Using patch-clamp techniques we studied several aspects of intracellular GABA(A) and glycine Cl- current regulation in cortical and spinal cord neurons, respectively. Activation of PKA with a permeable analog of cyclic AMP (cAMP) produced a potentiation of the Cl- current activated with glycine, but not of the current induced with GABA. The inactive analog was without effect. Activation of PKC with 1 microM PMA reduced the amplitude of the GABA(A) and glycine currents. Internal application of 1 mM cGMP, on the other hand, had no effect on the amplitude of either current. The amplitude of these inhibitory currents changed slightly during 20 min of patch-clamp recording. Internal perfusion of the neurons with 1 microM okadaic acid, a phosphatase inhibitor, induced potentiation in both currents. The amplitude of GABA(A) and glycine currents recorded with 1 mM internal CaCl2 and 10 mM EGTA (10 nM free Ca2+) decayed by less than 30% of control. Increasing the CaCl2 concentration to 10 mM (34 microM free Ca2+) induced a transient potentiation of the GABA(A) current. A strong depression of current amplitude was found with longer times of dialysis. The glycine current, on the contrary, was unchanged by increasing the intracellular Ca2+ concentration. Activation of G proteins with internal FAl4- induced an inhibition of the GABA(A) current, but potentiated the amplitude of the strychnine-sensitive Cl- current. These results indicate that GABA(A) and glycine receptors are differentially regulated by activation of protein kinases, G proteins and Ca2+. This conclusion supports the existence of selectivity in the intracellular regulation of these two receptor types.

Amyloid-β peptide (Aβ) is considered a key protein in the pathogenesis of Alzheimer's disease because of its neurotoxicity, resulting in impaired synaptic function and memory. On the other hand, it was demonstrated that low (picomolar) concentrations of Aβ enhance synaptic plasticity and memory, suggesting that in the healthy brain, physiological Aβ concentrations are necessary for normal cognitive functions. In the present study, we found that Aβ (1-42) in concentrations of 10 pМ - 100nМ enhanced desensitization of the glycine-activated current in isolated CA3 pyramidal neurons and also reversibly suppressed its peak amplitude during short (600ms) co-application with agonist. The effect was most prominent at low glycine concentrations. When glycine receptors were activated by other receptor agonists - taurine and β-alanine, the changes of current kinetics and amplitudes induced by Aβ had a similar character. When Aβ (100 pM) was added to the bath solution, it caused, besides acceleration of desensitization, more pronounced reduction of peak current amplitude. This effect developed slowly, during a few minutes, and was more prominent at saturating concentrations of agonists. The results suggest that Aβ interacts with glycine receptors through three different mechanisms - by enhancing receptor desensitization, by rapid inhibition of the receptor, and also by means of a slowly developing inhibition of the amplitude of the current, possibly through intracellular mechanisms. The observed changes in the activity of glycine receptors induced by Aβ can lead to suppression of the tonic inhibition of hippocampal neurons mediated by extrasynaptic glycine receptors.

GABAA receptors (GABAARs) and glycine receptors (GlyRs) are two principal inhibitory chloride ion channels in the central nervous system. The two receptors do not function independently but cross-talk to each other, i.e., the activation of one receptor would inhibit the other. This cross-talk is present in different patterns across various regions in the central nervous system; however, the factor that determines these patterns is not understood. Here, we show that the pattern of cross-talk between the two receptors is shaped by their relative expression level in a neuron: a higher expression level correlates with louder talk. In line with a tendency of decrease in expression level of GlyRs and increase in expression level of GABAARs from the spinal cord, the brainstem to the neocortex, GlyRs talked much louder (i.e. produced greater inhibition) than GABAARs (one-way pattern) in spinal cord neurons, about equally loud as GABAARs (symmetric pattern) in inferior colliculus neurons and less loud (i.e. less inhibition) than GABAARs (asymmetric pattern) in auditory cortex neurons. Overexpression of GlyRs in inferior colliculus neurons produced an asymmetric pattern that should otherwise have been observed in spinal cord neurons. These expression level-dependent patterns of cross-talk between the two receptors may suggest how the central nervous system uses an alternative mechanism to maintain a delicate level of inhibition through adjusting the proportion of the two receptors in a neuron along its pathway.

The responses of acutely dissociated medial preoptic neurons to application of GABA, and glycine were studied using the perforated-patch whole-cell recording technique under voltage-clamp conditions. GABA, at a concentration of 1 mM, evoked outward currents in all cells (n = 33) when studied at potentials positive to -80 mV. The I-V relation was roughly linear. The currents evoked by GABA were partially blocked by 25-75 microM picrotoxin and were also partially or completely blocked by 100-200 microM bicuculline. Glycine, at a concentration of 1 mM, did also evoke outward currents in all cells (n = 12) when studied at potentials positive to -75 mV. The I-V relation was roughly linear. The currents evoked by glycine were largely blocked by 1 microM strychnine. In conclusion, the present work demonstrates that neurons from the medial preoptic nucleus of rat directly respond to the inhibitory transmitters GABA and glycine with currents that can be attributed to GABAA receptors and glycine receptors respectively.

Glycine receptors (GlyRs) are pentameric membrane proteins in the form of either α-homomers or α-β heteromers. Four out of five subunits; α1-3 and β, have been found in the mammalian brain. Early studies investigating subunit composition and expression patterns of this receptor have proposed a developmental switch from α2 homomers to α1β heteromers as the CNS matures, a conclusion primarily based on results from the spinal cord. However, our previous results indicate that this might not apply to e.g. the forebrain regions. Here we examined alterations in GlyR expression caused by developmental changes in selected brain areas, focusing on reward-related regions. Animals of several ages (P2, P21 and P60) were included to examine potential changes over time. In accordance with previous reports, a switch in expression was observed in the spinal cord. However, the present results indicate that a decrease in α2 subunit expression is not replaced by α1 subunit expression since the generally low levels, and modest increases, of α1 could hardly replace the reduction in α2-mRNA. Instead mRNA measurements indicate that α2 continues to be the dominating α-subunit also in adult animals, usually in combination with high and stable levels of β-subunit expression. This indicates that alterations in GlyR subunit expression are not simply a maturation effect common for the entire CNS, but rather a unique pattern of transition depending on the region at hand.

N-methyl-D-aspartate receptors (NMDARs) are known for their role in the induction of long-term potentiation (LTP). Here we start by reviewing the early evidence for their role in LTP at CA1 synapses in the hippocampus. We then discuss more recent evidence that NMDAR dependent synaptic plasticity at these synapses can be separated into mechanistically distinct components. An initial phase of the synaptic potentiation, which is generally termed short-term potentiation (STP), decays in an activity-dependent manner and comprises two components that differ in their kinetics and NMDAR subtype dependence. The faster component involves activation of GluN2A and GluN2B subunits whereas the slower component involves activation of GluN2B and GluN2D subunits. The stable phase of potentiation, commonly referred to as LTP, requires activation of primarily triheteromeric NMDARs containing both GluN2A and GluN2B subunits. In new work, we compare STP with a rebound potentiation (RP) that is induced by NMDA application and conclude that they are different phenomena. We also report that NMDAR dependent long-term depression (NMDAR-LTD) is sensitive to a glycine site NMDAR antagonist. We conclude that NMDARs are not synonymous for either LTP or memory. Whilst important for the induction of LTP at many synapses in the CNS, not all forms of LTP require the activation of NMDARs. Furthermore, NMDARs mediate the induction of other forms of synaptic plasticity and are important for synaptic transmission. It is, therefore, not possible to equate NMDARs with LTP though they are intimately linked. This article is part of a Special Issue entitled SI: Brain and Memory.

Application of N-methyl-D-aspartate (NMDA) to the supraoptic nucleus of the hypothalamus (SON) generates clustered firing that may be important in hormone release. However, synaptically evoked EPSPs recorded from SON neurons exhibit varying contributions from NMDA receptors. We used the high resolution of single-channel recording to examine the receptor and ion channel properties of NMDA receptors expressed by SON neurons in 'punch' culture. Biocytin introduced into individual neurons during patch clamp recording revealed large (32.1+/-3.3 micron), oblong somas and bipolar extensions typical of magnocellular neuroendocrine cells (MNCs). Rapid application of NMDA (100-300 microM) in the presence of 10 microM glycine to outside-out macropatches resulted in openings with an average conductance of 46. 9 pS and reversal potential of +3.9 mV. Increasing glycine from 0.03 to 30 microM increased the apparent frequency, duration and occurrence of overlapping NMDA-elicited openings. NMDA responses were inhibited by Mg2+ in a voltage-dependent manner and by the NMDA-site antagonist, D-(-)-2-amino-5-phosphonovaleric acid (D-APV). Application of saturating NMDA or glycine alone with the glycine-site antagonist, 5,7-dichlorokynurenate (DCK) or with D-APV, respectively, did not result in agonist-induced openings. NR1 immunoreactivity was observed in large neurons (>25 micron) with MNC-like morphology. These single-channel and immunocytochemical data confirm the presence of functional NR1-containing NMDA receptors in MNCs.

Elimination of auditory nerve activity results in atrophy and death of nucleus magnocellularis (NM) neurons in the chick. One early event in the degeneration of NM neurons is a disruption of their ribosomes. This experiment examines the role of metabotropic glutamate receptors in afferent regulation of ribosomes. The auditory nerve on one side of a chick brainstem slice was stimulated in vitro. Rapid stimulation-dependent changes in ribosomes were visualized by immunolabeling using an antibody, called Y10B, that recognizes ribosomal RNA. In normal media, NM neurons on the stimulated side of the slice show greater Y10B labeling than the unstimulated NM neurons on the opposite side of the same slice. The role of metabotropic glutamate receptors was evaluated by unilaterally stimulating the auditory nerve in media containing the metabotropic glutamate receptor antagonist (RS)-alpha-methyl-4-carboxyphenyl-glycine (MCPG). Addition of MCPG to the bath did not block EPSPs produced by stimulating the auditory nerve. However, MCPG did prevent the stimulation-dependent regulation of ribosomes in NM neurons (as indexed by Y10B labeling). These data suggest that glutamate may play a trophic role in the young auditory system through activation of metabotropic glutamate receptors.

Group II metabotropic (mGlu II) receptor subtypes mGlu2 and mGlu3 are important modulators of synaptic plasticity and glutamate release in the brain. Accordingly, several pharmacological ligands have been designed to target these receptors for the treatment of neurological disorders characterized by anomalous glutamate regulation including epilepsy. In this study, we examine whether the expression level and function of mGlu2 and mGlu3 are altered in experimental epilepsy by using immunohistochemistry, Western blot analysis, RT-PCR and extracellular recordings. A down-regulation of mGlu2/3 protein expression at the mossy fiber pathway was associated with a significant reduction in mGlu2/3 protein expression in the hippocampus and cortex of chronically epileptic rats. Moreover, a reduction in mGlu2 and mGlu3 transcripts levels was noticed as early as 24 h after pilocarpine-induced status epilepticus (SE) and persisted during subsequent "latent" and chronic periods. In addition, a significant impairment of mGlu II-mediated depression of field excitatory postsynaptic potentials at mossy fiber-CA3 synapses was detected in chronically epileptic rats. Application of mGlu II agonists (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) induced a significant reduction of the fEPSP amplitude in control rats, but not in chronic epileptic rats. These data indicate a long-lasting impairment of mGlu2/3 expression that may contribute to abnormal presynaptic plasticity, exaggerate glutamate release and hyperexcitability in temporal lobe epilepsy.

Diminished synaptic inhibition in the superficial spinal dorsal horn contributes to exaggerated pain responses that accompany peripheral inflammation and neuropathy. α2GABAA receptors (α2GABAAR) constitute the most abundant GABAAR subtype at this site and are the targets of recently identified antihyperalgesic compounds. Surprisingly, hoxb8-α2-/- mice that lack α2GABAAR from the spinal cord and peripheral sensory neurons exhibit unaltered sensitivity to acute painful stimuli and develop normal inflammatory and neuropathic hyperalgesia. Here, we provide a comprehensive analysis of GABAergic neurotransmission, of behavioral phenotypes and of possible compensatory mechanisms in hoxb8-α2-/- mice. Our results confirm that hoxb8-α2-/- mice show significantly diminished GABAergic inhibitory postsynaptic currents (IPSCs) in the superficial dorsal horn but no hyperalgesic phenotype. We also confirm that the potentiation of dorsal horn GABAergic IPSCs by the α2-preferring GABAAR modulator HZ-166 is reduced in hoxb8-α2-/- mice and that hoxb8-α2-/- mice are resistant to the analgesic effects of HZ-166. Tonic GABAergic currents, glycinergic IPSCs, and sensory afferent-evoked EPSCs did not show significant changes in hoxb8-α2-/- mice rendering a compensatory up-regulation of other GABAAR subtypes or of glycine receptors unlikely. Although expression of serotonin and of the serotonin producing enzyme tryptophan hydroxylase (TPH2) was significantly increased in the dorsal horn of hoxb8-α2-/- mice, ablation of serotonergic terminals from the lumbar spinal cord failed to unmask a nociceptive phenotype. Our results are consistent with an important contribution of α2GABAAR to spinal nociceptive control but their ablation early in development appears to activate yet-to-be identified compensatory mechanisms that protect hoxb8-α2-/- mice from hyperalgesia.

The role of metabotropic glutamate receptors (mGluRs) in long-term potentiation (LTP) has remained controversial. However, it has recently been shown that group I mGluR activation, prior to high frequency stimulation (HFS), can facilitate or 'prime' LTP in the area CA1 of the hippocampus. Here we report that, in the dentate gyrus in vitro, activation of both group I and group II mGluRs primes LTP. Control LTP, 60 min after HFS was 145.4+/-3.6% of control. The group I mGluR agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, 100 microM), resulted in LTP of 180.1+/-12.1% of control, which was significantly greater than control LTP (n=4; P<0.05). The group I/II mGluR agonist 1S, 3R-1-aminocyclopentate-1,3-dicarboxylic acid (1S,3R-ACPD, 10 microM), and the group II mGluR agonist (2S,3S, 4S)-alpha-(carboxy-cyclopropyl)-glycine (L-CCG-1, 20 microM) also produced LTP that was significantly greater than control LTP (177. 7+/-11.5% and 183.2+/-9.1% of control respectively; n=5; P<0.05). The group III mGluR agonist l-2-amino-4-phosphonobutyric acid (L-AP4, 20 microM), failed to significantly prime LTP (153.8+/-5.9% of control; n=5). It also proved difficult to depotentiate the primed LTP. Following low frequency stimulation (LFS), control LTP was reduced to 101.1+/-3.6% of control, and to 145.0+/-2.1%, 141.2+/-14. 7% and 134.0+/-8.7% of control for CHPG, ACPD and L-CCG-1 primed LTP respectively. We conclude that LTP may be primed by mGluR activation in the dentate gyrus and that this priming is mediated through group I and II mGluRs.

After the superior salivatory nucleus (SSN) neurons were labeled by administration of cholera toxin B subunit (CTB) or wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) to the pterygopalatine ganglion, morphological interactions between SSN neurons and fibers afferent to SSN neurons were examined by light and electron microscopy with double-immunostaining techniques. Antibodies to either the neurotransmitters or its receptor were used to label glutamatergic, GABAergic and glycinergic synapses on these neurons. By light microscopy, SSN neurons were identified in the ipsilateral ventrolateral part of the rostral medulla oblongata, and rich distributions of glutamate- and GABA-immunoreactive (ir) axon varicosities were observed around SSN neurons. Electron microscopy revealed that dendrites of SSN neurons received asymmetric synapses from glutamate-ir axon varicosities. Somata as well as dendrites received symmetric synapses from GABA-ir varicosities, or showed immunoreactivity for glycine receptors. Quantitative analysis by electron microscopy showed that glutamate-ir axon varicosities comprised 45.3% of total axon profiles in the SSN region, while GABA-ir varicosities were 20.8% and varicosities presynaptic to glycine receptors were 19.9%. These findings suggest that glutamatergic, GABAergic and glycinergic inputs, originated from a variety of nuclei, directly affect the activity of SSN neurons, and play a role in the regulation of the pterygopalatine ganglion of the rat.

Metabotropic glutamate receptors (mGluRs) are thought to mediate diverse processes in brain including synaptic plasticity and excitotoxicity. These receptors are often divided into three groups by their pharmacological profiles. [3H]Glutamate binding in the presence of compounds selective for ionotropic glutamate receptors can be used as a general assay for these receptors; subtypes of this non-ionotropic [3H]glutamate binding differ in both pharmacology and anatomical distribution, and are differentially sensitive to quisqualate. The characteristics of these binding sites are consistent with those of group 1 (high-affinity quisqualate) and group 2 (low-affinity quisqualate) mGluRs. Under our assay conditions, no [3H]glutamate binding to group 3-like (L-AP4 sensitive) sites could be demonstrated. We have attempted to characterize particular agents which may selectively measure [3H]glutamate binding to mGluR subtypes. We used two isomers of 2-(carboxycyclopropyl)glycine, L-CCG-I and L-CCG-II, and the (2S,1'R,2'R,3'R) isomer of 2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) as competitors of non-ionotropic [3H]glutamate binding sites. DCG-IV clearly distinguishes two binding sites. Quantitative levels of DCG-IV binding by anatomic region correlate with quisqualate-defined binding subtypes: high-affinity DCG-IV binding correlates with low-affinity quisqualate binding, whereas low-affinity DCG-IV binding correlates with high-affinity quisqualate binding. L-CCG-II displaces only one type of non-ionotropic [3H]glutamate binding, corresponding to high-affinity quisqualate binding. Therefore DCG-IV and L-CCG-II at appropriate concentrations appear to distinguish binding to putative group 2 vs. group 1 mGluRs. L-CCG-I displaces both high- and low-affinity quisqualate binding sites, but unlike the other two compounds, does not clearly distinguish between them.

The embryonic development of synapses in the rostral nucleus of the solitary tract (rNST) was investigated in rat to determine when synapses begin to function. Using a brain slice preparation we studied appearance of synaptic receptors on second order rNST neurons and investigated the development of postsynaptic responses elicited by afferent nerve stimulation. Prenatal excitatory and inhibitory synaptic responses were recorded as early as E14. Glutamatergic and GABAergic postsynaptic responses were detected as early as E16. Both NMDA and AMPA receptors contributed to glutamatergic postsynaptic responses. GABAergic postsynaptic responses resulted primarily from activation of GABA(A) receptors. However, functional GABA(C) receptors were also demonstrated. A glycinergic postsynaptic response was not found although functional glycine receptors were demonstrated at E16. Solitary tract (ST) stimulation-evoked EPSCs, first detected at E16, were eliminated by glutamate receptor antagonists. ST-evoked IPSPs, also detected at E16, were eliminated by GABA(A) receptor antagonist. Thus, considerable prenatal development of rNST synaptic connections occurs and this will ensure postnatal function of central taste processing circuits.

GABA-A receptors mediate both phasic synaptic inhibition and more recently appreciated tonic currents in the vertebrate central nervous system. We addressed discrepancies in the literature regarding the pharmacology of tonic currents by examining tonic currents in a controlled environment of dissociated, solitary glutamatergic neurons. We describe a novel tonically active, bicuculline-sensitive chloride conductance that is insensitive to gabazine and to picrotoxin and thus not mediated by conventional GABA receptors. We exclude a significant contribution from small conductance calcium-gated potassium (SK) channels. We also pharmacologically exclude calcium-gated chloride channels, glycine receptors and the chloride current associated with glutamate transport. Finally, we demonstrate that, although small, this current modulates neuronal excitability. We speculate that this tonic current may provide a complementary mechanism for the regulation of neuronal excitability, particularly in regions with low ambient GABA concentrations. We conclude that this bicuculline-sensitive conductance needs to be accounted for in studies of GABA tonic currents, lest it be confused with currents associated with GABA overflow.

The effects of an antisense phosphodiester oligodeoxynucleotide (ODN) directed to the NR1 subunit of the NMDA receptor mRNA and of its corresponding sense ODN were investigated in mice. Treatment with the antisense ODN significantly increased the time mice spent in the open arms of an elevated maze while the total number of arm entries was unaltered. Furthermore, seizure latencies after the administration of an ED100 dose of NMDA (150 mg/kg) were significantly higher in antisense treated animals compared to vehicle controls. At the same time, treatment with NR1 antisense ODN significantly reduced the Bmax of [3H]CGS-19755 binding (2101 fmol/mg protein) compared to both vehicle (2787 fmol/mg protein) and sense (2832 +/- 39 fmol/mg protein) controls without any significant change in KD (33 nM). A corresponding reduction of [3H]CGP-39653 binding was also observed after treatment with NR1 antisense compared to both sense and vehicle controls. In contrast, neither antisense nor sense ODNs altered the proportion of high affinity glycine sites or the potency of glycine at either high or low affinity glycine binding sites to inhibit [3H]CGP-39653 binding. These results show that in vivo treatment with NR1 antisense ODNs to the NMDA receptor complex reduces antagonist binding at NMDA receptors and has pharmacological effects similar to those observed with some NMDA receptor antagonists. These results also suggest that treatment with antisense ODNs may provide another means to investigate allosteric modulation of receptor subtypes in vivo.

Calpains have been previously shown to regulate AMPA receptor properties by producing partial truncation of the C-terminal domains of several receptor subunits. We now report that NMDA receptor subunits, in particular NR2 subunits, are also subjected to calpain-mediated truncation. Treatment of synaptic membranes with calpain I resulted in truncation of both NR1 and NR2 subunits, with the appearance of NR2 species with lower mol.wt. than native subunits, but still recognized by antibodies directed at the C-terminal domain. This treatment did not modify the binding of several ligands of the NMDA receptors, such as glutamate, glycine or TCP. Incubation of thin frozen-thawed brain sections with calcium resulted in calpain-mediated selective degradation of NR2 subunits, as truncation into smaller fragments was totally blocked by calpain inhibitors. Under the same conditions, TCP binding to sections was decreased by about 50%, an effect also blocked by calpain inhibitors. Treatment of hippocampal slices in culture with the excitotoxin, kainic acid, also produced calpain-mediated truncation of the C-terminal domain of NR2 but not NR1 subunits of the NMDA receptors. The results indicate that calpain activation produces several modifications of NMDA receptors, including the truncation of the C-terminal domain of NR2 subunits, and changes in channel binding properties. They suggest that calpain-mediated regulation of NMDA receptors might represent a feed-back regulation of the receptors which could be used to limit receptor activation.

This study examined the acute effects of a variety of NMDA and non-NMDA antagonists on the activity of aromatic l-amino acid decarboxylase (AADC) in the corpus striatum (CS) and substantia nigra (SN) of the rat. Sixty min pretreatment with the high affinity NMDA receptor-channel blockers MK 801 (0.01, 0.1 and 1 mg/kg) and phencyclidine (4 mg/kg) elevated AADC activity in both the CS and SN (2- to 3-fold). Even more striking increases in AADC were noted with 40 mg/kg amantadine (3.8-fold for CS, 9.0-fold for SN), 40 mg/kg memantine (3.4-fold for CS, 3.1-fold for SN; 20 mg/kg no effect) and 40 mg/kg dextromethorphan (3.4-fold for CS, 6.2-fold for SN, in 6/10 'responders'). Similarly pronounced increases in AADC activity in CS (1.9-fold) and SN (2.8-fold) were detected after administering clonidine (2 mg/kg). R-HA 966 (5 mg/kg, not 1 mg/kg) modestly raised AADC activity in CS (0.45-fold) and not SN. Other drugs had no effect on the activity of the decarboxylase enzyme, including CGP 40116 (1 and 5 mg/g), eliprodil (10 mg/kg), NBQX (10 mg/kg, 30 min pretreatment) and atropine (1 mg/kg). These experiments indicate that blocking the NMDA receptor-channel (and to a lesser extent the glycine site) or stimulating alpha2-adrenoceptors, profoundly increases AADC activity, more especially in the SN than CS. By contrast, inhibiting the NMDA glutamate recognition or polyamine sites, AMPA or muscarinic receptors is without effect on AADC in either brain region. The ability of amantadine and memantine to potentiate the antiparkinsonian actions of l-DOPA in the clinic, may be due to facilitated decarboxylation of l-DOPA by the brain.