GABAergic Nerve Terminals Research Articles

Slc38a1 Conveys Astroglia-Derived Glutamine into GABAergic Interneurons for Neurotransmitter GABA Synthesis.

GABA signaling is involved in a wide range of neuronal functions, such as synchronization of action potential firing, synaptic plasticity and neuronal development. Sustained GABA signaling requires efficient mechanisms for the replenishment of the neurotransmitter pool of GABA. The prevailing theory is that exocytotically released GABA may be transported into perisynaptic astroglia and converted to glutamine, which is then shuttled back to the neurons for resynthesis of GABA—i.e., the glutamate/GABA-glutamine (GGG) cycle. However, an unequivocal demonstration of astroglia-to-nerve terminal transport of glutamine and the contribution of astroglia-derived glutamine to neurotransmitter GABA synthesis is lacking. By genetic inactivation of the amino acid transporter Solute carrier 38 member a1 (Slc38a1)—which is enriched on parvalbumin+ GABAergic neurons—and by intraperitoneal injection of radiolabeled acetate (which is metabolized to glutamine in astroglial cells), we show that Slc38a1 mediates import of astroglia-derived glutamine into GABAergic neurons for synthesis of GABA. In brain slices, we demonstrate the role of Slc38a1 for the uptake of glutamine specifically into GABAergic nerve terminals for the synthesis of GABA depending on demand and glutamine supply. Thus, while leaving room for other pathways, our study demonstrates a key role of Slc38a1 for newly formed GABA, in harmony with the existence of a GGG cycle.

Functional identification of neurons regulate sleep and wakefulness

Sleep/wakefulness state change is regulated by neurons, however, its regulatory mechanism is still unclear. Here we found that GABAergic neurons in the ventral tegmental area (VTA) have an important role in the regulation of sleep/wakefulness. Adeno-associate virus (AAV) vectors were injected into VTA of glutamic acid decarboxylase (GAD)-Cre mice in which GABAergic neurons are exclusively express Cre. To manipulate GABAergic neurons in the VTA, channelrhodopsin2 (ChR2), anion-channelrhodopsin2 (ACR2) or hM3Dq was expressed by AAV. Chemogenetical activation of these neurons significantly increased time in NREM sleep. To reveal neural mechanism, slice patch clamp was performed. AAV was injected to VTA to express ChR2 in the GABAergic neurons in the VTA. Orexin neurons expressing fluorescent protein were identified and recorded. Then, GABAergic nerve terminals from VTA were stimulated by blue light. Blue light significantly inhibited activity of orexin neurons. On the other hand, optogenetical inhibition of these neurons using ACR2 immediately induced wakefulness. To evaluate physiological importance of this response, these neurons were inhibited during recovery sleep after sleep deprivation for 4 hr. Inhibition of GABAergic neurons induced wakefulness even in the very sleepy condition. These results suggest that activity of GABAergic neurons in the VTA is critical to change sleep/wakefulness state especially in the regulation of NREM sleep and wakefulness.

Synaptic function and plasticity in identified inhibitory inputs onto VTA dopamine neurons.

Ventral tegmental area (VTA) dopaminergic neurons are key components of the reward pathway, and their activity is powerfully controlled by a diverse array of inhibitory GABAergic inputs. Two major sources of GABAergic nerve terminals within the VTA are local VTA interneurons and neurons in the rostromedial tegmental nucleus (RMTg). Here, using optogenetics, we compared synaptic properties of GABAergic synapses on VTA dopamine neurons using selective activation of afferents that originate from these two cell populations. We found little evidence of co-release of glutamate from either input, but RMTg-originating synaptic currents were reduced by strychnine, suggesting co-release of glycine and GABA. VTA-originating synapses displayed a lower initial release probability, and at higher frequency stimulation, short-term depression was more marked in VTA- but not RMTg-originating synapses. We previously reported that nitric oxide (NO)-induced potentiation of GABAergic synapses on VTA dopaminergic cells is lost after exposure to drugs of abuse or acute stress; in these experiments, multiple GABAergic afferents were simultaneously activated by electrical stimulation. Here we found that optogenetically-activated VTA-originating synapses on presumptive dopamine neurons also exhibited NO-induced potentiation, whereas RMTg-originating synapses did not. Despite providing a robust inhibitory input to the VTA, RMTg GABAergic synapses are most likely not those previously shown by our work to be persistently altered by addictive drugs and stress. Our work emphasises the idea that dopamine neuron excitability is controlled by diverse inhibitory inputs expected to exert varying degrees of inhibition and to participate differently in a range of behaviours.

Presynaptic GLP‐1 receptors enhance the depolarization‐evoked release of glutamate and GABA in the mouse cortex and hippocampus

Glucagon-like peptide-1 receptors (GLP-1Rs) have been shown to mediate cognitive-enhancing and neuroprotective effects in the central nervous system. However, little is known about their physiological roles on central neurotransmission, especially at the presynaptic level. Using purified synaptosomal preparations and immunofluorescence techniques, here we show for the first time that GLP-1Rs are localized on mouse cortical and hippocampal synaptic boutons, in particular on glutamatergic and GABAergic nerve terminals. Their activation by the selective agonist exendin-4 (1-100 nM) was able to increase the release of either [3 H]d-aspartate or [3 H]GABA. These effects were abolished by 10 nM of the selective GLP1-R antagonist exendin-3 (9-39) and were prevented by the selective adenylyl cyclase inhibitor 2',5'-dideoxyadenosine (10 µM), indicating the involvement of classic GLP-1Rs coupled to Gs protein stimulating cAMP synthesis. Our data demonstrate the existence and activity of presynaptic receptors for GLP-1 that could represent additional mechanisms by which this neurohormone exerts its effects in the CNS. © 2017 BioFactors, 44(2):148-157, 2018.

Conditional Deletion of the Prolactin Receptor Reveals Functional Subpopulations of Dopamine Neurons in the Arcuate Nucleus of the Hypothalamus.

Tuberoinfundibular dopamine (TIDA) neurons, known as neuroendocrine regulators of prolactin secretion from the pituitary gland, also release GABA within the hypothalamic arcuate nucleus. As these neurons express prolactin receptors (Prlr), prolactin may regulate GABA secretion from TIDA neurons, potentially mediating actions of prolactin on hypothalamic function. To investigate whether GABA is involved in feedback regulation of TIDA neurons, we examined the physiological consequences of conditional deletion of Prlr in GABAergic neurons. For comparison, we also examined mice in which Prlr were deleted from most forebrain neurons. Both neuron-specific and GABA-specific recombination of the Prlr gene occurred throughout the hypothalamus and in some extrahypothalamic regions, consistent with the known distribution of Prlr expression, indicative of knock-out of Prlr. This was confirmed by a significant loss of prolactin-induced phosphorylation of STAT5, a marker of prolactin action. Several populations of GABAergic neurons that were not previously known to be prolactin-sensitive, notably in the medial amygdala, were identified. Approximately 50% of dopamine neurons within the arcuate nucleus were labeled with a GABA-specific reporter, but Prlr deletion from these dopamine/GABA neurons had no effect on feedback regulation of prolactin secretion. In contrast, Prlr deletion from all dopamine neurons resulted in profound hyperprolactinemia. The absence of coexpression of tyrosine hydroxylase, a marker for dopamine production, in GABAergic nerve terminals in the median eminence suggested that rather than a functional redundancy within the TIDA population, the dopamine/GABA neurons in the arcuate nucleus represent a subpopulation with a functional role distinct from the regulation of prolactin secretion. Using a novel conditional deletion of the prolactin receptor, we have identified functional subpopulations in hypothalamic dopamine neurons. Although commonly considered a uniform population of neuroendocrine neurons involved in the control of prolactin secretion, we have shown that approximately half of these neurons express GABA as well as dopamine, but these neurons are not necessary for the feedback regulation of prolactin secretion. The absence of tyrosine hydroxylase in GABAergic nerve terminals in the median eminence suggests that only the non-GABAergic dopamine neurons are involved in the control of pituitary prolactin secretion, and the GABAergic subpopulation may function as interneurons within the arcuate nucleus to regulate other aspects of hypothalamic function.

Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca2+ influx, not Ca2+-exocytosis coupling

Identifying presynaptic mechanisms of general anesthetics is critical to understanding their effects on synaptic transmission. We show that the volatile anesthetic isoflurane inhibits synaptic vesicle (SV) exocytosis at nerve terminals in dissociated rat hippocampal neurons through inhibition of presynaptic Ca(2+) influx without significantly altering the Ca(2+) sensitivity of SV exocytosis. A clinically relevant concentration of isoflurane (0.7 mM) inhibited changes in [Ca(2+)]i driven by single action potentials (APs) by 25 ± 3%, which in turn led to 62 ± 3% inhibition of single AP-triggered exocytosis at 4 mM extracellular Ca(2+) ([Ca(2+)]e). Lowering external Ca(2+) to match the isoflurane-induced reduction in Ca(2+) entry led to an equivalent reduction in exocytosis. These data thus indicate that anesthetic inhibition of neurotransmitter release from small SVs occurs primarily through reduced axon terminal Ca(2+) entry without significant direct effects on Ca(2+)-exocytosis coupling or on the SV fusion machinery. Isoflurane inhibition of exocytosis and Ca(2+) influx was greater in glutamatergic compared with GABAergic nerve terminals, consistent with selective inhibition of excitatory synaptic transmission. Such alteration in the balance of excitatory to inhibitory transmission could mediate reduced neuronal interactions and network-selective effects observed in the anesthetized central nervous system.

Immunogold characteristics of VGLUT3-positive GABAergic nerve terminals suggest corelease of glutamate.

There is compelling evidence that glutamate can act as a cotransmitter in the mammalian brain. Interestingly, the third vesicular glutamate transporter (VGLUT3) is primarily found in neurons that were anticipated to be nonglutamatergic. Whereas the function of VGLUT3 in acetylcholinergic and serotoninergic neurons has been elucidated, the role of VGLUT3 in neurons releasing gamma-aminobutyric acid (GABA) is not settled. We have previously shown that VGLUT3 is found together with the vesicular GABA transporter (VIAAT) on synaptic vesicle membranes in the hippocampus. Now we provide novel electron microscopic data from the rat hippocampus suggesting that glutamate is enriched in inhibitory nerve terminals containing VGLUT3 compared to those lacking VGLUT3. The opposite was found for GABA; VGLUT3-positive inhibitory terminals contained lower density of GABA labeling compared to VGLUT3-negative inhibitory terminals. In addition, semiquantitative confocal immunofluorescence showed that N-methyl-D-aspartate (NMDA)-receptor labeling was present more frequently in VGLUT3-positive/VIAAT-positive synapses versus in VGLUT3-negative/VIAAT-positive synapses. Electron microscopic immunogold data further suggest that NMDA receptors are enriched in VGLUT3 containing inhibitory terminals. Our data reveal new chemical characteristics of a subset of GABAergic interneurons in the hippocampus. The analyses suggest that glutamate is coreleased with GABA from hippocampal basket cell-synapses to act on NMDA receptors.

Zolpidem and eszopiclone prime α1β2γ2 GABAA receptors for longer duration of activity.

GABAA receptors mediate neuronal inhibition in the brain. They are the primary targets for benzodiazepines, which are widely used to treat neurological disorders including anxiety, epilepsy and insomnia. The mechanism by which benzodiazepines enhance GABAA receptor activity has been extensively studied, but there is little mechanistic information on how non-benzodiazepine drugs that bind to the same site exert their effects. Eszopiclone and zolpidem are two non-benzodiazepine drugs for which no mechanism of action has yet been proposed, despite their clinical importance as sleeping aids. Here we investigate how both drugs enhance the activity of α1β2γ2 GABAA receptors. We used rapid ligand application onto macropatches and single-channel kinetic analysis to assess rates of current deactivation. We also studied synaptic currents in primary neuronal cultures and in heterosynapses, whereby native GABAergic nerve terminals form synapses with HEK293 cells expressing α1β2γ2 GABAA receptors. Drug binding and modulation was quantified with the aid of an activation mechanism. At the single-channel level, the drugs prolonged the duration of receptor activation, with similar KD values of ∼80 nM. Channel activation was prolonged primarily by increasing the equilibrium constant between two connected shut states that precede channel opening. As the derived mechanism successfully simulated the effects of eszopiclone and zolpidem on ensemble currents, we propose it as the definitive mechanism accounting for the effects of both drugs. Importantly, eszopiclone and zolpidem enhanced GABAA receptor currents via a mechanism that differs from that proposed for benzodiazepines.

Muscarinic M1 receptors regulate propofol modulation of GABAergic transmission in rat ventrolateral preoptic neurons.

GABAergic neurons within the ventrolateral preoptic area (VLPO) play an important role in sleep-wakefulness regulation. Propofol, a widely used systemic anesthetic, has lately been reported to excite noradrenaline (NA)-inhibited type of VLPO neurons. Present study tested if acetylcholine system takes part in the propofol modulation of GABAergic spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in mechanically dissociated rat VLPO neurons using a conventional whole-cell patch clamp technique. Propofol reversibly decreased mIPSC frequency without affecting the current amplitude, indicating that propofol acts presynaptically to decrease the probability of spontaneous GABA release. The propofol action on GABAergic mIPSC frequency was completely blocked by atropine, a nonselective muscarinic acetylcholine (mACh) receptor antagonist, and pirenzepine, a selective M1 receptor antagonist. These results suggest that propofol acts on M1 receptors on GABAergic nerve terminals projecting to VLPO neurons to inhibit spontaneous GABA release. The M1 receptor-mediated modulation of GABAergic transmission onto VLPO neurons may contribute to the regulation of loss of consciousness induced by propofol.

Pharmacological actions of thymol and an analogue at GABAB autoreceptors.

GABAB autoreceptors inhibit release of GABA from GABAergic nerve terminals. Agonists of these receptors (e.g. baclofen) inhibit, whereas antagonists (e.g. (+)-(S)-5,5-dimethylmorpholinyl-2-acetic acid; Sch50911) enhance release of the transmitter. The actions of thymol (2-isopropyl-5-methylphenol) and the structurally related compound 2-tert-butyl-4-methylphenol, (4MP) on the release of [(3) H]-GABA were examined in rat neocortical slices where the GABAergic nerves had been preloaded with [(3) H]-GABA and subsequently stimulated electrically on two occasions (S1 and S2 ). Test agents, baclofen and Sch50911 were added to the superfusion medium prior to the second period of stimulation (S2 ). Stimulation-induced overflow (SIO) of [(3) H]-GABA as a consequence of these stimulations (SIO1 and SIO2 ) were calculated and the effects of agents determined by comparing the SIO2 /SIO1 ratio in the presence of each agent with that in control tissue. Thymol potentiated the release of [(3) H]-GABA (EC50 170μmol/L), an action reversed by baclofen (2μmol/L). Baclofen alone had little effect on GABA release. Release of [(3) H]-GABA was inhibited by 4MP (IC50 3μmol/L) and this effect was blocked by Sch50911 (10μmol/L). Alone, Sch50911 markedly potentiated the release of GABA. These results imply that 4MP is an agonist of GABAB autoreceptors; however, further studies are needed to confirm that thymol is indeed a GABAB autoreceptor antagonist. Of interest are structural differences in these agents. Thymol has a propyl group in the ortho position relative to the phenolic hydroxyl, whereas in 4MP this is a butyl group and the methyl group moves from position 5 to 4. Whether one or both of these changes was responsible for the above actions is unknown.

Functional selectivity and time-dependence of μ-opioid receptor desensitization at nerve terminals in the mouse ventral tegmental area.

BACKGROUND AND PURPOSEThe majority of studies examining desensitization of the μ-opioid receptor (MOR) have examined those located at cell bodies. However, MORs are extensively expressed at nerve terminals throughout the mammalian nervous system. This study is designed to investigate agonist-induced MOR desensitization at nerve terminals in the mouse ventral tegmental area (VTA).EXPERIMENTAL APPROACHMOR function was measured in mature mouse brain slices containing the VTA using whole-cell patch-clamp electrophysiology. Presynaptic MOR function was isolated from postsynaptic function and the functional selectivity, time-dependence and mechanisms of agonist-induced MOR desensitization were examined.KEY RESULTSMORs located at GABAergic nerve terminals in the VTA were completely resistant to rapid desensitization induced by the high-efficacy agonists DAMGO and Met-enkephalin. MORs located postsynaptically on GABAergic cell bodies readily underwent rapid desensitization in response to DAMGO. However, after prolonged (>7 h) treatment with Met-enkephalin, profound homologous MOR desensitization was observed. Morphine could induce rapid MOR desensitization at nerve terminals when PKC was activated.CONCLUSIONS AND IMPLICATIONSAgonist-induced MOR desensitization in GABAergic neurons in the VTA is compartment-selective as well as agonist-selective. When MORs are located at cell bodies, higher-efficacy agonists induce greater levels of rapid desensitization than lower-efficacy agonists. However, the converse is true at nerve terminals where agonists that induce MOR desensitization via PKC are capable of rapid agonist-induced desensitization while higher-efficacy agonists are not. MOR desensitization induced by higher-efficacy agonists at nerve terminals only takes place after prolonged receptor activation.LINKED ARTICLESThis article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2

Presynaptic GABAB autoreceptor regulation of nicotinic acetylcholine receptor mediated [3H]-GABA release from mouse synaptosomes

Activation of nicotinic acetylcholine receptors (nAChRs) can elicit neurotransmitter release from presynaptic nerve terminals. Mechanisms contributing to cell-and-terminal specific regulation of nAChR-mediated neurotransmitter exocytosis are not fully understood. The experiments discussed here examine how activation of GABAB auto- and hetero-receptors suppress nAChR-mediated release of [3H]-GABA and [3H]-dopamine (3H-DA) from mouse striatal synaptosomes. Activation of presynaptic GABAB receptors with (R)-baclofen decreased both [3H]-GABA and [3H]-DA release evoked by potassium depolarization. However, when nAChRs were activated with ACh to evoke neurotransmitter release, (R)-baclofen had no effect on [3H]-DA release, but potently inhibited ACh-evoked [3H]-GABA release. Inhibition of nAChR-evoked [3H]-GABA release by (R)-baclofen was time sensitive and the effect was lost after prolonged exposure to the GABAB agonist. The early inhibitory effect of GABAB activation on ACh-evoked [3H]-GABA release was partially attenuated by antagonists of the phosphatase, calcineurin. Furthermore, antagonists of protein kinase C (PKC) prevented the time-dependent loss of the inhibitory (R)-baclofen effect on [3H]-GABA release. These results suggest that α4β2*-nAChRs present on GABAergic nerve terminals in the striatum are subject to functional regulation by GABAB autoreceptors that is apparently cell-type specific, since it is absent from DAergic striatal nerve terminals. In addition, the functional modulation of α4β2*-type nAChRs on striatal GABAergic nerve terminals by GABAB autoreceptor activation is time-sensitive and appears to involve opposing actions of calcineurin and PKC.

NDRG2 as a marker protein for brain astrocytes.

The protein NDRG2 (N-myc downregulated gene 2) is expressed in astrocytes. We show here that NDRG2 is located in the cytosol of protoplasmic and fibrous astrocytes throughout the mammalian brain, including Bergmann glia as observed in mouse, rat, tree shrew, marmoset and human. NDRG2 immunoreactivity is detectable in the astrocytic cell bodies and excrescencies including fine distal processes. Glutamatergic and GABAergic nerve terminals are associated with NDRG2 immunopositive astrocytic processes. Müller glia in the retina displays no NDRG2 immunoreactivity. NDRG2 positive astrocytes are more abundant and more evenly distributed in the brain than GFAP (glial fibrillary acidic protein) immunoreactive cells. Some regions with very little GFAP such as the caudate nucleus show pronounced NDRG2 immunoreactivity. In white matter areas, NDRG2 is less strong than GFAP labeling. Most NDRG2 positive somata are immunoreactive for S100ß but not all S100ß cells express NDRG2. NDRG2 positive astrocytes do not express nestin and NG2 (chondroitin sulfate proteoglycan 4). The localization of NDRG2 overlaps only partially with that of aquaporin 4, the membrane-bound water channel that is concentrated in the astrocytic endfeet. Reactive astrocytes at a cortical lesion display very little NDRG2, which indicates that expression of the protein is reduced in reactive astrocytes. In conclusion, our data show that NDRG2 is a specific marker for a large population of mature, non-reactive brain astrocytes. Visualization of NDRG2 immunoreactive structures may serve as a reliable tool for quantitative studies on numbers of astrocytes in distinct brain regions and for high-resolution microscopy studies on distal astrocytic processes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00441-014-1837-5) contains supplementary material, which is available to authorized users.

Large Conductance Calcium-Activated Potassium Channels: Their Expression and Modulation of Glutamate Release from Nerve Terminals Isolated from Rat Trigeminal Caudal Nucleus and Cerebral Cortex

Large conductance, calcium-activated potassium channels [big potassium (BK) channel] consist of a tetramer of pore-forming α-subunit and distinct accessory β-subunits (β1-4) that modify the channel's properties. In this study, we analyzed the effects of BK channel activators and blockers on glutamate and γ-aminobutyric acid (GABA) release from synaptosomes isolated from the cerebral cortices or trigeminal caudal nuclei (TCN) of rats. Real-time polymerase chain reaction was used to characterize BK channel α and β(1-4) subunit expression in the cortex and in the trigeminal ganglia (TG), whose neurons project primary terminal afferents into the TCN. Immunocytochemistry was used to localize these subunits on cortical and TCN synaptosomes. The BK channels regulating [(3)H]D-aspartate release from primary afferent nerve terminals projecting into the TCN displayed limited sensitivity to iberiotoxin, whereas those expressed on cortical synaptosomes were highly sensitive to this toxin. BK channels did not appear to be present on GABAergic nerve terminals from the TCN since [(3)H]-γ-aminobutyric acid release in this model was unaffected by BK channel activators or blockers. Gene expression studies revealed expression levels of the α subunit in the TG that were only 31.2 ± 2.1% of those found in cortical tissues. The β4 subunit was the accessory subunit expressed most abundantly in both the cortex and TG. Levels of β1 and β2 were low in both these areas although β2 expression in the TG was higher than that found in the cortex. Immunocytochemistry experiments showed that co-localization of α and β4 subunits (the accessory subunit most abundantly expressed in both brain areas) was more common in TCN synaptosomes than in cortical synaptosomes. On the basis of these findings, it is reasonable to hypothesize that BK channels expressed on glutamatergic terminals in the TCN and cortex have distinct pharmacological profiles, which probably reflect different α and β subunit combinations. Channels in the cortex seem to be composed mainly of α subunits and to a lesser degree by α and β4 subunits, whereas in the TG the α + β4 combination seems to prevail (although α and/or α + β2 channels cannot be excluded). In light of the BK channels' selective control of excitatory transmission and their pharmacological diversity, their effects on primary glutamatergic afferents projecting to TCN represent a potential target for drug therapy of migraines and other types of orofacial pain.

ApoE4 induces Aβ42, tau, and neuronal pathology in the hippocampus of young targeted replacement apoE4 mice

BackgroundRecent findings suggest that the pathological effects of apoE4, the most prevalent genetic risk factor for Alzheimer’s disease (AD), start many years before the onset of the disease and are already detectable at a young age. In the present study we investigated the extent to which such pathological and cognitive impairments also occur in young apoE4 mice.ResultsThis study revealed that the levels of the presynaptic glutamatergic vesicular transporter, VGlut, in the CA3, CA1, and DG hippocampal subfields were lower in hippocampal neurons of young (4-month-old) apoE4-targeted replacement mice than in those of the apoE3 mice. In contrast, the corresponding inhibitory GABAergic nerve terminals and perikarya were not affected by apoE4.This synaptic effect was associated with hyperphosphorylation of tau in these neurons. In addition, apoE4 increased the accumulation of neuronal Aβ42 and induced mitochondrial changes, both of which were specifically pronounced in CA3 neurons. Spatial navigation behavioral studies revealed that these hippocampal pathological effects of apoE4 are associated with corresponding behavioral impairments. Time-course studies revealed that the effects of apoE4 on tau hyperphosphorylation and the mitochondria were already apparent at the age of 1 month and that the apoE4-driven accumulation of neuronal Aβ and reduced VGlut levels evolve later and are apparent at the age of 2–4 months. Furthermore, the levels of tau phosphorylation decrease in apoE3 mice and increase in apoE4 mice between 1 and 4 months, whereas the levels of Aβ42 decrease in apoE3 mice and are not affected in apoE4 mice over the same time period.ConclusionsThese findings show that apoE4 stimulates the accumulation of Aβ42 and hyperphosphorylated tau and reduces the levels of VGlut in hippocampal neurons of young apoE4-targeted replacement mice and that these neurochemical effects are associated with cognitive impairments. This model is not associated with hypothesis-driven mechanistic manipulations and is thus most suitable for unbiased studies of the mechanisms underlying the pathological effects of apoE4.

α2A adrenoceptor‐mediated presynaptic inhibition of GABAergic transmission in rat tuberomammillary nucleus neurons

Histaminergic neurons within the tuberomammillary nucleus (TMN) play an important role in the regulation of sleep-wakefulness. Here, we report the adrenergic modulation of GABAergic transmission in rat TMN histaminergic neurons using a conventional whole-cell patch clamp technique. Norepinephrine (NE) reversibly decreased the amplitude of action potential-dependent GABAergic inhibitory post-synaptic currents (IPSCs) and increased the paired pulse ratio. The NE-induced inhibition of GABAergic IPSCs was mimicked by clonidine, a selective α2 adrenoceptor agonist. However, cirazoline and isoproterenol, nonselective α1 and β adrenoceptor agonists, respectively, had no effect on GABAergic IPSCs. The NE-induced inhibition of GABAergic IPSCs was significantly blocked by BRL44408, a selective α2A adrenoceptor antagonist, but not imiloxan or JP1302, a selective α2B and α2C adrenoceptor antagonists. The extent of NE-induced inhibition of GABAergic IPSCs was inversely proportional to the extracellular Ca(2+) concentration. Pharmacological agents affecting the activities of adenylyl cyclase or G-protein-coupled inwardly rectifying K(+) channels did not affect the NE-induced inhibition of GABAergic IPSCs. However, NE had no effect on the frequency and amplitude of GABAergic miniature IPSCs. These results suggest that NE acts on presynaptic α2A adrenoceptor to inhibit action potential-dependent GABA release via the inhibition of Ca(2+) influx from the extracellular space to GABAergic nerve terminals, and that this α2A adrenoceptor-mediated modulation of GABAergic transmission may be involved in regulating the excitability of TMN histaminergic neurons.

Cellular Targets of Nitric Oxide in the Hippocampus

In the hippocampus, as in many other CNS areas, nitric oxide (NO) participates in synaptic plasticity, manifested as changes in pre- and/or postsynaptic function. While it is known that these changes are brought about by cGMP following activation of guanylyl cyclase-coupled NO receptors attempts to locate cGMP by immunocytochemistry in hippocampal slices in response to NO have failed to detect the cGMP elevation where expected, i.e. in the pyramidal neurones. Instead, astrocytes, unidentified varicose fibres and GABA-ergic nerve terminals are reported to be the prominent NO targets, raising the possibility that NO acts indirectly via other cells. We have re-investigated the distribution of cGMP generated in response to endogenous and exogenous NO in hippocampal slices using immunohistochemistry and new conditions designed to optimise cGMP accumulation and, hence, its detectability. The conditions included use of tissue from the developing rat hippocampus, a potent inhibitor of phosphodiesterase-2, and an allosteric enhancer of the NO-receptive guanylyl cyclase. Under these conditions, cGMP was formed in response to endogenous NO and was found in a population of pyramidal cell somata in area CA3 and subiculum as well as in structures described previously. The additional presence of exogenous NO resulted in hippocampal cGMP reaching the highest level recorded for brain tissue (1700 pmol/mg protein) and in cGMP immunolabelling throughout the pyramidal cell layer. Populations of axons and interneurones were also stained. According with these results, immunohistochemistry for the common NO receptor β1-subunit indicated widespread expression. A similar staining pattern for the α1-subunit with an antibody used previously in the hippocampus and elsewhere, however, proved to be artefactual. The results indicate that the targets of NO in the hippocampus are more varied and extensive than previous evidence had suggested and, in particular, that the pyramidal neurones participating in NO-dependent synaptic plasticity are direct NO targets.

Muscarinic M4 receptors regulate GABAergic transmission in rat tuberomammillary nucleus neurons

Histaminergic neurons within the tuberomammillary nucleus (TMN) play an important role in sleep-wakefulness regulation. Here, we report the muscarinic modulation of GABAergic spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in mechanically dissociated rat histaminergic neurons using a conventional whole-cell patch clamp technique. Muscarine, a nonselective muscarinic acetylcholine (mACh) receptor agonist, reversibly decreased mIPSC frequency without affecting the current amplitude, indicating that muscarine acts presynaptically to decrease the probability of spontaneous GABA release. The muscarine action on GABAergic mIPSC frequency was completely blocked by atropine, a nonselective mACh receptor antagonist, and tropicamide, an M4 receptor antagonist. The muscarine-induced decrease in mIPSC frequency was completely occluded in the presence of Cd2+, a general voltage-dependent Ca2+ channel blocker, or in a Ca2+-free external solution. However, pharmacological agents affecting adenylyl cyclase or G-protein coupled inwardly rectifying K+ channel activity did not prevent the inhibitory action of muscarine on GABAergic mIPSCs. These results suggest that muscarine acts on M4 receptors on GABAergic nerve terminals projecting to histaminergic neurons to inhibit spontaneous GABA release via the inhibition of Ca2+ influx from the extracellular space. Muscarine also inhibited action potential-dependent GABA release by activating presynaptic M4 receptors in more physiological conditions. The M4 receptor-mediated modulation of GABAergic transmission onto TMN neurons may contribute to the regulation of sleep-wakefulness.

Ontogenetic changes in the distribution of the vesicular GABA transporter VGAT correlate with the excitation/inhibition shift of GABA action

GABA is the major inhibitory neurotransmitter in the adult CNS and is among others involved in the synchronization of large neuronal networks. During development, GABA acts as a morphogenetic factor and has transient excitatory actions in many brain regions. One distinct protein, the vesicular GABA transporter (VGAT), has been identified accumulating GABA into presynaptic vesicles prior to its exocytotic release. The function of VGAT and its distribution is well defined in the adult, but its contribution to the transient excitatory action at putative GABAergic nerve terminals in the immature brain and its potential roles in putative glutamatergic nerve terminals remain elusive. We have studied VGAT expression in the brain from late embryonic stages through several postnatal stages until adulthood. Quantitative immunoblotting and immunolabeling of tissue sections at the light microscope and the electron microscope levels show an abrupt augmentation in VGAT staining in the cerebral cortex during the first three postnatal weeks, resembling the increase in other proteins involved in GABA synthesis and recycling in the same time frame – such as GAD65, GAD67, GAT1 (Slc6a1) and SN1 (Slc38a3) – and coincides with the synaptogenetic spurt. Dynamic changes in the expression of VGAT are seen in many cellular populations and in several layers in different brain regions. However, mossy fiber terminals (MFT) elude staining for VGAT. We also demonstrate that VGAT+ nerve terminals undergo a developmental reorganization so that from targeting primarily the dendrites of the principal neurons in several brain regions in the immature brain, they target the soma of the same cells in the adult. This shift in the targeted subcellular compartment coincides with the conversion of the chloride gradient across neuronal membranes and suggests that it may be important for the shift of GABA action from excitation to inhibition and for the establishment of the potent synchronization of neuronal networks.

Molecular and pharmacological evidence for a facilitatory functional role of pre‐synaptic GLUK2/3 kainate receptors on GABA release in rat trigeminal caudal nucleus

BackgroundGamma-aminobutyric acid (GABA) and glutamate (GLU) are involved in nociceptive signals processing in the trigeminal system. In this study, we investigated the influence of excitatory transmission on GABA release in nerve terminals isolated from the rat trigeminal caudal nucleus (TCN).MethodsWe utilize biochemical (superfused synaptosomes loaded with [3H]GABA) and morphological (immunofluorescence experiments with specific antibody) techniques.ResultsOur results show that GLU potentiates the release of [3H]GABA evoked by 9, 15 and 30 mM [K+]e; 15 mM [K+]e-evoked [3H]GABA release was also reinforced by domoate and kainate (KA), two naturally occurring GLU-receptor agonists. The enhancement of 15 mM [K+]e-evoked [3H]GABA release produced by 100 μM KA was abolished by NBQX, a mixed AMPA/KA receptor antagonist, but was not affected by GYKI52466, a selective AMPA receptor antagonist. ATPA, a selective agonist for KA receptors containing the GLUK1 subunit, had no effect on depolarization-induced [3H]GABA release, and UBP310, which selectively antagonizes these same receptors, failed to reverse the KA-induced potentiation of 15 mM [K+]e-evoked [3H]GABA release. The KA-induced potentiation was also unaffected by concanavalin A (10 μM), a positive allosteric modulator of GLUK1- and GLUK2-containing KA receptors.Immunofluorescence experiments revealed that GABAergic nerve terminals in the TCN differentially expressed GLUK subunits, with GLUK2/3-positive terminals being twice more abundant than GLUK1-containing synaptosomes.ConclusionsThese findings indicate that pre-synaptic KA receptors facilitating GABA release from TCN nerve terminals mainly express GLUK2/GLUK3 subunits, supporting the notion that different types of KA receptors are involved in the various stages of pain transmission.