GABA(A) receptors in the primate basal ganglia: an autoradiographic and a light and electron microscopic immunohistochemical study of the alpha1 and beta2,3 subunits in the baboon brain

The distribution of gamma-aminobutyric acid(A) (GABA(A)) receptors was investigated in the basal ganglia in the baboon brain by using receptor autoradiography and the immunohistochemical localisation of the alpha1 and beta2,3 subunits of the GABA(A) receptor by light and electron microscopy. In the caudate-putamen, the alpha1 subunit was distributed in high densities in the matrix compartment, and the beta2,3 subunits were more homogeneously distributed; the globus pallidus showed lower levels of the alpha1 and beta2,3 subunits. Four types of alpha1 subunit immunoreactive neurons were identified in the baboon striatum: the most numerous (75%) were type 1 medium-sized aspiny neurons; type 2 (2%) were large aspiny neurons with an indented nuclear membrane located in the ventral striatum; type 3 neurons were the least numerous (1%) and were comprised of large neurons in the ventromedial regions of the striatum; and type 4 (22%) neurons were medium to large aspiny neurons located in striosomes. At the ultrastructural level, alpha1 and beta2,3 subunit immunoreactivity was localised in the neuropil of the striatum in both symmetrical and asymmetrical synaptic contacts. In the globus pallidus, alpha1 and beta2,3 subunits were localised on large neurons and were found in three types of synaptic terminals: type 1 terminals were small and established symmetrical synapses; type 2 terminals were large; and type 3 terminals formed small synaptic terminals with subjunctional dense bodies. These results show that the subunit composition of GABA(A) receptors varies between the striosome and the matrix compartments in the striatum and that there is receptor subunit homogeneity in the globus pallidus.     

Neurochemistry of the mammalian cone 'synaptic complex'

The cone 'synaptic complex' is a unique structure in which a single presynaptic axon secretes glutamate onto processes of bipolar cells (both ON and OFF) and horizontal cells. In turn, the horizontal cell processes antagonize cone and bipolar responses to glutamate (probably by GABA). What still remains largely unknown is the molecular identity of the postsynaptic receptors and their exact locations. We identified several subunits of the glutamate receptor and the GABAA receptor expressed at the cone synaptic complex and localized them ultrastructurally. Glutamate receptors: (i) Invaginating (probably ON) bipolar dendrites in the monkey and rat express the metabotropic glutamate receptor, mGluR6. The stain is intense on the dendritic membrane where it first enters the invagination, and weak at the tip nearest to the ribbon. The cone membrane is electron-dense where it apposes the intense stain for mGluR6. Surprisingly, invaginating bipolar dendrites in the cat also express the AMPA receptor subunits, GluR2/3 and GluR4. (ii) Dendrites forming basal contacts in the cat (probably OFF) express the AMPA subunits GluR2/3, GluR4, and also the kainate subunit, GluR6/7. The stain is especially intense at the dendritic tips in apposition to electron-dense regions of cone membrane. (iii) Horizontal cells in the cat express the AMPA subunits GluR2/3, GluR4 and the kainate subunit, GluR6/7. The stain is strongest in the cytosol of somas and primary dendrites, but is also present in the invaginating terminals where it localizes to the membrane subjacent to the ribbon. GABAA receptors: (i) ON and OFF bipolar dendrites in the monkey express the alpha 1 and beta 2/3 subunits. The stain is localized to the bipolar cell membrane in apposition to horizontal cell processes. (ii) Cones did not express the GABAA subunits tested by immunocytochemistry, but beta 3 mRNA was amplified by RT-PCR from rat photoreceptors. Conclusions: (i) mGluR6 receptors concentrate on dendrites at the base of the invagination rather than at the apex. This implies that receptors at both 'invaginating' and 'basal' contacts lie roughly equidistant from the release sites and should therefore receive similar spatiotemporal concentrations of glutamate. (ii) The 'cone' membrane is electron-dense opposite to the receptor sites on both ON and OFF bipolar cells. This suggests a special role for this region in synaptic transmission. Possibly, these densities signify a transporter that would regulate glutamate concentration at sites remote (> 200 nm) from the locus of vesicle release.     

Light and electron microscopy of the ground squirrel retina: functional considerations

Light and electron microscopy of Golgi-impregnated ground squirrel retinas have revealed a range of morphological subtypes of bipolar, amacrine, and ganglion cells. There are at least seven subtypes of bipolar cells. Those subtypes in which the somata were high (sclerad) in the inner nuclear layer (3 subtypes) had axon terminals low (vitread) in the inner plexiform layer, and those with somata low in the inner nuclear layer (4 subtypes) had axon terminals high in the inner plexiform layer. The bipolar subtypes with high axon terminals made flat contacts with receptor cells, whereas all but one of the bipolar subtypes with low axon terminals made ribbon-related contacts with receptor cells. There are at least five subtypes of amacrine cells. The two subtypes which the Golgi method revealed most frequently were a broad-field, unistratified neuron with a dendritic spread in excess of 1,000 mum and a narrow-field, diffuse neuron with a dendritic spread of about 30 mum. The broad-field, unistratified cell had the lowest proportion of amacrine vs. bipolar cell synaptic input of the amacrine subtypes (43%), whereas the narrow-field, diffuse cell had one of the greatest proportions of amacrine cell input (96%). There are at least 15 subtypes of ganglion cells. The proportion of synaptic inputs to these cells ranged from 21% to 100% amacrine cell synapses. An attempt has been made to relate this new knowledge of retinal circuitry to the physiological output of the ganglion cells.     

Heterodimerization is required for the formation of a functional GABA(B) receptor

GABA (gamma-aminobutyric acid) is the main inhibitory neurotransmitter in the mammalian central nervous system, where it exerts its effects through ionotropic (GABA(A/C)) receptors to produce fast synaptic inhibition and metabotropic (GABA(B)) receptors to produce slow, prolonged inhibitory signals. The gene encoding a GABA(B) receptor (GABA(B)R1) has been cloned; however, when expressed in mammalian cells this receptor is retained as an immature glycoprotein on intracellular membranes and exhibits low affinity for agonists compared with the endogenous receptor on brain membranes. Here we report the cloning of a complementary DNA encoding a new subtype of the GABAB receptor (GABA(B)R2), which we identified by mining expressed-sequence-tag databases. Yeast two-hybrid screening showed that this new GABA(B)R2-receptor subtype forms heterodimers with GABA(B)R1 through an interaction at their intracellular carboxy-terminal tails. Upon expression with GABA(B)R2 in HEK293T cells, GABA(B)R1 is terminally glycosylated and expressed at the cell surface. Co-expression of the two receptors produces a fully functional GABA(B) receptor at the cell surface; this receptor binds GABA with a high affinity equivalent to that of the endogenous brain receptor. These results indicate that, in vivo, functional brain GABA(B) receptors may be heterodimers composed of GABA(B)R1 and GABA(B)R2.     

Membrane currents evoked by ionotropic glutamate receptor agonists in rod bipolar cells in the rat retinal slice preparation

1. With the use of the whole cell voltage-clamp technique, I have recorded the current responses to ionotropic glutamate receptor agonists of rod bipolar cells in vertical slices of rat retina. Rod bipolar cells constitute a single population of cells and were visualized by infrared differential interference contrast video microscopy. They were targeted by the position of their cell bodies in the inner nuclear layer and, after recording, were visualized in their entirety by labeling with the fluorescent dye Lucifer yellow, which was included in the recording pipette. To study current-voltage relationships of evoked currents, voltage-gated potassium currents were blocked by including Cs+ and tetraethylammonium+ in the recording pipette. 2. Pressure application of the non-N-methyl-D-aspartate (non-NMDA) receptor agonists kainate and (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) from puffer pipettes evoked a long-latency conductance increase selective for chloride ions. When the intracellular chloride concentration was increased, the reversal potential changed, corresponding to the change in equilibrium potential for chloride. The response was evoked in the presence of 5 mM Co2+ and nominally O mM Ca2+ in the extracellular solution, presumably blocking all external Ca2(+)-dependent release of neurotransmitter. 3. The long latency of kainate-evoked currents in bipolar cells contrasted with the short-latency currents evoked by gamma-aminobutyric acid (GABA) and glycine in rod bipolar cells and by kainate in amacrine cells. 4. Application of NMDA evoked no response in rod bipolar cells. 5. Coapplication of AMPA with cyclothiazide, a blocker of agonist-evoked desensitization of AMPA receptors, enhanced the conductance increase compared with application of AMPA alone. Coapplication of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked the response to kainate and AMPA, indicating that the response was mediated by conventional ionotropic glutamate receptors. 6. The conductance increase evoked by non-NMDA receptor agonists could not be blocked by a combination of 100 microM picrotoxin and 10 microM strychnine. Application of the GABAC receptor antagonist 3-aminopropyl (methyl)phosphinic acid (3-APMPA) strongly reduced the response, and coapplication of 500 microM 3-APMPA and 100 microM picrotoxin completely blocked the response. These results suggested that the conductance increase evoked by non-NMDA receptor agonists was mediated by release of GABA and activation of GABAC receptors, and most likely also GABAA receptors, on rod bipolar cells. 7. Kainate responses like those described above could not be evoked in bipolar cells in which the axon had been cut somewhere along its passage to the inner plexiform layer during the slicing procedure. This suggests that the response was dependent on the integrity of the axon terminal in the inner plexiform layer, known to receive GABAergic synaptic input from amacrine cells. 8. The results indicate that ionotropic glutamate receptors are not involved in mediating synaptic input from photoreceptors to rod bipolar cells and that an unconventional mechanism of GABA release from amacrine cells might operate in the inner plexiform layer.     

Immunocytochemical localization of the postsynaptic density protein PSD-95 in the mammalian retina

Synapse-associated proteins are the scaffold for the selective aggregation of ion channels at synapses; they provide the link to cytoskeletal elements and possibly are involved with the regulation of synaptic efficacy by electrical activity. The localization of the postsynaptic density protein PSD-95 was studied in different mammalian retinae (rat, monkey, and tree shrew) by using immunocytochemical methods. Immunofluorescence for PSD-95 was most prominent in the outer plexiform layer (OPL). The axon terminals of rods and cones, the rod spherules and cone pedicles, were strongly labeled. Electron microscopy, using preembedding immunocytochemistry, showed PSD-95 localized presynaptically within the photoreceptor terminals. Distinct PSD-95 labeling was also present in the inner plexiform layer (IPL). It had a punctate appearance suggesting the synaptic clustering of PSD-95 in the IPL. Electron microscopy showed that PSD-95 was concentrated in processes that were postsynaptic at bipolar cell ribbon synapses (dyads). As a rule, only one of the two postsynaptic members of the dyad was labeled for PSD-95. Double-labeling experiments were performed for PSD-95 and for SAP 102 or PSD-93, respectively, two other members of the family of synapse-associated proteins. All three were found to be colocalized in the synaptic hot spots in the IPL. In the OPL, however, PSD-95 and PSD-93 were found presynaptically, whereas SAP 102 was located postsynaptically at photoreceptor synapses. Double-labeling experiments also were performed for PSD-95 and for the NR1 subunit of the NMDA receptor. They were found to be colocalized in synaptic hot spots in the IPL.     

The gamma 2 subunit of the GABAA receptor is concentrated in synaptic junctions containing the alpha 1 and beta 2/3 subunits in hippocampus, cerebellum and globus pallidus

The gamma 2 subunit is necessary for the expression of the full benzodiazepine pharmacology of GABAA receptors and is one of the major subunits in the brain. In order to determine the location of channels containing the gamma 2 subunit in relation to GABA-releasing terminals on the surface of neurons, a new polyclonal antipeptide antiserum was developed to the gamma 2 subunit and used in high resolution, postembedding, immunoelectron-microscopic procedures. Dual immunogold labelling of the same section for two subunits, and up to three sections of the same synapse reacted for different subunits, were used to characterize the subunit composition of synaptic receptors. The gamma 2 subunit was present in type 2, "symmetrical" synapses in each of the brain areas studied, with the exception of the granule cell layer of the cerebellum. The gamma 2 subunit was frequently co-localized in the same synaptic junction with the alpha 1 and beta 2/3 subunits. The immunolabelling of synapses was coincident with the junctional membrane specialization of the active zone. Immunolabelling for the receptor often occurred in multiple clusters in the synapses. In the hippocampus, the gamma 2 subunit was present in basket cell synapses on the somata and proximal dendrites and in axo-axonic cell synapses on the axon initial segment of pyramidal and granule cells. Some synapses on the dendrites of GABAergic interneurones were densely labelled for the gamma 2, alpha 1 and beta 2/3 subunits. In the cerebellum, the gamma 2 subunit was present in both distal and proximal Purkinje cell dendritic synapses established by stellate and basket cell, respectively. On the soma of Purkinje cells, basket cell synapses were only weakly labelled. Synapses on interneuron dendrites were more densely labelled for the gamma 2, alpha 1 and beta 2/3 subunits than synapses on Purkinje or granule cells. Although immunoperoxidase and immunofluorescence methods show an abundance of the gamma 2 subunit in granule cells, the labelling of Golgi synapses was much weaker with the immunogold method than that of the other cell types. In the globus pallidus, many type 2 synapses were labelled for the gamma 2 subunit together with alpha 1 and beta 2/3 subunits. The results show that gamma 2 and beta 2/3 subunits receptor channels are highly concentrated in GABAergic synapses that also contain the alpha 1 and beta 2/3 subunits. Channels containing the gamma 2 subunit are expressed in synapses on functionally distinct domains of the same neuron receiving GABA from different presynaptic sources. There are quantitative differences in the density of GABAA receptors at synapses on different cell types in the same brain area.     

Synaptic inputs mediating bipolar cell responses in the tiger salamander retina

Postsynaptic receptors in bipolar cells were studied by focal application of glutamate and GABA to the outer and inner plexiform layers (OPL and IPL) under visual guidance in living retinal slices of the tiger salamander. Two different types of conductance change could be elicited in bipolar cells by applying glutamate to the OPL. In off-center cells, which had axon telodendria ramifying in the distal 55% of the IPL, glutamate elicited a conductance increase associated with a reversal potential near -5 mV. In on-center cells, which had telodendria stratified in the proximal 45% of the IPL, glutamate caused a conductance decrease associated with a reversal potential near -11 mV. These observations suggest that glutamate gates relatively nonspecific cation channels at synapses between photoreceptors and bipolar cell dendrites. Application of glutamate to the IPL elicited no conductance change in Co2+ Ringer's solution, but in normal Ringer's it generated a conductance increase associated with a reversal potential near the chloride equilibrium potential (ECl). These findings are consistent with the notion that glutamate receptors exist in GABAergic and/or glycinergic amacrine cells, and that glutamate in the IPL depolarizes these cells, causing GABA and/or glycine release and the opening of chloride channels in bipolar cell axon terminals. In Co2+ Ringer's, application of GABA at the OPL elicited no conductance changes in bipolar cells, suggesting that GABA receptors do not exist on bipolar cell dendrites. Applied at the IPL, GABA elicited large conductance increases associated with a reversal potential near ECl. Implications of these results for the functional circuitry of the tiger salamander retina are discussed.     

Cat intraamygdaloid inhibitory network: ultrastructural organization of parvalbumin-immunoreactive elements

Projection neurons of the basolateral (BL) amygdaloid complex are regulated by an intrinsic inhibitory network. To improve our understanding of this inhibitory circuit, we studied the synaptology of parvalbumin-immunopositive (PV+) elements as this calcium-binding protein is localized in a subpopulation of gamma-aminobutyric acid (GABA)-ergic interneurons. Two populations of PV+ cells were identified on the basis of soma shape (ovoid, type A vs. polygonal, type B). In the lateral and BL nuclei, the majority of boutons in contact with PV+ cells formed asymmetric synapses (types 1-3; 94%), whereas a minority (type 4, 6%) established symmetric synaptic contacts and resembled GABAergic terminals. In both nuclei, type B PV+ perikarya were more densely innervated than were type A neurons. However, the pattern of synaptic innervation of type B PV+ neurons differed in the two nuclei: in the lateral nucleus, they were almost exclusively innervated by a population of small, presumed excitatory terminals (type 1), whereas the four categories of terminals contributed more equally to their innervation in the BL nucleus. PV+ boutons belonged to a single category of terminals that was enriched with GABA and formed symmetric synapses mostly with the proximal part of PV neurons. The proportion of axosomatic synapses was significantly higher in the lateral nucleus than in the BL nucleus (33% vs. 18%). The reverse was true for the contacts with proximal dendrites (33% in the lateral nucleus vs. 46% in the BL nucleus). The remaining terminals formed synapses with distal dendrites (23-28%) and spines (8-12%). These results indicate that PV+ interneurons receive massive excitatory inputs and that PV+ terminals are strategically located to exert a powerful inhibitory control of amygdala neurons.     

Identification and localization of an immunoreactive AMPA-type glutamate receptor subunit (GluR4) with respect to identified photoreceptor synapses in the outer plexiform layer of goldfish retina

L-glutamate, the main excitatory synaptic transmitter in the retina, is released from photoreceptors and evokes responses in second-order retinal neurons (horizontal, bipolar cells) which utilize both ionotropic and metabotropic types of glutamate receptors. In the present study, to elucidate the functional roles of glutamate receptors in synaptic transmission, we have identified a specific ionotropic receptor subunit (GluR4) and determined its localization with respect to photoreceptor cells in the outer plexiform layer of the goldfish retina by light and pre-embedding electron-microscopical immunocytochemistry. We screened antisera to mammalian AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate)-preferring ionotropic glutamate receptors (GluR 1-4) of goldfish retina by light- and electron-microscopical immunocytochemistry. Only immunoreactive (IR) GluR4 was found in discrete clusters in the outer plexiform layer. The cones contacted in this manner were identified as long-wavelength ("red") and intermediate-wavelength ("green") cones, which were strongly immunoreactive to monoclonal antibody FRet 43 and antisera to goldfish red and green-cone opsins; and short-wavelength ("blue") cones, which were weakly immunoreactive to FRet 43 but strongly immunoreactive with antiserum to blue-cone opsin. Immunoblots of goldfish retinal homogenate with anti-GluR4 revealed a single protein at M(r) = 110 kDa. Preadsorption of GluR4 antiserum with either the immunizing rat peptide, or its goldfish homolog, reduced or abolished staining in retinal sections and blots. Therefore, we have detected and localized genuine goldfish GluR4 in the outer plexiform layer of the goldfish retina. We characterized contacts between photoreceptor cells and GluR4-IR second-order neurons in the electron microscope. IR-GluR4 was localized to invaginating central dendrites of triads in ribbon synapses of red cones, semi-invaginating dendrites in other cones and rods, and dendrites making wide-cleft basal junctions in rods and cones; the GluR4-IR structures are best identified as dendrites of OFF-bipolar cells. The results of our studies indicate that in goldfish retina GluR4-expressing neurons are postsynaptic to all types of photoreceptors and that transmission from photoreceptors to OFF-bipolars is mediated at least in part by AMPA-sensitive receptors containing GluR4 subunits.     

Synthesis of amidrazones using an engineered papain nitrile hydratase

Diadenosine polyphosphates present at the cytosol can be transported to secretory granules allowing their exocytotic release. Extracellularly, they can act through specific metabotropic or ionotropic receptors, or as analogues of P2X and P2Y nucleotide receptors. The specific ionotropic receptor P4 is present in synaptic terminals, and modulated by protein kinases (PK) A and C and protein phosphatases. Activation of PKA or PKC, directly or through membrane receptors, results in a decrease of affinity or in reduction of the Ca2+ transient respectively. Adenosine and ATP, both products of the extracellular destruction of diadenosine polyphosphates, acting through A1 or P2Y receptors respectively, are important physiological modulators at the P4 receptor.     

A single point mutation decreases picrotoxinin sensitivity of the human GABA receptor rho 1 subunit

The GABA receptor rho subunits are thought to form bicuculline-insensitive and picrotoxinin-sensitive GABAC receptors. We have investigated the role of the amino acid at position 309 in transmembrane segment M2 of the human rho 1 subunit as a determinant for picrotoxinin sensitivity. The mutant rho 1P309S was constructed by exchanging proline 309 for serine, the corresponding amino acid of the human rho 2 subunit. Whole-cell recordings from HEK-293 cells transfected with rho 1P309S cDNA revealed that the sensitivity of the rho 1P309S channels for picrotoxinin was four-fold lower than that of the wild type rho 1 subunit. The affinity of the mutant receptor for GABA was only slightly changed. These results provide direct evidence that the amino acid at position 309 is an important determinant for the picrotoxinin sensitivity of GABA receptors formed by the rho subunits.     

Recruitment of functional GABA(A) receptors to postsynaptic domains by insulin

Modification of synaptic strength in the mammalian central nervous system (CNS) occurs at both pre- and postsynaptic sites. However, because postsynaptic receptors are likely to be saturated by released transmitter, an increase in the number of active postsynaptic receptors may be a more efficient way of strengthening synaptic efficacy. But there has been no evidence for a rapid recruitment of neurotransmitter receptors to the postsynaptic membrane in the CNS. Here we report that insulin causes the type A gamma-aminobutyric acid (GABA[A]) receptor, the principal receptor that mediates synaptic inhibition in the CNS, to translocate rapidly from the intracellular compartment to the plasma membrane in transfected HEK 293 cells, and that this relocation requires the beta2 subunit of the GABA(A) receptor. In CNS neurons, insulin increases the expression of GABA(A) receptors on the postsynaptic and dendritic membranes. We found that insulin increases the number of functional postsynaptic GABA(A) receptors, thereby increasing the amplitude of the GABA(A)-receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) without altering their time course. These results provide evidence for a rapid recruitment of functional receptors to the postsynaptic plasma membrane, suggesting a fundamental mechanism for the generation of synaptic plasticity.     

Adrenoceptor-mediated elevation of ambient GABA levels activates presynaptic GABA(B) receptors in rat sensorimotor cortex

At inhibitory synapses in the mature neocortex and hippocampus in vitro, spontaneous action-potential-dependent and -independent release of gamma-aminobutyric acid (GABA) activates postsynaptic GABA(A) receptors but not pre- or postsynaptic GABA(B) receptors. Elevation of synaptic GABA levels with pharmacological agents or electrical stimulation can cause activation of GABA(B) receptors, but the physiological conditions under which such activation occurs need further elucidation. In rodent sensorimotor cortex, epinephrine produced a depression in the amplitude of evoked monosynaptic inhibitory postsynaptic currents (IPSCs) and a concomitant, adrenoceptor-mediated increase in the frequency of spontaneous IPSCs. Blockade of GABA(B) receptors prevented the depression of evoked IPSC amplitude by epinephrine but did not affect the increase in spontaneous IPSC frequency. These data show that adrenoceptor-mediated increases in spontaneous IPSCs can cause activation of presynaptic GABA(B) receptors and indirectly modulate impulse-related GABA release, presumably through elevation of synaptic GABA levels.     

Presynaptic and postsynaptic localization of GABA(B) receptors in neurons of the rat retina

The recently cloned GABA(B) receptors were localized in rat retina using specific antisera. Immunolabelling was detected in the inner and outer plexiform layers (IPL, OPL), and in a number of cells in the inner nuclear layer and the ganglion cell layer. Double-labelling experiments for GABA (gamma-aminobutyric acid) and GABA(B) receptors, respectively, demonstrated a co-localization in horizontal cells and amacrine cells. Electron microscopy showed that GABA(B) receptors of the OPL were localized presynaptically in horizontal cell processes invaginating into photoreceptor terminals. In the IPL, GABA(B) receptors were present presynaptically in amacrine cells, as well as postsynaptically in amacrine and ganglion cells. The postnatal development of GABA(B) receptors was also studied, and immunoreactivity was observed well before morphological and synaptic differentiation of retinal neurons. The present results suggest a presynaptic (autoreceptor) as well as postsynaptic role for GABA(B) receptors. In addition, the extrasynaptic localization of GABA(B) receptors could indicate a paracrine function of GABA in the retina.     

Distinct deactivation and desensitization kinetics of recombinant GABAA receptors

The functional role of the large heterogeneity in GABAA receptor subunit genes and its role in setting the properties of inhibitory synapses in the CNS is poorly understood. A kinetic comparison between currents elicited by ultra-rapid application with a piezoelectric translator of 1 mM GABA to mammalian cells transfected with cDNAs encoding distinct GABAA receptor subunits revealed that the intrinsic deactivation and desensitization properties depend on subunit combination. In particular, receptors containing alpha 6 with beta 2 gamma 2 subunits were endowed with a significantly slower deactivation as compared to those receptors containing alpha 1 with beta 2 gamma 2 subunits. While desensitization produced by prolonged GABA applications on alpha 1 beta 2 gamma 2 receptors was characterized by a rapid exponential decay followed by a slower decay and a steady state response, alpha 6 beta 2 gamma 2 receptors lacked desensitization. Furthermore, GABAA receptors lacking the gamma 2 subunit were characterized by a much larger non-desensitization component and a very rapid deactivation. Lastly, analysis of GABA-activated currents in cells cotransfected with alpha 1 and alpha 6 together with beta 2 gamma 2 subunit revealed unique kinetic properties. Our results suggest that distinct subunit composition confers specific deactivation and desensitization properties that may profoundly affect synaptic decay kinetics and the capability to sustain high frequency synaptic inputs.     

GABAA, NMDA and AMPA receptors: a developmentally regulated 'ménage à trois'

The main ionotropic receptors (GABAA, NMDA and AMPA) display a sequential participation in neuronal excitation in the neonatal hippocampus. GABA, the principal inhibitory transmitter in the adult CNS, acts as an excitatory transmitter in early postnatal stage. Glutamatergic synaptic transmission is first purely NMDA-receptor based and lacks functional AMPA receptors. Therefore, initially glutamatergic synapses are 'silent' at resting membrane potential, NMDA channels being blocked by Mg2+. However, when GABA and glutamatergic synapses are coactivated during the physiological patterns of activity, GABAA receptors can facilitate the activation of NMDA receptors, playing the role conferred to AMPA receptors later on in development. Determining the mechanisms underlying the development of this 'ménage à trois' will shed light not only on the wide range of trophic roles of glutamate and GABA in the developing brain, but also on the significance of the transition from neonatal to adult forms of plasticity.     

Immunocytochemical localization of the NMDA-R2A receptor subunit in the cat retina

Immunocytochemical studies were performed to determine the distribution and cellular localization of the NMDA-R2A receptor subunit (R2A) in the cat retina. R2A-immunoreactivity (R2A-IR) was noted in all layers of the retina, with specific localizations in the outer segments of red/green and blue cone photoreceptors, B-type horizontal cells, several types of amacrine cells, Müller cells and the majority of cells in the ganglion cell layer. In the inner nuclear layer, 48% of all cells residing in the amacrine cell layer were R2A-IR including a cell resembling the GABAergic A17 amacrine cell. Interestingly, the AII rod amacrine cell was devoid of R2A-IR. Although the localization of the R2A subunit was anticipated in ganglion cells, amacrines and Müller cells, the presence of this receptor subunit to the cells in the outer retina was not expected. Here, both the R2A and the R2B subunits were found to be present in the outer segments of cone photoreceptors and to the tips of rod outer segments. Although the function of these receptor subunits in rod and cone photoreceptors remains to be determined, the fact that both R2A and R2B receptor subunits are localized to cone outer segments suggests a possible alternative pathway for calcium entry into a region where this cation plays such a crucial role in the process of phototransduction. To further classify the cells that display NR2A-IR, we performed dual labeling experiments showing the relationship between R2A-labeled cells with GABA. Results showed that all GABAergic-amacrines and displaced amacrines express the R2A-subunit protein. In addition, approximately 11% of the NR2A-labeled amacrines, did not stain for GABA. These findings support pharmacological data showing that NMDA directly facilitates GABA release in retina and retinal cultures [I.L. Ferreira, C.B. Duarte, P.F. Santos, C.M. Carvalho, A.P. Carvalho, Release of [3H]GABA evoked by glutamate receptor agonist in cultured chick retinal cells: effect of Ca2+, Brain Res. 664 (1994) 252-256; G.D. Zeevalk, W.J. Nicklas, Action of the anti-ischemic agent ifenprodil on N-methyl-d-aspartate and kainate-mediated excitotoxicity, Brain Res. 522 (1990) 135-139; R. Huba, H.D. Hofmann, Transmitter-gated currents of GABAergic amacrine-like cells in chick retinal cultures, Vis. Neurosci. 6 (1991) 303-314; M. Yamashita, R. Huba, H.D. Hofmann, Early in vitro development of voltage- and transmitter-gated currents in GABAergic amacrine cells, Dev. Brain Res. 82 (1994) 95-102; R. Ientile, S. Pedale, V. Picciurro, V. Macaione, C. Fabiano, S. Macaione, Nitric oxide mediates NMDA-evoked [3H]GABA release from chick retina cells, FEBS Lett. 417 (1997) 345-348; R.C. Kubrusly, M.C. deMello, F.G. deMello, Aspartate as a selective NMDA agonist in cultured cells from the avian retina, Neurochem. Intl. 32 (1998) 47-52] or reduction of GABA in vivo [N.N. Osborn, A.J. Herrera, The effect of experimental ischaemia and excitatory amino acid agonist on the GABA and serotonin immunoreactivities in the rabbit retina, Neurosci. 59 (1994) 1071-1081]. Since the majority of GABAergic synapses in the inner retina are onto both rod and cone bipolar axon terminals [R.G. Pourcho, M.T. Owzcarzak, Distribution of GABA immunoreactivity in the cat retina: A light and electron-microscopic study, Vis. Neurosci. 2 (1989) 425-435], we hypothesize that the NMDA-receptor plays a crucial role in providing feedback inhibition onto rod and cone bipolar cells.