Glycinergic Neurons Research Articles

Selective Modulation of Histaminergic Inputs on Projection Neurons of Cerebellum Rapidly Promotes Motor Coordination via HCN Channels.

Insights into function of central histaminergic system, a general modulator originating from the hypothalamus for whole brain activity, in motor control are critical for understanding the mechanism underlying somatic-nonsomatic integration. Here, we show a novel selective role of histamine in the cerebellar nuclei, the final integrative center and output of the cerebellum. Histamine depolarizes projection neurons but not interneurons in the cerebellar nuclei via the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels coupled to histamine H2 receptors, which are exclusively expressed on glutamatergic and glycinergic projection neurons. Furthermore, blockage of HCN channels to block endogenous histaminergic afferent inputs in the cerebellar nuclei significantly attenuates motor balance and coordination. Therefore, through directly and quickly modulation on projection neurons but not interneurons in the cerebellar nuclei, central histaminergic system may act as a critical biasing force to not only promptly regulate ongoing movement but also realize a rapid integration of somatic and nonsomatic response.

Antinociceptive effect of intracerebroventricular administration of glycine transporter-2 inhibitor ALX1393 in rat models of inflammatory and neuropathic pain

Glycinergic transmission has an important role in regulating nociception in the spinal cord. The glycine transporter-2 (GlyT2) is localized at presynaptic terminals of glycinergic neurons and eliminates glycine from the synaptic cleft to terminate glycinergic transmission. Systemic and intrathecal administration of GlyT2 inhibitors alleviate various types of pain. Although the GlyT2s and glycine receptors are widely distributed in the central nervous system, little is known about the role of glycinergic transmission in pain perception at supraspinal regions. The present study examined the antinociceptive effect of intracerebroventricular (i.c.v.) administration of the selective GlyT2 inhibitor ALX1393 on inflammatory and neuropathic pain in experimental models. For i.c.v. administration, a guide cannula was implanted into the right lateral ventricle of male Sprague–Dawley rats. Normal rats were used to assess inflammatory nociception using the formalin test and motor function using the rotarod test. Chronic constriction injury (CCI) to the sciatic nerve was induced in the rats. The CCI rats were then used to assess mechanical, cold, and thermal hyperalgesia using the electronic von Frey test, cold plate test, and the plantar test, respectively. ALX1393 (25, 50, and 100μg) was administered i.c.v. to examine its effects on supraspinal antinociception. Supraspinal ALX1393 in normal rats suppressed the late-phase response in the formalin test but did not affect motor performance. In the CCI rats, ALX1393 inhibited mechanical and cold hyperalgesia in a dose-dependent manner. The antihyperalgesic effects of ALX1393 (100μg) were reversed completely by i.c.v. pretreatment with a glycine receptor antagonist strychnine (10μg). These results suggest that GlyT2 contributes to nociceptive transmission at supraspinal level and that the selective GlyT2 inhibitor is a promising candidate for the treatment of inflammatory and neuropathic pain without causing motor dysfunction.

Defining inhibitory neurone function in respiratory circuits: opportunities with optogenetics?

Pharmacological and mathematical modelling studies support the view that synaptic inhibition in mammalian brainstem respiratory circuits is essential for generating normal and stable breathing movements. GABAergic and glycinergic neurones are known components of these circuits but their precise functional roles have not been established, especially within key microcircuits of the respiratory pre-Bötzinger (pre-BötC) and Bötzinger (BötC) complexes involved in phasic control of respiratory pump and airway muscles. Here, we review briefly current concepts of relevant complexities of inhibitory synapses and the importance of synaptic inhibition in the operation of these microcircuits. We highlight results and limitations of classical pharmacological studies that have suggested critical functions of synaptic inhibition. We then explore the potential opportunities for optogenetic strategies that represent a promising new approach for interrogating function of inhibitory circuits, including a hypothetical wish list for optogenetic approaches to allow expedient application of this technology. We conclude that recent technical advances in optogenetics should provide a means to understand the role of functionally select and regionally confined subsets of inhibitory neurones in key respiratory circuits such as those in the pre-BötC and BötC.

Linear coding of complex sound spectra by discharge rate in neurons of the medial nucleus of the trapezoid body (MNTB) and its inputs

The interaural level difference (ILD) cue to sound location is first encoded in the lateral superior olive (LSO). ILD sensitivity results because the LSO receives excitatory input from the ipsilateral cochlear nucleus and inhibitory input indirectly from the contralateral cochlear nucleus via glycinergic neurons of the ipsilateral medial nucleus of the trapezoid body (MNTB). It is hypothesized that in order for LSO neurons to encode ILDs, the sound spectra at both ears must be accurately encoded via spike rate by their afferents. This spectral-coding hypothesis has not been directly tested in MNTB, likely because MNTB neurons have been mostly described and studied recently in regards to their abilities to encode temporal aspects of sounds, not spectral. Here, we test the hypothesis that MNTB neurons and their inputs from the cochlear nucleus and auditory nerve code sound spectra via discharge rate. The Random Spectral Shape (RSS) method was used to estimate how the levels of 100-ms duration spectrally stationary stimuli were weighted, both linearly and non-linearly, across a wide band of frequencies. In general, MNTB neurons, and their globular bushy cell inputs, were found to be well-modeled by a linear weighting of spectra demonstrating that the pathways through the MNTB can accurately encode sound spectra including those resulting from the acoustical cues to sound location provided by head-related directional transfer functions (DTFs). Together with the anatomical and biophysical specializations for timing in the MNTB-LSO complex, these mechanisms may allow ILDs to be computed for complex stimuli with rapid spectrotemporally-modulated envelopes such as speech and animal vocalizations and moving sound sources.

Spinal mechanism of micturition reflex inhibition by naftopidil in rats

AimWe investigated the spinal mechanism through which naftopidil inhibits the micturition reflex by comparing the effects of noradrenaline and naftopidil in rats. MethodsThe following were investigated: the influence of oral naftopidil on plasma monoamine and amino acid levels, the distribution of oral 14C-naftopidil, the effects of intravenous (IV) or intrathecal (IT) injection of noradrenaline or naftopidil on isovolumetric bladder contractions, amino acid levels in the lumbosacral spinal cord after IT noradrenaline or naftopidil, and the effects of IT naftopidil and strychnine and/or bicuculline on isovolumetric bladder contractions. Key findingsOral naftopidil decreased the plasma adrenaline level, while it increased the serotonin and glycine levels. After oral administration, 14C-naftopidil was detected in the spinal cord and cerebrum, as well as in plasma and the prostate gland. When the bladder volume was below the threshold for isovolumetric reflex contractions, IV (0.1mg) or IT (0.1μg) noradrenaline evoked bladder contractions, but IV (1mg) or IT (0.01–1μg) naftopidil did not. When the bladder volume was above the threshold for isovolumetric reflex contractions, IV or IT noradrenaline transiently abolished bladder contractions. IT noradrenaline decreased the levels of glycine and gamma-aminobutyric acid (GABA) in the lumbosacral cord, while IT naftopidil increased the GABA level. IT strychnine and/or bicuculline blocked the inhibitory effect of IT naftopidil on bladder contractions. SignificanceNaftopidil inhibits the micturition reflex by blocking α1 receptors, as well as by the activation of serotonergic, glycinergic, and GABAergic neurons in the central nervous system.

The effect of tracheal occlusion on respiratory load compensation: Changes in neurons containing inhibitory neurotransmitter in the nucleus of the solitary tract in conscious rats

Respiratory load compensation volume–time (Vt–T) relationships have been extensively studied in anesthetized animals. There are only a few studies in conscious animals although consciousness and behavior play a critical role in modulation of breathing. The aims of the study were to determine the effect of intermittent and transient tracheal occlusions (ITTO) elicited load compensation responses and the changes in activation of inhibitory glycinergic neurons in the nucleus of solitary tract (NTS) in conscious rats. The results showed that ITTO elicited an increase in expiratory time (Te) but did not affect inspiratory time (Ti) and diaphragm activity (EMGdia). An increase in total breathing time (Ttot) was due exclusively to the increase in Te. In addition, glycinergic neurons were activated in the intermediate NTS (iNTS) but not in the caudal NTS (cNTS). These results suggest that the activated glycinergic neurons in the iNTS may be important for the neurogenesis of load compensation responses in conscious animals.

GABAergic and glycinergic inhibitory synaptic transmission in the ventral cochlear nucleus studied in VGAT channelrhodopsin-2 mice.

Both glycine and GABA mediate inhibitory synaptic transmission in the ventral cochlear nucleus (VCN). In mice, the time course of glycinergic inhibition is slow in bushy cells and fast in multipolar (stellate) cells, and is proposed to contribute to the processing of temporal cues in both cell types. Much less is known about GABAergic synaptic transmission in this circuit. Electrical stimulation of the auditory nerve or the tuberculoventral pathway evokes little GABAergic synaptic current in brain slice preparations, and spontaneous GABAergic miniature synaptic currents occur infrequently. To investigate synaptic currents carried by GABA receptors in bushy and multipolar cells, we used transgenic mice in which channelrhodopsin-2 and EYFP is driven by the vesicular GABA transporter (VGAT-ChR2-EYFP) and is expressed in both GABAergic and glycinergic neurons. Light stimulation evoked action potentials in EYFP-expressing presynaptic cells, and evoked inhibitory postsynaptic potentials (IPSPs) in non-expressing bushy and planar multipolar cells. Less than 10% of the IPSP amplitude in bushy cells arose from GABAergic synapses, whereas 40% of the IPSP in multipolar neurons was GABAergic. In voltage clamp, glycinergic IPSCs were significantly slower in bushy neurons than in multipolar neurons, whereas there was little difference in the kinetics of the GABAergic IPSCs between two cell types. During prolonged stimulation, the ratio of steady state vs. peak IPSC amplitude was significantly lower for glycinergic IPSCs. Surprisingly, the reversal potentials of GABAergic IPSCs were negative to those of glycinergic IPSCs in both bushy and multipolar neurons. In the absence of receptor blockers, repetitive light stimulation was only able to effectively evoke IPSCs up to 20 Hz in both bushy and multipolar neurons. We conclude that local GABAergic release within the VCN can differentially influence bushy and multipolar cells.

Genetic ablation of VIAAT in glycinergic neurons causes a severe respiratory phenotype and perinatal death.

Both glycinergic and GABAergic neurons require the vesicular inhibitory amino acid transporter (VIAAT) for synaptic vesicle filling. Presynaptic GABA concentrations are determined by the GABA-synthesizing enzymes glutamate decarboxylase (GAD)65 and GAD67, whereas the presynaptic glycine content depends on the plasma membrane glycine transporter 2 (GlyT2). Although severely impaired, glycinergic transmission is not completely absent in GlyT2-knockout mice, suggesting that other routes of glycine uptake or de novo synthesis of glycine exist in presynaptic terminals. To investigate the consequences of a complete loss of glycinergic transmission, we generated a mouse line with a conditional ablation of VIAAT in glycinergic neurons by crossing mice with loxP-flanked VIAAT alleles with a GlyT2-Cre transgenic mouse line. Interestingly, conditional VIAAT knockout (VIAAT cKO) mice were not viable at birth. In addition to the dominant respiratory failure, VIAAT cKO showed an umbilical hernia and a cleft palate. Immunohistochemistry revealed an almost complete depletion of VIAAT in the brainstem. Electrophysiology revealed the absence of both spontaneous glycinergic and GABAergic inhibitory postsynaptic currents from hypoglossal motoneurons. Our results demonstrate that the deletion of VIAAT in GlyT2-Cre expressing neurons also strongly affects GABAergic transmission and suggest a large overlap of the glycinergic and the GABAergic neuron population during early development in the caudal parts of the brain.

Changes in spinal inhibitory networks induced by furosemide in humans.

During neural development in animals, GABAergic and glycinergic neurons are first excitatory, and then become inhibitory in the mature state. This developmental shift is due mainly to strong expression of the cation-chloride K-Cl cotransporter 2 (KCC2) and down-regulation of Na-K-Cl cotransporter 1 (NKCC1) during maturation. The down-regulation of co-transporter KCC2 after spinal cord transection in animals leads to the depolarising (excitatory) action of GABA and glycine and thus results in a reduction of inhibitory synaptic efficiency. Furosemide, a loop diuretic, has been shown to selectively and reversibly block inhibitory postsynaptic potentials without affecting excitatory postsynaptic potentials in animal spinal neurons. Moreover, this diuretic has been also demonstrated to block the cation-chloride co-transporters. Here, we used furosemide to demonstrate changes in spinal inhibitory networks in healthy human subjects. Non-invasive electrophysiological techniques were used to assess presynaptic inhibition, postsynaptic inhibition and the efficacy of synaptic transmission between muscle afferent terminals and soleus motoneurons in the spinal cord. Orally administered furosemide, at doses commonly used in the clinic (40 mg), significantly reduced spinal inhibitory interneuronal activity for at least 70 min from intake compared to control experiments in the same subjects while no changes were observed in the efficacy of synaptic transmission between muscle afferent terminals and soleus motoneurons. The reduction of inhibition was dose-dependent. Our results provide indirect evidence that reversible changes in the cation-chloride transport system induce modulations of inhibitory neuronal activity at spinal cord level in humans.

Intravenous or Local Injections of Flavoxate in the Rostral Pontine Reticular Formation Inhibit Urinary Frequency Induced by Activation of Medial Frontal Lobe Neurons in Rats

The rostral pontine reticular formation has a strong inhibitory effect onmicturition by facilitating lumbosacral glycinergic neurons. We assessed the influence of the rostral pontine reticular formation on the micturition reflex after noradrenaline injection in the medial frontal lobe. We also examined the relation between the medial frontal lobe and the rostral pontine reticular formation. Continuous cystometry was performed in 28 female rats. After the interval between bladder contractions was shortened by noradrenaline injection in the medial frontal lobe we injected glutamate or flavoxate hydrochloride in the rostral pontine reticular formation or intravenously injected flavoxate or propiverine. The change in bladder activity was examined. Noradrenaline injection in the medial frontal lobe shortened the interval between bladder contractions. In contrast to the bladder contraction interval before and after noradrenaline injection in the medial frontal lobe, the interval was prolonged after noradrenaline injection when glutamate or flavoxate was injected in the rostral pontine reticular formation, or flavoxate was injected intravenously. Noradrenaline injection in the medial frontal lobe plus intravenous propiverine injection also prolonged the interval compared to that after noradrenaline injection alone. However, the interval after noradrenaline injection in the medial frontal lobe plus intravenous injection of propiverine was shorter than that before noradrenaline injection only. Medial frontal lobe neurons excited by noradrenaline may facilitate the micturition reflex via activation of inhibitory interneurons, which inhibit descending rostral pontine reticular formation neurons that innervate the lumbosacral glycinergic inhibitory neurons. Therefore, the mechanism of micturition reflex facilitation by the activation of medial frontal lobe neurons involves the rostral pontine reticular formation.

Functional connectivity between Bötzinger complex glycinergic neurons and parafacial late‐expiratory neurons for expiratory and sympathetic control (712.17)

Our computational model predicts that post‐inspiratory (post‐I) neurons expressing the glycine transporter 2 (GLYT2) located in the Bötzinger Complex (BötC) suppress generation of abdominal and sympathetic late‐expiratory (late‐E) activities in eucapnia. Hypercapnia allows parafacial late‐E neurons to escape this inhibition. To test functional connectivity between glycinergic BötC and late‐E neurons, we used cell‐type specific viral vector expression of channelrhodopsin (ChETA) or hallorhodopsin (eNpHR3.0) to excite/inhibit GLTY2 neurons in the BötC of rats. Intracellular recordings of parafacial late‐E neurons (n=9) confirmed that inhibition during post‐I prevents generation of late‐E activity under eucapnia. eNpHR3.0‐mediated inhibition of GLYT2 neurons in the BötC evoked abdominal and sympathetic late‐E activities and increased arterial pressure (n=4).In contrast, ChETA‐mediated excitation of GLYT2 neurons in the BötC abolished the CO2‐induced abdominal and sympathetic late‐E activities (n=4), due to inhibition of parafacial late‐E neurons (n=3). These results indicate an emergence of parafacial late‐E activity with hypercapnia which is normally suppressed by BötC glycinergic neurons.Grant Funding Source: Supported by NIH R01 NS069220 and FAPESP

Tracheal occlusion-evoked respiratory load compensation and inhibitory neurotransmitter expression in rats

Respiratory load compensation is a sensory-motor reflex generated in the brain stem respiratory neural network. The nucleus of the solitary tract (NTS) is thought to be the primary structure to process the respiratory load-related afferent activity and contribute to the modification of the breathing pattern by sending efferent projections to other structures in the brain stem respiratory neural network. The sensory pathway and motor responses of respiratory load compensation have been studied extensively; however, the mechanism of neurogenesis of load compensation is still unknown. A variety of studies has shown that inhibitory interconnections among the brain stem respiratory groups play critical roles for the genesis of respiratory rhythm and pattern. The purpose of this study was to examine whether inhibitory glycinergic neurons in the NTS were activated by external and transient tracheal occlusions (ETTO) in anesthetized animals. The results showed that ETTO produced load compensation responses with increased inspiratory, expiratory, and total breath time, as well as elevated activation of inhibitory glycinergic neurons in the caudal NTS (cNTS) and intermediate NTS (iNTS). Vagotomized animals receiving transient respiratory loads did not exhibit these load compensation responses. In addition, vagotomy significantly reduced the activation of inhibitory glycinergic neurons in the cNTS and iNTS. The results suggest that these activated inhibitory glycinergic neurons in the NTS might be essential for the neurogenesis of load compensation responses in anesthetized animals.

Abundant Expression of Guidance and Synaptogenic Molecules in the Injured Spinal Cord

BackgroundSpinal interneurons have emerged as crucial targets of supraspinal input during post-injury axonal remodelling. For example, lesioned corticospinal projections use propriospinal neurons as relay stations to form intraspinal detour circuits that circumvent the lesion site and contribute to functional recovery. While a number of the molecules that determine the formation of neuronal circuits in the developing nervous system have been identified, it is much less understood which of these cues are also expressed in the injured spinal cord and can thus guide growing collaterals and initiate synaptogenesis during circuit remodelling.Methodology/Principal FindingsTo address this question we characterized the expression profile of a number of guidance and synaptogenic molecules in the cervical spinal cord of healthy and spinal cord-injured mice by in situ hybridization. To assign the expression of these molecules to distinct populations of interneurons we labeled short and long propriospinal neurons by retrograde tracing and glycinergic neurons using a transgenically expressed fluorescent protein. Interestingly, we found that most of the molecules studied including members of slit-, semaphorin-, synCAM-, neuroligin- and ephrin- families as well as their receptors are also present in the adult CNS. While many of these molecules were abundantly expressed in all interneurons examined, some molecules including slits, semaphorin 7a, synCAM4 and neuroligin 1 showed preferential expression in propriospinal interneurons. Overall the expression pattern of guidance and synaptogenic molecules in the cervical spinal cord appeared to be stable over time and was not substantially altered following a midthoracic spinal cord injury.ConclusionsTaken together, our study indicates that many of the guidance and synaptogenic cues that regulate neuronal circuit formation in development are also present in the adult CNS and therefore likely contribute to the remodelling of axonal connections in the injured spinal cord.

Identification of initially appearing glycine‐immunoreactive neurons in the embryonic zebrafish brain

Glycine is a major inhibitory neurotransmitter in the central nervous system of vertebrates. Here, we report the initial development of glycine-immunoreactive (Gly-ir) neurons and fibers in zebrafish. The earliest Gly-ir cells were found in the hindbrain and rostral spinal cord by 20 h post-fertilization (hpf). Gly-ir cells in rhombomeres 5 and 6 that also expressed glycine transporter 2 (glyt2) mRNA were highly stereotyped; they were bilaterally located and their axons ran across the midline and gradually turned caudally, joining the medial longitudinal fascicles in the spinal cord by 24 hpf. Gly-ir neurons in rhombomere 5 were uniquely identified, since there was one per hemisegment, whereas the number of Gly-ir neurons in rhombomere 6 were variable from one to three per hemisegment. Labeling of these neurons by single-cell electroporation and tracing them until the larval stage revealed that they became MiD2cm and MiD3cm, respectively. The retrograde labeling of reticulo-spinal neurons in Tg(glyt2:gfp) larva, which express GFP in Gly-ir cells, and a genetic mosaic analysis with glyt2:gfp DNA construct also supported this notion. Gly-ir cells were also distributed widely in the anterior brain by 27 hpf, whereas glyt2 was hardly expressed. Double staining with anti-glycine and anti-GABA antibodies demonstrated distinct distributions of Gly-ir and GABA-ir cells, as well as the presence of doubly immunoreactive cells in the brain and placodes. These results provide evidence of identifiable glycinergic (Gly-ir/glyt2-positive) neurons in vertebrate embryos, and they can be used in further studies of the neurons' development and function at the single-cell level.

Morphological, biophysical and synaptic properties of glutamatergic neurons of the mouse spinal dorsal horn

Interneurons of the spinal dorsal horn are central to somatosensory and nociceptive processing. A mechanistic understanding of their function depends on profound knowledge of their intrinsic properties and their integration into dorsal horn circuits. Here, we have used BAC transgenic mice expressing enhanced green fluorescent protein (eGFP) under the control of the vesicular glutamate transporter (vGluT2) gene (vGluT2::eGFP mice) to perform a detailed electrophysiological and morphological characterisation of excitatory dorsal horn neurons, and to compare their properties to those of GABAergic (Gad67::eGFP tagged) and glycinergic (GlyT2::eGFP tagged) neurons. vGluT2::eGFP was detected in about one-third of all excitatory dorsal horn neurons and, as demonstrated by the co-expression of vGluT2::eGFP with different markers of subtypes of glutamatergic neurons, probably labelled a representative fraction of these neurons. Three types of dendritic tree morphologies (vertical, central, and radial), but no islet cell-type morphology, were identified in vGluT2::eGFP neurons. vGluT2::eGFP neurons had more depolarised action potential thresholds and longer action potential durations than inhibitory neurons, while no significant differences were found for the resting membrane potential, input resistance, cell capacitance and after-hyperpolarisation. Delayed firing and single action potential firing were the single most prevalent firing patterns in vGluT2::eGFP neurons of the superficial and deep dorsal horn, respectively. By contrast, tonic firing prevailed in inhibitory interneurons of the dorsal horn. Capsaicin-induced synaptic inputs were detected in about half of the excitatory and inhibitory neurons, and occurred more frequently in superficial than in deep dorsal horn neurons. Primary afferent-evoked (polysynaptic) inhibitory inputs were found in the majority of glutamatergic and glycinergic neurons, but only in less than half of the GABAergic population. Excitatory dorsal horn neurons thus differ from their inhibitory counterparts in several biophysical properties and possibly also in their integration into the local neuronal circuitry.

Mitochondrial Abnormality Associates with Type-Specific Neuronal Loss and Cell Morphology Changes in the Pedunculopontine Nucleus in Parkinson Disease

Cholinergic neuronal loss in the pedunculopontine nucleus (PPN) associates with abnormal functions, including certain motor and nonmotor symptoms. This realization has led to low-frequency stimulation of the PPN for treating patients with Parkinson disease (PD) who are refractory to other treatment modalities. However, the molecular mechanisms underlying PPN neuronal loss and the therapeutic substrate for the clinical benefits following PPN stimulation remain poorly characterized, hampering progress toward designing more efficient therapies aimed at restoring the PPN's normal functions during progressive parkinsonism. Here, we investigated postmortem pathological changes in the PPN of PD cases. Our study detected a loss of neurons producing gamma-aminobutyric acid (GABA) as their output and glycinergic neurons, along with the pronounced loss of cholinergic neurons. These losses were accompanied by altered somatic cell size that affected the remaining neurons of all neuronal subtypes studied here. Because studies showed that mitochondrial dysfunction exists in sporadic PD and in PD animal models, we investigated whether altered mitochondrial composition exists in the PPN. A significant up-regulation of several mitochondrial proteins was seen in GABAergic and glycinergic neurons; however, cholinergic neurons indicated down-regulation of the same proteins. Our findings suggest an imbalance in the activity of key neuronal subgroups of the PPN in PD, potentially because of abnormal inhibitory activity and altered cholinergic outflow.

Amino acid immunoreactivity in normal human retina and after brachytherapy

We localised amino acids in the mid‐peripheral aged human retina and a retina that had undergone radiation treatment 10 years earlier. The distribution pattern of glutamate, γ‐amino butyric acid (GABA), glycine, glutamine and taurine, reflected patterns established in the primate retina. The retina that had undergone radiation exposure displayed both anatomical and neurochemical remodelling. The proximal retina comprised around 40 to 45 per cent of the total retina and neuronal kinesis and aberrant neuronal projections were also present. Amino acid neurochemistry was strikingly different with Müller cells displaying GABA loading, glycinergic neurons displaced and displaying a very high level of glycine labelling. We conclude that radiation exposure triggered these changes in the human retina and likely reflects general remodelling of structure and function following ischaemic damage to endothelial cells.

Vesicular GABA transporter (VGAT) transports β‐alanine

Vesicular GABA transporter (VGAT) is expressed in GABAergic and glycinergic neurons, and is responsible for vesicular storage and subsequent exocytosis of these inhibitory amino acids. In this study, we show that VGAT recognizes β-alanine as a substrate. Proteoliposomes containing purified VGAT transport β-alanine using Δψ but not ΔpH as a driving force. The Δψ-driven β-alanine uptake requires Cl(-). VGAT also facilitates Cl(-) uptake in the presence of β-alanine. A previously described VGAT mutant (Glu213Ala) that disrupts GABA and glycine transport similarly abrogates β-alanine uptake. These findings indicated that VGAT transports β-alanine through a mechanism similar to those for GABA and glycine, and functions as a vesicular β-alanine transporter. Vesicular GABA transporter (VGAT) is expressed in GABAergic and glycinergic neurons, and is responsible for vesicular storage and subsequent exocytosis of these inhibitory amino acids. In the present study, we showed that proteoliposomes containing purified VGAT transport β-alanine using Δψ as a driving force. VGAT also facilitates Cl(-) uptake. Our findings indicated that VGAT functions as a vesicular β-alanine transporter.

The effect of tracheal occlusion on the load compensation response and the changes in inhibitory neurotransmitter in the brainstem of conscious rats

Respiratory load compensation in conscious animals is mediated by cortical, subcortical modulation and brainstem mediated reflexes. We hypothesized that consciousness behavior would reconfigure the brainstem mediated respiratory load compensation reflex in respond to tracheal occlusion (TO). We also hypothesized that glycinergic neurons in the NTS would not be activated in conscious rats as we found in anesthetized rats in a previous study. A tracheal occluder and diaphragm electrodes were implanted in six experimental (E) and six sham (S) rats for the measurement of respiratory parameters. Seven days after recovery, the E rats had 2–3 seconds TOs separated by 10–15 unloaded breaths for 10 minutes. Ninety minutes after TO, the rats were anesthetized, brainstem were perfused and harvested for the double immnofluorescence staining of c‐Fos and glycine transporter 2. The results showed that conscious rats prolonged expiratory time (Te) in respond to TO but inspiratory time (Te) was unaffected by the load. The glycinergic neurons in the NTS were activated in conscious rats. In conclusion, conscious rat load compensation responses are characterized by maintaining normal Ti and modulating Te which differs from anesthetized rats, but glycinergic neurons in the NTS were activated which are similar to anesthetized rats.

Mixed miniature postsynaptic currents resulting from co‐release of glycine and GABA recorded from glycinergic neurons in the neonatal respiratory network

Inhibitory neurons are involved in the generation and patterning of the respiratory rhythm in the adult animal. However, the role of glycinergic neurons in the respiratory rhythm in the developing network is still not understood. Although the complete loss of glycinergic transmission in vivo is lethal, the blockade of glycinergic transmission in slices of the medulla has little effect on pre-Bötzinger complex network activity. As 50% of the respiratory rhythmic neurons in this slice preparation are glycinergic, they have to be considered as integrated parts of the network. We aimed to investigate whether glycinergic neurons receive mixed miniature inhibitory postsynaptic currents (mIPSCs) that result from co-release of GABA and glycine. Quantification of mixed mIPSCs by the use of different objective detection methods resulted in a wide range of results. Therefore, we generated traces of mIPSCs with a known distribution of mixed mIPSCs and mono-transmitter-induced mIPSCs, and tested the detection methods on the simulated data. We found that analysis paradigms, which are based on fitting the sum of two mIPSC templates, to be most acceptable. On the basis of these protocols, 20-40% of all mIPSCs recorded from respiratory glycinergic neurons are mixed mIPSCs that result from co-release of GABA and glycine. Furthermore, single-cell reverse transcriptase polymerase chain reaction revealed that 46% of glycinergic neurons co-express mRNA of glycine transporter 2 together with at least one marker protein of GABAergic neurons. Our data suggest that significant co-transmission occurs in the pre-Bötzinger complex that might be involved in the shaping of synaptic inhibition of respiratory glycinergic neurons.