmitter receptor plasticity is a mechanism that can regulate the temporal and intensity encoding of a synapse. While this has been extensively studied as a mechanism of learning, less is known about such processes in sensory systems. This study examines modulation of glycine receptor function at the first synapse in the retina. It was found that horizontal cells, which are postsynaptic to photoreceptors, have glycine receptor currents that are enhanced when internal calcium is elevated. This can be achieved by glutamatergic synaptic input or by activation of voltage-gated calcium channels. When the retina was maintained in a dark-adapted state, the calcium levels in horizontal cells were relatively low. After a series of brief light stimuli, the internal calcium concentration in horizontal cells was elevated, and the glycine currents were faster and greater in amplitude. The increase of internal calcium levels was caused by increased transmitter release from photoreceptors. Thus glycine receptor function is state dependent and can be rapidly altered by synaptic input from photoreceptors. Light stimulation drives glycine receptor plasticity in the retinal neural network.

Maturation of some brain stem and spinal inhibitory systems is characterized by a shift from GABAergic to glycinergic transmission. Little is known about how this transition is expressed in terms of individual axonal inputs and synaptic sites. We have explored this issue in the rat medial nucleus of the trapezoid body (MNTB). Synaptic responses at postnatal days 5–7 (P5–P7) were small, slow, and primarily mediated by GABAA receptors. By P8–P12, an additional, faster glycinergic component emerged. At these ages, GABAA, glycine, or both types of receptors mediated transmission, even at single synaptic sites. Thereafter, glycinergic development greatly accelerated. By P25, evoked inhibitory postsynaptic currents (IPSCs) were 10 times briefer and 100 times larger than those measured in the youngest group, suggesting a proliferation of synaptic inputs activating fast-kinetic receptors. Glycinergic miniature IPSCs (mIPSCs) increased markedly in size and decay rate with age. GABAergic mIPSCs also accelerated, but declined slightly in amplitude. Overall, the efficacy of GABAergic inputs showed little maturation between P5 and P20. Although gramicidin perforated-patch recordings revealed that GABA or glycine depolarized P5–P7 cells but hyperpolarized P14–P15 cells, the young depolarizing inputs were not suprathreshold. In addition, vesicle-release properties of inhibitory axons also matured: GABAergic responses in immature rats were highly asynchronous, while in older rats, precise, phasic glycinergic IPSCs could transmit even with 500-Hz stimuli. Thus development of inhibition is characterized by coordinated modifications to transmitter systems, vesicle release kinetics, Cl− gradients, receptor properties, and numbers of synaptic inputs. The apparent switch in GABA/glycine transmission was predominantly due to enhanced glycinergic function.

Previously, we showed that ethanol increases synaptic glycine currents, an effect that depends on ethanol concentration and developmental age of the preparation. Glycine receptor (GlyR) subunits undergo a shift from α2/β to α1/β from neonate to juvenile ages, with synaptic glycine currents from neonate hypoglossal motoneurons (HMs) being less sensitive to ethanol than those from juvenile HMs. Here we investigate whether these dose and developmental effects are also present in excised membrane patches containing GlyRs and if ethanol changes response kinetics. We excised outside-out patches from rat HM somata and applied glycine using either a picospritzer or piezo stack translator. Ethanol (100 mM) increased the response to glycine (200 μM) of patches from neonate and juvenile HMs. However, 30 mM ethanol increased the response from only juvenile HM patches. Using a lower concentration of glycine (30 μM) to observe single channel openings, we found that 100 mM ethanol increased the number of GlyRs that open in response to glycine and decreased first latency to channel opening. To investigate GlyR kinetic properties, we rapidly applied 1 mM glycine for 1 ms and found that glycine currents were increased by ethanol (100 mM) at both ages. For patches from juvenile HMs, ethanol consistently decreased response rise-time and increased response decay time. Using kinetic modeling, we determined that ethanol's potentiation of the glycine response arises from an increase in the glycine association ( kon) and a decrease in the dissociation ( koff) rate constants, resulting in increased glycine affinity of the GlyR.

We have investigated the fundamental properties of central auditory glycinergic synapses in early postnatal development in normal and congenitally deaf ( dn/dn) mice. Glycinergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded using patch-clamp methods in neurons from a brain slice preparation of the medial nucleus of the trapezoid body (MNTB), at 12-14 days postnatal age. Our results show a number of significant differences between normal and deaf mice. The frequency of mIPSCs is greater (50%) in deaf versus normal mice. Mean mIPSC amplitude is smaller in deaf mice than in normal mice (mean mIPSC amplitude: deaf, 64 pA; normal, 106 pA). Peak-scaled fluctuation analysis of mIPSCs showed that mean single channel conductance is greater in the deaf mice (deaf, 64 pS; normal, 45 pS). The mean decay time course of mIPSCs is slower in MNTB neurons from deaf mice (mean half-width: deaf, 2.9 ms; normal, 2.3 ms). Light- and electron-microscopic immunolabeling results showed that MNTB neurons from deaf mice have more (30%) inhibitory synaptic sites (postsynaptic gephyrin clusters) than MNTB neurons in normal mice. Our results demonstrate substantial differences in glycinergic transmission in normal and congenitally deaf mice, supporting a role for activity during development in regulating both synaptic structure (connectivity) and the fundamental (quantal) properties of mIPSCs at central glycinergic synapses.

Although functional glycine receptors (GlyRs) are present in the mature nucleus accumbens (NAcc), an important area of the mesolimbic dopamine system involved in drug addiction, their role has been unclear because the NAcc contains little glycine. However, taurine, an agonist of GlyRs, is abundant throughout the brain, especially during early development. In the present study on freshly dissociated NAcc neurons from young Sprague-Dawley rats (12- to 21-day old), we found that both glycine and taurine can strongly depolarize NAcc neurons and modulate their excitability. In voltage-clamped NAcc neurons, glycine and taurine elicited chloride currents ( IGly and ITau) with an EC50 of 0.12 and 1.25 mM, respectively. The reversal potential of IGly or ITau was 0 mV in conventional whole cell mode and –30 mV in gramicidin-perforated mode. At concentrations 50 of 60 nM and 36.5 μM for IGly, and 40 nM and 42.2 μM for ITau) but were insensitive to 10 μM bicuculline. The currents elicited by taurine (≤1 mM) showed complete cross-desensitization with IGly, but none with γ-aminobutyric acid (GABA)-induced currents ( IGABA). However, ITau elicited by very concentrated taurine (10 mM) showed partial cross-desensitization with IGABA, and it was substantially antagonized by 10 μM bicuculline. These results indicate that taurine binds mainly to GlyRs in NAcc, but it could be a partial agonist of GABAA receptors. By activating GlyRs, taurine may play an important physiological role in the control of NAcc function, especially during development.

The effect of adenosine on inhibitory postsynaptic currents (IPSCs) was examined in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by using the whole cell patch-clamp technique. Adenosine reversibly reduced the amplitude of GABAergic and glycinergic electrically evoked IPSCs (eIPSCs) in a dose-dependent manner (EC50 = 14.5 and 19.1 μM, respectively). The A1 adenosine-receptor agonist N6-cyclopentyladenosine also reduced the eIPSCs, whereas the A1 antagonist 8-cyclopentyl-1,3-dimethylxanthine reversed the inhibition produced by adenosine. In paired-pulse experiments, the ratio of the second to first GABAergic or glycinergic eIPSC amplitude was increased by adenosine, whereas the response of SG neurons to exogenous GABA or glycine was unaffected. Adenosine reduced the frequency of GABAergic and glycinergic spontaneous IPSCs without changing their amplitude. This reduction in frequency disappeared in the presence of a K+-channel blocker (4-aminopyridine) but not in the absence of Ca2+. The inhibition by adenosine disappeared in the presence of cyclic-AMP analog (8-Br-cyclic AMP) and adenylate-cyclase activator (forskolin) but not protein-kinase C (PKC) activator (phorbol-12,13-dibutyrate). We conclude that adenosine suppresses inhibitory transmission in SG neurons by activating presynaptic A1 receptors and that this action is mediated by K+ channels and cyclic AMP but not by Ca2+ channels and PKC. This inhibitory action of adenosine probably contributes to the modulation of pain transmission in the SG.

An inhibitory role for strychnine-sensitive glycine-gated chloride channels (GlyRs) in mature hippocampus has been overlooked, largely due to the misconception that GlyR expression ceases early during development and to few functional studies demonstrating their presence. As a result, little is known regarding the physiological and pharmacological properties of native GlyRs expressed by hippocampal neurons. In this study, we used pharmacological tools and whole cell patch-clamp recordings of CA1 pyramidal cells and interneurons in acutely prepared hippocampal slices from 3- to 4-wk old rats to characterize these understudied receptors. We show that glycine application to recorded pyramidal cells and interneurons elicited strychnine-sensitive chloride-mediated currents ( I gly) that did not completely desensitize in the continued presence of agonist but reached a steady state at 45–60% of the peak amplitude. Additionally, the inhibitory amino acid, taurine, which has been shown to activate GlyRs in other systems, activated GlyRs expressed by both pyramidal cells and interneurons, although with much less potency than glycine, having an EC50 10-fold higher. To examine the potential subunit composition of hippocampal GlyRs, we tested the effect of the GABAA receptor antagonist, picrotoxin, on I gly recorded from both cell types. At low micromolar concentrations of picrotoxin (≤100 μM), which selectively block α homomeric GlyRs, I gly was partially attenuated in both cell types, indicating that α homomeric receptors are expressed by pyramidal cells and interneurons. At picrotoxin concentrations ≤1 mM, ∼10–20% of the whole cell current remained, suggesting that αβ heteromeric GlyRs are also expressed because this subtype of GlyR is relatively resistant to picrotoxin antagonism. Finally, we examined whether hippocampal GlyRs are modulated by zinc. Consistent with previous reports in other preparations, zinc elicited a bidirectional modulation of GlyRs, with physiological zinc concentrations (1–100 μM) increasing whole cell currents and concentrations >100 μM depressing them. Furthermore, the same concentration of zinc that potentiates I gly suppressed currents mediated by the N-methyl-d-aspartate subtype of the glutamate receptor. Thus we provide a pharmacological characterization of native GlyRs expressed by both major neuron types in hippocampus and show that these receptors can be activated by taurine, an amino acid that is highly concentrated in hippocampus. Furthermore, our data suggest that at least two GlyR subtypes are present in hippocampus and that GlyR-mediated currents can be potentiated by zinc at concentrations that suppress glutamate-mediated excitability.

Ethanol increased the frequency of miniature glycinergic currents [miniature inhibitory postsynaptic currents (mIPSCs)] in cultured spinal neurons. This effect was dependent on intracellular calcium augmentation, since preincubation with BAPTA (an intracellular calcium chelator) or thapsigargin [a sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor] significantly attenuated this effect. Similarly, U73122 (a phospholipase C inhibitor) or 2-aminoethoxydiphenyl borate [2-APB, an inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) inhibitor] reduced this effect. Block of ethanol action was also achieved after preincubation with Rp-cAMPS, inhibitor of the adenylate cyclase (AC)/PKA signaling pathway. These data suggest that there is a convergence at the level of IP3R that accounts for presynaptic ethanol effects. At the postsynaptic level, ethanol increased the decay time constant of mIPSCs in a group of neurons (30 ± 10% above control, n = 13/26 cells). On the other hand, the currents activated by exogenously applied glycine were consistently potentiated (55 ± 10% above control, n = 11/12 cells), which suggests that ethanol modulates synaptic and nonsynaptic glycine receptors (GlyRs) in a different fashion. Supporting the role of G protein modulation on ethanol responses, we found that a nonhydrolyzable GTP analog [guanosine 5′- O-(3-thiotriphosphate) (GTPγS)] increased the decay time constant in ∼50% of the neurons (28 ± 12%, n = 11/19 cells) but potentiated the glycine-activated Cl− current in most of the neurons examined (83 ± 29%, n = 7/9 cells). In addition, confocal microscopy showed that α1-containing GlyRs colocalized with Gβ and Piccolo (a presynaptic cytomatrix protein) in ∼40% of synaptic receptor clusters, suggesting that colocalization of Gβγ and GlyRs might account for the difference in ethanol sensitivity at the postsynaptic level.

The role of glycinergic and GABAergic systems in mediating spontaneous synaptic transmission in newly formed neural networks was examined in motoneurons in the developing rat spinal cord. Properties of action potential–independent miniature inhibitory postsynaptic currents (mIPSCs) mediated by glycine and GABAA receptors (GlyR and GABAAR) were studied in spinal cord slices of 17- to 18-day-old embryos ( E17–18) and 1- to 3-day-old postnatal rats ( P1–3). mIPSC frequency and amplitude significantly increased after birth, while their decay time decreased. To determine the contribution of glycinergic and GABAergic synapses to those changes, GlyR- and GABAAR-mediated mIPSCs were isolated based on their pharmacological properties. Two populations of pharmacologically distinct mIPSCs were recorded in the presence of glycine or GABAA receptors antagonists: bicuculline-resistant, fast-decaying GlyR-mediated mIPSCs, and strychnine-resistant, slow-decaying GABAAR-mediated mIPSCs. The frequency of GABAAR-mediated mIPSCs was fourfold higher than that of GlyR-mediated mIPSCs at E17–18, indicating that GABAergic synaptic sites were functionally dominant at early stages of neural network formation. Properties of GABAAR-mediated mIPSC amplitude fluctuations changed from primarily unimodal skewed distribution at E17–18 to Gaussian mixtures with two to three discrete components at P1–3. A developmental shift from primarily long-duration GABAergic mIPSCs to short-duration glycinergic mIPSCs was evident after birth, when the frequency of GlyR-mediated mIPSCs increased 10-fold. This finding suggested that either the number of glycinergic synapses or the probability of vesicular glycine release increased during the period studied. The increased frequency of GlyR-mediated mIPSCs was associated with more than a twofold increase in their mean amplitude, and in the number of motoneurons in which mIPSC amplitude fluctuations were best fitted by multi-component Gaussian curves. A third subpopulation of mIPSCs was apparent in the absence of glycine and GABAA receptor antagonists: mIPSCs with both fast and slow decaying components. Based on their dual-component decay time and their suppression by either strychnine or bicuculline, we assumed that these were generated by the activation of co-localized postsynaptic glycine and GABAA receptors. The contribution of mixed glycine-GABA synaptic sites to the generation of mIPSCs did not change after birth. The developmental switch from predominantly long-duration GABAergic inhibitory synaptic currents to short-duration glycinergic currents might serve as a mechanism regulating neuronal excitation in the developing spinal networks.

Spontaneous miniature glycinergic inhibitory postsynaptic currents (mIPSCs) in mechanically dissociated rat sacral dorsal commissural nucleus (SDCN) neurons attached with intact glycinergic presynaptic nerve terminals and evoked IPSCs (eIPSCs) in the slice preparation were investigated using nystatin-perforated patch and conventional whole cell recording modes under the voltage-clamp conditions. Trans-ACPD (tACPD) reversibly reduced the mIPSC frequency without affecting the mean amplitude. The effect was mimicked by a specific metabotropic glutamate receptor (mGluR) II subtype agonist, (2S, 1′S, 2′S)-2-(carboxycyclo propyl) glycine (L-CCG-I), and a specific mGluRIII subtype agonist, 2-amino-4-phosphonobutyrate (L-AP4). These inhibitory effects on mIPSC frequency were blocked by the specific antagonists for mGluRII, α-methyl-1-(2S, 1′S, 2′S)-2-(carboxycyclo propyl) glycine and (RS)-α-cyclopropyl-4-phosphonophenylglycine. In the slice preparation, eIPSC amplitude and mIPSC frequency were decreased reversibly by L-CCG-I (10−6M) and L-AP4 (10−6M). In K+-free or K+-free external solution with Ba2+and Cs+, Ca2+-free or Cd2+external solution, the inhibitory effect of tACPD on mIPSC frequency was unaltered. Forskolin and 8-Br-cAMP significantly increased presynaptic glycine release, and prevented the inhibitory action of tACPD on mIPSC frequency. Sp-cAMP, however, did not prevent the inhibitory action of tACPD on mIPSC frequency. It was concluded that the activation of mGluRs inhibits glycine release by reducing the action of cAMP/PKA pathway.

We used whole cell and outside-out patch-clamp techniques with reticulospinal Mauthner neurons of zebrafish embryos to investigate the developmental changes in the properties of glycinergic synaptic currents in vivo from the onset of synaptogenesis. Miniature inhibitory postsynaptic currents (mIPSCs) were isolated and recorded in the presence of TTX (1 microM), kynurenic acid (1 mM), and bicuculline (10 microM) and were found to be sensitive to strychnine (1 microM). The mIPSCs were first observed in 26-29 h postfertilization (hpf) embryos at a very low frequency of approximately 0.04 Hz, which increased to approximately 0.5 Hz by 30-40 hpf, and was approximately 10 Hz in newly hatched (50 hpf) larvae, indicating an accelerated increase in synaptic activity. At all embryonic stages, the amplitudes of the mIPSCs were variable but their means were similar ( approximately 100 pA), suggesting rapid formation of the postsynaptic matrix. The 20-80% rise times of mIPSCs in embryos were longer (0.6-1.2 ms) than in larvae (approximately 0.3 ms), likely due to slower diffusion of glycine at the younger, immature synapses. The mIPSCs decayed with biexponential (tau(off1) and tau(off2)) time courses with a half-width in 26-29 hpf embryos that was longer and more variable than in older embryos and larvae. In 26- to 29-hpf embryos, tau(off1) was approximately 15 ms and tau(off2) was approximately 60 ms, representing events of intermediate duration; but occasionally long mIPSCs were observed in some cells where tau(off1) was approximately 40 ms and tau(off2) was approximately 160 ms. In 30-40 hpf embryos, the events were faster, with tau(off1) approximately 9 ms and tau(off2) approximately 40 ms, and in larvae, events declined somewhat further to tau(off1) approximately 4 ms and tau(off2) approximately 30 ms. Point-per-point amplitude histograms of the decay of synaptic events at all stages resulted in the detection of similar single channel conductances estimated as approximately 45 pS, indicating the presence of heteromeric glycine receptors (GlyRs) from the onset of synaptogenesis. Fast-flow (1 ms) application of a saturating concentration of glycine (3-10 mM) to outside-out patches obtained at 26-29 hpf revealed GlyR currents that decayed biexponentially with time constants resembling the values found for intermediate and long mIPSCs; by 30-40 hpf, the GlyR currents resembled fast mIPSCs. These observations indicate that channel kinetics limited the mIPSC duration. Our data suggest that glycinergic mIPSCs result from the activation of a mixture of fast and slow GlyR subtypes, the properties and proportion of which determine the decay of the synaptic events in the embryos.

Whole cell voltage-clamp electrophysiology was used to examine interactions between GABA and glycine at inhibitory amino acid receptors on rat olfactory bulb neurons in primary culture. Membrane currents evoked by GABA and glycine were selectively inhibited by low concentrations of bicuculline and strychnine, respectively, suggesting that they activate pharmacologically distinct receptors. However, GABA- and glycine-mediated currents showed cross-inhibition when the two amino acids were applied sequentially. Application of one amino acid inhibited the response to immediate subsequent application of the other. In the majority of neurons, GABA inhibited subsequent glycine-evoked currents and glycine inhibited subsequent GABA-evoked currents. In a small proportion of neurons, however, GABA inhibited glycine-evoked currents but glycine had little effect on GABA-evoked currents. The reverse was true in other neurons, suggesting that alterations in chloride gradients alone did not account for the cross-inhibition. Furthermore, no cross-inhibition was observed between GABA- and glycine-evoked currents in some neurons. The amplitude of the current evoked by the coapplication of saturating concentrations of GABA and glycine in these neurons was nearly the sum of the currents evoked by GABA and glycine alone. In contrast, the currents were not additive in neurons demonstrating cross-inhibition. These results suggest that olfactory bulb neurons heterogeneously express a population of inhibitory amino acid receptors that can bind either GABA or glycine. Interactions between GABA and glycine at inhibitory amino acid receptors may provide a mechanism to modulate inhibitory synaptic transmission.

Electrophysiological recordings of outside-out patches to fast-flow applications of glycine were made on patches derived from the Mauthner cells of the 50-h-old zebrafish larva. As for glycinergic miniature inhibitory postsynaptic currents (mIPSCs), depolarizing the patch produced a broadening of the transient outside-out current evoked by short applications (1 ms) of a saturating concentration of glycine (3 mM). When the outside-out patch was depolarized from -50 to +20 mV, the peak current varied linearly with voltage. A 1-ms application of 3 mM glycine evoked currents that activated rapidly and deactivated biexponentially with time constants of approximately 5 and approximately 30 ms (holding potential of -50 mV). These two decay time constants were increased by depolarization. The fast deactivation time constant increased e-fold per 95 mV. The relative amplitude of the two decay components did not significantly vary with voltage. The fast component represented 64.2 +/- 2.8% of the total current at -50 mV and 54.1 +/- 10% at +20 mV. The 20-80% rise time of these responses did not show any voltage dependence, suggesting that the opening rate constant is insensitive to voltage. The 20-80% rise time was 0.2 ms at -70 mV and 0.22 ms at +20 mV. Responses evoked by 100-200 ms application of a low concentration of glycine (0.1 mM) had a biphasic rising phase reflecting the complex gating behavior of the glycine receptor. The time constant of these two components and their relative amplitude did not change with voltage, suggesting that modal shifts in the glycine-activated channel gating mode are not sensitive to the membrane potential. Using a Markov model to simulate glycine receptor gating behavior, we were able to mimic the voltage-dependent change in the deactivation time course of the responses evoked by 1-ms application of 3 mM glycine. This kinetics model incorporates voltage-dependent closing rate constants. It provides a good description of the time course of the onset of responses evoked by the application of a low concentration of glycine at all membrane potentials tested.

Contribution of single-channel properties to the time course and amplitude variance of quantal glycine currents recorded in rat motoneurons. The amplitude of spontaneous, glycinergic miniature inhibitory postsynaptic currents (mIPSCs) recorded in hypoglossal motoneurons (HMs) in an in vitro brain stem slice preparation increased over the first 3 postnatal weeks, from 42 ± 6 pA in neonate (P0–3) to 77 ± 11 pA in juvenile (P11–18) HMs. Additionally, mIPSC amplitude distributions were highly variable: CV 0.68 ± 0.05 (means ± SE) for neonates and 0.83 ± 0.06 for juveniles. We wished to ascertain the contribution of glycine receptor (GlyR)-channel properties to this change in quantal amplitude and to the amplitude variability and time course of mIPSCs. To determine whether a postnatal increase in GlyR-channel conductance accounted for the postnatal change in quantal amplitude, the conductance of synaptic GlyR channels was determined by nonstationary variance analysis of mIPSCs. It was 48 ± 8 pS in neonate and 46 ± 10 pS in juvenile HMs, suggesting that developmental changes in mIPSC amplitude do not result from a postnatal alteration of GlyR-channel conductance. Next we determined the open probability ( P open) of GlyR channels in outside-out patches excised from HMs to estimate the contribution of stochastic channel behavior to quantal amplitude variability. Brief (1 ms) pulses of glycine (1 mM) elicited patch currents that closely resembled mIPSCs. The GlyR channels’ P open, calculated by nonstationary variance analysis of these currents, was ∼0.70 (0.66 ± 0.09 in neonates and 0.72 ± 0.05 in juveniles). The decay rate of patch currents elicited by brief application of saturating concentrations of glycine (10 mM) increased postnatally, mimicking previously documented changes in mIPSC time course. Paired pulses of glycine (10 mM) were used to determine if rapid GlyR-channel desensitization contributed to either patch current time course or quantal amplitude variability. Because we did not observe any fast desensitization of patch currents, we believe that fast desensitization of GlyRs underlies neither phenomenon. From our analysis of glycinergic patch currents and mIPSCs, we draw three conclusions. First, channel deactivation is the primary determinant of glycinergic mIPSC time course, and postnatal changes in channel deactivation rate account for observed developmental changes in mIPSC decay rate. Second, because GlyR-channel P openis high, differences in receptor number between synapses rather than stochastic channel behavior are likely to underlie the majority of quantal variability seen at glycinergic synapses throughout postnatal development. We estimate the number of GlyRs available at a synapse to be on average 27 in neonate neurons and 39 in juvenile neurons. Third, this change in the calculated number of GlyRs at each synapse may account for the postnatal increase in mIPSC amplitude.

Singer, Joshua H., Edmund M. Talley, Douglas A. Bayliss, and Albert J. Berger. Development of glycinergic synaptic transmission to rat brain stem motoneurons. J. Neurophysiol. 80: 2608–2620, 1998. Using an in vitro rat brain stem slice preparation, we examined the postnatal changes in glycinergic inhibitory postsynaptic currents (IPSCs) and passive membrane properties that underlie a developmental change in inhibitory postsynaptic potentials (IPSPs) recorded in hypoglossal motoneurons (HMs). Motoneurons were placed in three age groups: neonate (P0–3), intermediate (P5–8), and juvenile (P10–18). During the first two postnatal weeks, the decay time course of both unitary evoked IPSCs [mean decay time constant, τdecay= 17.0 ± 1.6 (SE) ms in neonates and 5.5 ± 0.4 ms in juveniles] and spontaneous miniature IPSCs (τdecay= 14.2 ± 2.4 ms in neonates and 6.3 ± 0.7 ms in juveniles) became faster. As glycine uptake does not influence IPSC time course at any postnatal age, this change most likely results from a developmental alteration in glycine receptor (GlyR) subunit composition. We found that expression of fetal (α2) GlyR subunit mRNA decreased, whereas expression of adult (α1) GlyR subunit mRNA increased postnatally. Single GlyR-channels recorded in outside-out patches excised from neonate motoneurons had longer mean burst durations than those from juveniles (18.3 vs. 11.1 ms). Concurrently, HM input resistance ( RN) and membrane time constant (τm) decreased ( RNfrom 153 ± 12 MΩ to 63 ± 7 MΩ and τmfrom 21.5 ± 2.7 ms to 9.1 ± 1.0 ms, neonates and juveniles, respectively), and the time course of unitary evoked IPSPs also became faster (τdecay= 22.4 ± 1.8 and 7.7 ± 0.9 ms, neonates vs. juveniles, respectively). Simulated synaptic currents were used to probe more closely the interaction between IPSC time course and τm, and these simulations demonstrated that IPSP duration was reduced as a consequence of postnatal changes in both the kinetics of the underlying GlyR channel and the membrane properties that transform the IPSC into a postsynaptic potential. Additionally, gramicidin perforated-patch recordings of glycine-evoked currents reveal a postnatal change in reversal potential, which is shifted from −37 to −73 mV during this same period. Glycinergic PSPs are therefore depolarizing and prolonged in neonate HMs and become faster and hyperpolarizing during the first two postnatal weeks.

Tian, Ning, Thomas N. Hwang, and David R. Copenhagen. Analysis of excitatory and inhibitory spontaneous synaptic activity in mouse retinal ganglion cells. J. Neurophysiol. 80: 1327–1340, 1998. Spontaneous inhibitory and excitatory postsynaptic currents (sIPSCs and sEPSCs) were identified and characterized with whole cell and perforated patch voltage-clamp recordings in adult mouse retinal ganglion cells. Pharmacological dissection revealed that all cells were driven by spontaneous synaptic inputs mediated by glutamate and γ-aminobutyric acid-A (GABAA) receptors. One-half (7/14) of the cells also received glycinergic spontaneous synaptic inputs. Both GABAA and glycine receptor–mediated sIPSCs had rise times (10–90%) of A receptor–mediated sIPSCs were comparable with those of the glycine receptor–mediated sIPSCs. The average decay time constant for monoexponentially fitted sIPSCs was 63.2 ± 74.1 ms (mean ± SD, n = 3278). Glutamate receptor–mediated sEPSCs had an average rise time of 0.50 ± 0.20 ms ( n = 109) and an average monoexponential decay time constant of 5.9 ± 8.6 ms ( n = 2705). Slightly more than two-thirds of the spontaneous synaptic events were monoexponential (68% for sIPSCs and 76% for sEPSCs). The remainder of the events was biexponential. The amplitudes of the spontaneous synaptic events were not correlated with rise times, suggesting that the electrotonic filtering properties of the neurons and/or differences in the spatial location of synaptic inputs could not account for the difference between the decay time constants of the glutamate and GABAA/glycine receptor–mediated spontaneous synaptic events. The amplitudes of sEPSCs were similar to those recorded in tetrodotoxin (TTX), consistent with the events measured in control saline being the response to the release of a single quantum of transmitter. The range of the sIPSC amplitudes in control saline was wider than that recorded in TTX, consistent with some sIPSCs being evoked by presynaptic spikes having an average quantal size greater than one. The rates of sIPSCs and sEPSCs were determined under equivalent conditions by recording with perforated patch electrodes at potentials at which both types of event could be identified. Two groups of ganglion cell were observed; one group had an average sEPSCs/sIPSCs frequency ratio of 0.96 ± 0.77 ( n = 28) and another group had an average ratio of 6.63 ± 0.82 ( n = 7). These findings suggest that a subset of cells is driven much more strongly by excitatory synaptic inputs. We propose that this subset of cells could be off ganglion cells, consistent with the higher frequency of spontaneous action potentials found in off ganglion cells in other studies.

Legendre, P. Pharmacological evidence for two types of postsynaptic glycinergic receptors on the Mauthner cell of 52-h-old zebrafish larvae. J. Neurophysiol. 77: 2400–2415, 1997. The presence of homooligomeric and heterooligomeric glycine receptors (GlyRs) on the Mauthner (M) cell in the isolated medulla of 52-h-old zebrafish larvae was investigated by analysis of the effects of picrotoxin on glycine-gated channels and on glycinergic miniature inhibitory postsynaptic currents (mIPSCs). Two functionally different GlyRs have been previously described on the M cell. The effects of picrotoxin on these two GlyRs were first analyzed by measuring the relative change in their total open probability ( NPo) with picrotoxin concentration. Picrotoxin had no significant effect on the glycine channel with a single conductance level of 40–46 pS. In contrast, picrotoxin application decreased the NPoof the GlyR with multiple subconductance levels. On this GlyR, picrotoxin decreased in a concentration-dependent manner the occurrence of the 80- to 86-pS substate (median inhibiting concentration = 0.89 μM) and had no apparent effect on the 40- to 46-pS opening probability. Opening frequency and the mean open times of the 80- to 88-pS main conductance state were reduced in the presence of 10 μM picrotoxin, but their relative weight remained unchanged. These effects of picrotoxin were not voltage dependent. Picrotoxin also modified 40- to 46-pS kinetics. At 100 μM, picrotoxin evoked voltage-independent flickering during channel openings. Short and long mean open times were significantly decreased, whereas the relative proportion of long mean open times was increased. The medium closed time was decreased, whereas medium burst duration was increased. The burst frequency remained unchanged. Spontaneous glycinergic mIPSCs were recorded in the presence of 1 μM tetrodotoxin + 25 μM bicuculline (holding potential = −50 mV). Application of 10 μM picrotoxin did not change the frequency of the synaptic activity, whereas it decreased the amplitude of large mIPSCs. No effect was observed on the time to peak (0.8 ms) or the mean decay time constant (τd= 7.7 ms). Increasing picrotoxin concentration to 100 μM resulted in a decrease of mIPSC frequency (35.6%), amplitude (39.8%), and τd(from 7.7 to 5 ms). These results suggest that these two functionally different GlyRs correspond to α1homooligomeric-like and α1/β-heterooligomeric-like GlyRs, and that both are synaptically activated. Variation in the proportions of GlyR subtypes from one synapse to another could partly account for the broad amplitude distribution of mIPSCs recorded from the zebrafish M cell.

Allosteric modulators exert actions on neurotransmitter receptors by positively or negatively altering the effective response of these receptors to their respective neurotransmitter. γ-Aminobutyric acid (GABA) type A ionotropic receptors (GABAARs) are major targets for allosteric modulators such as benzodiazepines, neurosteroids, and barbiturates. Analysis of substances that produce similar effects has been hampered by the lack of techniques to assess the localization and function of such agents in brain slices. Here we describe measurement of the sniffer patch laser uncaging response (SPLURgE), which combines the sniffer patch recording configuration with laser photolysis of caged GABA. This methodology enables the detection of allosteric GABAAR modulators endogenously present in discrete areas of the brain slice and allows for the application of exogenous GABA with spatiotemporal control without altering the release and localization of endogenous modulators within the slice. Here we demonstrate the development and use of this technique for the measurement of allosteric modulation in different areas of the thalamus. Application of this technique will be useful in determining whether a lack of modulatory effect on a particular category of neurons or receptors is due to insensitivity to allosteric modulation or a lack of local release of endogenous ligand. We also demonstrate that this technique can be used to investigate GABA diffusion and uptake. This method thus provides a biosensor assay for rapid detection of endogenous GABAAR modulators and has the potential to aid studies of allosteric modulators that exert effects on other classes of neurotransmitter receptors, such as glutamate, acetylcholine, or glycine receptors.

1. Although glycine has been identified as a required coagonist with glutamate at N-methyl-D-aspartate (NMDA) receptors, the understanding of glycine's role in excitatory synaptic neurotransmission is quite limited. In the present study, we used the whole cell patch-clamp technique to examine the ability of glycine to regulate current flow through synaptic NMDA receptors at excitatory synapses between cultured hippocampal neurons and in acutely isolated hippocampal slices. 2. These studies demonstrate that the glycine modulatory site on the synaptic NMDA receptor is not saturated under baseline conditions and that increased glycine concentrations can markedly increased NMDA-receptor-mediated excitatory postsynaptic currents (EPSCs) in hippocampal neurons in both dissociated cell culture and in slice. Saturation of the maximal effect of glycine takes place at different concentrations for different cells in culture, suggesting the presence of heterogenous NMDA receptor subunit compositions. 3. Bath-applied glycine had no effect on the time course of EPSCs in either brain slice or culture, indicating that desensitization of the NMDA receptor is not prevented by glycine over the time course of an EPSC. 4. When extracellular glycine concentration is high, all miniature EPSCs recorded in the cultured hippocampal neurons contained NMDA components, indicating that segregation of non-NMDA receptors at individual synaptic boutons does not occur.

1. The modulatory effect of alpha 2 adrenoceptor on the taurine response was investigated in substantia nigra (SN) neurons acutely dissociated from the rat using a nystatin perforated-patch recording mode under voltage-clamp conditions. 2. Complete cross-desensitization was observed between 10(-3) M glycine and 3 x 10(-3) M taurine-induced currents. Both currents were antagonized by 10(-6) M strychnine, thus indicating that taurine acts on strychnine-sensitive glycine receptor on the SN neurons. 3. The simultaneous application of norepinephrine (NE) with prazosin (10(-5) M) and propranolol (10(-5) M) potentiated the taurine response (Itau) in an NE concentration-dependent manner at a holding potential (VH) of -40 mV. Clonidine mimicked the NE effect on the Itau, thus indicating the involvement of alpha 2 adrenoceptor activation in the potentiation of Itau. 4. Alpha 2 adrenoceptor activation by NE with prazosin and propranolol significantly potentiated the peak amplitude of Itau without shifting the taurine concentration-response relationships either to left or right side. The respective concentrations of taurine for the threshold, half maximal and maximal responses in the presence of 10(-4) M NE with prazosin (10(-5) M) and propranolol (10(-5) M) were 3 x 10(-5) M, 3.1 x 10(-4) M, and 3 x 10(-3) M. The same concentrations in the absence of NE were 3 x 10(-5) M, 3.2 x 10(-4) M, and 3 x 10(-3) M, respectively. 5. The reversal potentials of Itau with and without NE were very close to the theoretical Cl- equilibrium potential, thus indicating that the potentiation of Itau by alpha 2 adrenoceptor activation was due to an increase in the taurine-induced Cl- currents. 6. Forskolin (3 x 10(-5) M) and isobutylmethylxanthine (3 x 10(-5) M) suppressed the peak amplitude of Itau. In the presence of dibutyryl cyclic AMP (10(-4) M), which also suppressed Itau, alpha 2 adrenoceptor activation failed to potentiate Itau. 7. N-[2(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H-89) mimicked the effect of alpha 2 adrenoceptor activation on Itau. In addition, the potentiation of Itau by alpha 2 adrenoceptor was not observed in the presence of 10(-6) M H-89. 8. The treatment of SN neurons with pertussis toxin (500 ng/ ml) for 18 h completely abolished the facilitatory effect of alpha 2 adrenoceptor on Itau. 9. These results suggest that the activation of alpha 2 adrenoceptor coupled with IAP-sensitive GTP binding protein decreases the intracellular cyclic AMP and cyclic AMP-dependent protein kinase activity, thus resulting in the potentiation of glycine receptor-mediated taurine response in rat SN neurons.

1. We have studied the effects of locally applying the glycinergic antagonist strychnine to rhythmically active spinal neurons in amphibian embryos during fictive swimming. Intracellular recordings were made from motoneurons and premotor interneurons in Xenopus laevis, a well-studied model system, and from motoneurons in three other species (Rana temporaria, Bufo bufo, and Triturus vulgaris). Overall, these embryos cover a range of swimming patterns from the short-cycle-period, brief-motor-root bursts of Xenopus, to the long-cycle-period, long-motor-root bursts of Rana, which are more typical of adult patterns. 2. Local strychnine application had no significant effect on the gross pattern of swimming; episode duration and the burst duration in rostral ventral roots away from the application site were unaltered, and left-right alternation was preserved. We have therefore been able to examine the effects of inhibition on individual neurons, uninfluenced by overall changes in the operation of the swimming neural circuitry. 3. In all cases strychnine blocked midcycle inhibition and significantly increased the peak on-cycle depolarization during swimming. In Rana, Bufo, and Triturus motoneurons, and in Xenopus interneurons, strychnine significantly increased the reliability of firing during swimming. In Xenopus motoneurons, where spiking was 100% reliable anyway, the timing of the spikes was advanced relative to rostral ventral root activity. These results do not provide support for postinhibitory rebound as a factor in the spike-generating process during swimming. In addition to midcycle inhibition, Xenopus motoneurons can also show a smaller, additional on-cycle inhibition that is blocked by strychnine. 4. In both Rana and Bufo the duration of caudal ventral root bursts close to the site of drug application was increased by strychnine, showing that the increased motoneuron reliability not only leads to more intense, but also more extensive, ventral root activity. 5. At the level of single neurons, glycinergic inhibition effectively reduces on-cycle excitation and in turn controls the reliability, extent, and precise timing of motoneuron firing. These changes may be the individual components underlying broader effects of inhibition described previously, such as locomotor frequency control. They also show how any modulation of inhibition in localized regions of the spinal cord could produce localized control of neuronal firing properties.

1. In lamprey, stretch receptor neurons (SRNs), also referred to as edge cells, are located along the lateral margin of the spinal cord. They sense the lateral movements occurring in each swim cycle during locomotion. The isolated lamprey spinal cord in vitro was used to investigate the activity of SRNs during fictive locomotion induced by bath-applied N-methyl-D-aspartate (NMDA). Intracellular recordings with potassium acetate filled electrodes showed that 63% of SRNs had a clear locomotor-related modulation of their membrane potential. 2. Of the modulated SRNs, two-thirds had periods of alternating excitation and inhibition occurring during the ipsilateral and the contralateral ventral root bursts, respectively. The phasic hyperpolarization could be reversed into a depolarizing phase after the injection of chloride ions into the cells; this revealed a chloride-dependent synaptic drive. The remaining modulated SRNs were inhibited phasically during ipsilateral motor activity. 3. Experiments with barriers partitioning the recording chamber with the spinal cord into three pools, allowed an inactivation of the locomotor networks within one pool by washing out NMDA from the pool in which the SRN was recorded. This resulted in a marked reduction, but not an abolishment, of the amplitude of the membrane potential oscillations. Both the excitatory and the inhibitory phases were reduced, resulting from removal of input from inhibitory and excitatory interneurons projecting from the adjacent pools. If the glycine receptor antagonist strychnine (1 microM) was applied in one pool, the phasic hyperpolarizing phase disappeared without affecting the excitatory phase. 4. Bath application of the gamma-aminobutyric acid (GABA)A receptor antagonist, bicuculline (50-100 microM) blocked the spontaneous large unitary inhibitory postsynaptic potentials, which occurred without a clear phasic pattern. Bicuculline had no significant effect on the peak to peak amplitude of the locomotor-related membrane potential oscillations. The inhibition in SRNs therefore has a dual origin: glycinergic interneurons provide phasic inhibition, while the GABA system can exert a tonic inhibition via GABAA receptors. 5. These data show that, in addition to the stretch-evoked excitation, which SRNs receive during each locomotor cycle, most of them also receive excitation from the central pattern generator network during the ipsilateral contraction, which may ensure a maintained high level of sensitivity to stretch during the shortening phase of the locomotor cycle. This arrangement is analogous to the efferent control of muscle spindles exerted by gamma-motoneurons in mammals, which as a rule are coactivated with alpha-motoneurons to the same muscle (alpha-gamma linkage).

1. To identify the type(s) and properties of inhibitory postsynaptic receptor(s) involved in synaptic transmission in cultured rat embryonic spinal cord and medullary neurons, we have used whole cell patch-clamp techniques to record miniature inhibitory postsynaptic currents (mIPSCs) in the presence of tetrodotoxin, DL-2-amino-5-phosphonovaleric acid, and 6-cyano-7-nitroquinoxaline-2,3-dione. 2. The mIPSCs recorded from both spinal cord and medullary neurons had skewed amplitude distributions. 3. The glycinergic antagonist strychnine and the GABAergic antagonist bicuculline each decreased both the frequency and mean peak amplitudes of mIPSCs. We conclude that both glycine and gamma-aminobutyric acid (GABA) are neurotransmitters at inhibitory synapses in our cultured cells. 4. Most (approximately 96-97%) mIPSCs decay with single-exponential time constants, and decay time distributions were consistently best fitted by the sum of four Gaussians with decay constants as follows: D1 = 5.8 +/- 0.1 (SE) ms (n = 63), D2 = 12.2 +/- 0.2 ms (n = 61), D3 = 23.2 +/- 0.4 ms (n = 54), and D4 = 44.7 +/- 1.0 ms (n = 57). We conclude that the four classes of decay times represent kinetically different inhibitory postsynaptic receptor populations. 5. Strychnine and bicuculline usually had one of two different effects on the mIPSC decay time constant distributions; either selective decreases in the frequency of mIPSCs with decay times in certain classes (i.e., the D1 class was reduced by bicuculline, the D2 class by strychnine, and the D3 and D4 classes by both antagonists) or a nonselective depression in the frequency of mIPSCs with decay times in all four classes. The particular effect observed in a given neuron was correlated with the presence or absence of ATP and guanosine 5'-triphosphate (GTP) in the patch pipette. Namely, in 71% of the antagonist applications where the pipette contained ATP and GTP, the result was a nonselective decrease in mIPSCs in all decay time constant classes. Conversely, in 54% of the antagonist applications in their absence, the result was a selective decrease in the frequency of mIPSCs in specific decay time constant classes. 6. In some experiments, mIPSCs reappeared in antagonist solution after an essentially complete block. Recovery from block in the continued presence of antagonist was never observed in the absence of ATP and GTP (8 neurons), and, at the same time, 5 of 9 neurons patched with ATP and GTP in the pipette did show recovery (56%).

1. Membrane currents induced by glycine (Iglys) were investigated in cholinergic neurons in primary culture, isolated from the septal region of fetal rat brains, by use of the whole cell voltage-clamp recording configuration. A glycine response was observed in 131 of 150 neurons examined. 2. Igly was accompanied by an increase in conductance and reversed the direction at the Cl- equilibrium potential. The Igly-voltage relation was a little outwardly rectifying in a range of -40 to +30 mV in a nearly symmetrical Cl- solution system. 3. Glycine applied by local perfusion produced an Igly, which increased in amplitude sigmoidally with increasing concentrations (EC50 = 110 microM; nH = 1.75) and desensitized at higher concentrations. There was no nondesensitizing component in Igly (100 microM). Strychnine inhibited Igly in a dose-dependent and competitive manner; it took at most 1 min for this action to be attained to a steady level. Schild analysis yielded the dissociation constant for strychnine to be 0.062 microM. Igly was depressed noncompetitively by a gamma-aminobutyric acid-A (GABAA) receptor-channel blocker, picrotoxin, in a dose-dependent manner (IC50 = 45 microM for peak Igly at 100 microM). On the contrary, another GABAA inhibitor, bicuculline (10 microM), did not affect Igly. 4. Igly was unaffected by spermine (1 mM), ethanol (1-100 mM), and diazepam (1 microM) and was depressed by 14% by pentobarbital (100 microM). On the other hand, Zn2+ potentiated Igly in a dose-dependent (EC50 = 9.1 microM; nH = 1.3; 38% increase for Igly at 50 microM by 100 microM Zn2+) and reversible manner. The facilitatory action was due to an increase in the apparent affinity for glycine (by 139% by 100 microM Zn2+), and did not depend on holding potentials. Pb2+ (100 microM) and La3+ (100 microM) also enhanced Igly (50 microM) by 23 and 14%, respectively; these actions were abolished in the presence of Zn2+ (100 microM). Igly was not affected by Ba2+, Sr2+, Co2+, Mn2+, Cd2+, and Al3+ (100 microM each). 5. Rat septal cholinergic neurons in culture were endowed with a Cl(-)-selective glycine receptor channel whose activation was sensitive to strychnine and picrotoxin. Zn2+ induced a positive modulation of the glycine response, possibly through an allosteric interaction with the glycine-binding site; this action was shared by Pb2+ and La3+ with a potency sequence of Zn2+ > Pb2+ > La3+. This metal-ion binding site could serve to enhance a glycine action.

1. Glycine was applied to acutely dissociated neurons of the guinea pig ventral cochlear nucleus (VCN) with the use of iontophoresis. With approximately equal chloride concentrations in the extra- and intracellular solutions (i.e., chloride equilibrium potential = 0 mV), cells held at -60 mV responded with inward currents that were 1-10 nA in amplitude, had rise times of approximately 50 ms, and decayed to half of the peak amplitude in 50-600 ms. More than 95% of cells with diameters > 12 microns responded to glycine. Response amplitude and area increased with increasing duration of the iontophoretic pulse. Response amplitude saturated at pulse durations of 60-80 ms, whereas response area did not exhibit saturation for pulse durations of 10-100 ms. 2. The glycine antagonist strychnine was added to the extracellular solution at concentrations of 0.5-500 nM to evaluate its effect on glycine-evoked responses. Strychnine produced a 50% reduction in the response at a concentration of 12 nM and the dose-response function had a limiting slope (Hill coefficient) of 1.4. 3. Changes in glycine-evoked currents as a function of cell membrane potential were examined in the presence of tetrodotoxin, tetraethylammonium chloride, and 4-aminopyridine, which block sodium and potassium conductances activated by depolarization. Both the amplitude and the decay of glycine-evoked currents displayed a voltage dependence. Under conditions where the glycine currents reversed at -35 mV, the amplitudes of responses evoked at membrane potentials of 0 mV were 2.3 times larger than those of responses evoked at -70 mV. The decay time constant at 0 mV was 1.49 times longer than that at -70 mV. 4. Acutely dissociated neurons of the VCN previously have been classified on the basis of the absence (type I) or presence (type II) of a low-threshold outward current. Type I cells fire repetitively in response to current pulses, whereas type II cells fire transiently. Glycine-evoked responses were compared in cells identified electrophysiologically as type I or type II on the basis of previously established criteria under voltage clamp. The average amplitudes of responses recorded at a membrane potential of -70 mV were 1.1 and 1.3 nA for type I and type II cells, respectively. The rise time of the glycine current for the two groups of cells was similar (52 ms for type I and 57 ms for type II), but the decay of currents to half-maximum amplitude following the offset of the iontophoretic pulse was longer in type II cells (340 ms) than in type I cells (173 ms). No differences between the two groups were noted with regard to the outward rectification of peak currents or the voltage dependence of current decay. 5. The reversal potential of glycine-evoked responses was determined in extracellular solutions with varying chloride concentrations. The change in the glycine reversal potential (54 mV) for a 10-fold change in chloride concentration was similar to the change in the chloride equilibrium potential (58 mV) over the same range of extracellular chloride concentrations. A similar result was obtained by maintaining the extracellular chloride concentration constant and varying the chloride concentration in the intracellular solution. Glycine-evoked responses were not affected by changes in the potassium or sodium equilibrium potentials. The glycine receptors are therefore principally permeable to chloride. 6. In the VCN, glycine-mediated currents are readily evoked from the majority of larger neurons, indicating an abundance of glycine receptors on the somata and proximal processes of these neurons. The properties of glycine receptors in VCN and other areas of the nervous system are generally similar. The voltage dependence of glycine-evoked currents implies that the inhibitory effectiveness of glycine receptors in VCN increases nonlinearly with depolarization.