Correlated size variations in human visual cortex, lateral geniculate nucleus, and optic tract

We have examined several components of the human visual system to determine how the dimensions of the optic tract, lateral geniculate nucleus (LGN), and primary visual cortex (V1) vary within the same brain. Measurements were made of the cross-sectional area of the optic tract, the volumes of the magnocellular and parvocellular layers of the LGN, and the surface area and volume of V1 in one or both cerebral hemispheres of 15 neurologically normal human brains obtained at autopsy. Consistent with previous observations, there was a two- to threefold variation in the size of each of these visual components among the individuals studied. Importantly, this variation was coordinated within the visual system of any one individual. That is, a relatively large V1 was associated with a commensurately large LGN and optic tract, whereas a relatively small V1 was associated with a commensurately smaller LGN and optic tract. This relationship among the components of the human visual system indicates that the development of its different parts is interdependent. Such coordinated variation should generate substantial differences in visual ability among humans.     

Benzodiazepine modulation of recombinant alpha1beta3gamma2 GABA(A) receptor function efficacy determination using the Cytosensor microphysiometer

The nonsteroidal antioestrogen tamoxifen has been shown to block a number of voltage-gated cation-selective channels but its effect on ligand-gated cation-selective channels has not been studied. We have investigated the action of tamoxifen and the related derivative toremifene on ligand-gated cationic nicotinic acetylcholine and 5-HT3 receptor channels. Tamoxifen and toremifene both inhibited cationic currents of adult-type human muscle nicotinic acetylcholine receptors expressed in Xenopus oocytes with similar IC50 values of 1.2 +/- 0.03 microM (nH = 0.84 +/- 0.02) and 1.2 +/- 0.1 microM (nH = 1.1 +/- 0.1), respectively. Tamoxifen could also block native 5-HT3 receptors in NG108-15 neuroblastoma/glioma hybrid cells with IC50 = 0.81 +/- 0.15 microM and nH of 1.3 +/- 0.3. The characteristics of block by tamoxifen at the 5-HT3 receptor were voltage- and use-independent. The inhibition of the 5-HT-evoked currents were not overcome by increasing concentrations of 5-HT consistent with a noncompetitive mechanism of block.     

Activation kinetics and single channel properties of recombinant alpha1beta2gamma2L GABA(A) receptor channels

Recombinant alpha1beta2gamma2L GABA(A) receptor channels, transiently expressed in HEK 293 cells, were investigated using the patch-clamp technique in combination with a device for ultra-fast solution exchange. The dose-response relationship revealed an EC50 of 11.6 +/- 0.9 microM and saturated with 3 mM GABA. The slope between 0.001 and 0.01 mM GABA was 2.2 +/- 0.4, indicating at least three binding sites for GABA. The rise time decreased from about 120 ms at 0.001 mM GABA to about 0.8 ms at 10 mM GABA. Single channel openings were grouped in bursts with an average duration of 10.3 +/- 3.0 ms. More than 95% of the current was represented by a single channel slope conductance of about 29 pS.     

Synchronization of visual responses between the cortex, lateral geniculate nucleus, and retina in the anesthetized cat

Synchronization of spatially distributed responses in the cortex is often associated with periodic activity. Recently, synchronous oscillatory patterning was described for visual responses in retinal ganglion cells that is reliably transmitted by the lateral geniculate nucleus (LGN), raising the question of whether oscillatory inputs contribute to synchronous oscillatory responses in the cortex. We have made simultaneous multi-unit recordings from visual areas 17 and 18 as well as the LGN and the retina to examine the interactions between subcortical and cortical synchronization mechanisms. Strong correlations of oscillatory responses were observed between retina, LGN, and cortex, indicating that cortical neurons can become synchronized by oscillatory activity relayed through the LGN. This feedforward synchronization occurred with oscillation frequencies in the range of 60-120 Hz and was most pronounced for responses to stationary flashed stimuli and more frequent for cells in area 18 than in area 17. In response to moving stimuli, by contrast, subcortical and cortical oscillations dissociated, proving the existence of independent subcortical and cortical mechanisms. Subcortical oscillations maintained their high frequencies but became transient. Cortical oscillations were now dominated by a cortical synchronizing mechanism operating in the 30-60 Hz frequency range. When the cortical mechanism dominated, LGN responses could become phase-locked to the cortical oscillations via corticothalamic feedback. In summary, synchronization of cortical responses can result from two independent but interacting mechanisms. First, a transient feedforward synchronization to high-frequency retinal oscillations, and second, an intracortical mechanism, which operates in a lower frequency range and induces more sustained synchronization.     

Exposure to prenatal nicotine transiently increases neuronal nicotinic receptor subunit alpha7, alpha4 and beta2 messenger RNAs in the postnatal rat brain

This study determined the effects of prenatal nicotine exposure (2 mg/kg/day) in Sprague Dawley CD rats via subcutaneously implanted osmotic minipumps, during gestational days 7-21, on postnatal levels of neuronal nicotinic receptor alpha4, alpha7 and beta2 subunit messenger RNAs. Northern analysis of postnatal day 1, 7, 14 and 28 hippocampal/septal and cortical total RNA using alpha-[32P]dCTP-labeled alpha4, alpha7 and beta2 complementary DNA probes identified a single (5.7-kb) alpha7 messenger RNA, three (2.4-, 3.8- and 8.0-kb) alpha4 messenger RNAs and four (3.7-, 5.0-, 7.5- and 10.0-kb) beta2 messenger RNAs. In comparison to prenatal saline, prenatal nicotine produced several significantly higher messenger RNA levels (cortical: 5.7-kb alpha7, 2.4-, 3.8- and 8.0-kb alpha4, 10.0-kb beta2; hippocampal/septal: 2.4- and 8.0-kb alpha4); these increases occurred predominantly on, but were not restricted to, postnatal day 14. Effects of nicotine were generally resolved by postnatal day 28. Collapsing the data across sex and age, a significant treatment effect indicated that hippocampal/septal and cortical 8.0-kb alpha4 messenger RNA levels and 10.0-kb beta2 messenger RNA levels were significantly higher following prenatal nicotine exposure. This is the first study indicating that prenatal nicotine produces alterations in developing postnatal rat neuronal nicotinic receptor messenger RNA levels, possibly by premature stimulation of neuronal nicotinic receptors. These results further implicate the teratogenic potential of nicotine in postnatal neuronal development.     

Na,K-ATPase subunit isoforms in human reticulocytes: evidence from reverse transcription-PCR for the presence of alpha1, alpha3, beta2, beta3, and gamma

The objective of this study has been to determine which Na,K-ATPase isoforms are expressed in red blood cells and whether kinetic differences in the uncoupled sodium efflux mode between the human red blood cell Na,K-ATPase and other preparations can be explained by differences in the underlying subunit composition. To this end, human reticulocyte RNA was isolated, reverse transcribed, amplified by PCR and appropriate primers, and sequenced. Primers from highly conserved areas as well as isoform-specific primers were used. The alpha1 and alpha3 isoforms of the alpha subunit, and the beta2 and beta3 isoforms of the beta subunit were found. The complete coding regions of the cDNAs for the reticulocyte subunits were sequenced from overlapping PCR fragments. No difference was found between the reticulocyte isoforms and the ones already known. The fact that we found beta2 but not beta1 in reticulocyte single-stranded cDNA, and beta1 but not beta2 in a leukocyte library indicates that leukocyte contamination of our reticulocyte preparation was negligible. Analysis of a human bone marrow library showed that alpha1, alpha2, and alpha3 as well as all three beta isoforms were present. The extent to which the kinetic properties of uncoupled sodium efflux might depend on different isoform combinations is not yet known.     

The role of the GABA(A) receptor alpha1 subunit N-terminal extracellular domain in propofol potentiation of chloride current

Propofol (2,6-diisopropylphenol), an intravenous general anesthetic in active clinical use today, potentiates the action of gamma-aminobutyric acid (GABA) at the type-A receptor and also directly induces current in the absence of GABA. We expressed different combinations of murine GABA(A) receptor alpha1, beta3 and gamma2 subunits in Xenopus oocytes to investigate the subunit dependence of propofol potentiation of pentobarbital-induced current. Pentobarbital induces current in all beta3-subunit-containing receptors, whereas current gating by GABA requires the presence of both alpha1 and beta3 subunits. Therefore, pentobarbital rather than GABA was used to induce current in order to separate the subunit dependence of current gating from the subunit dependence of potentiating action of propofol. alpha1beta3gamma2, alpha1beta3, beta3gamma2, or beta3 subunit combinations all responded to pentobarbital in a dose-dependent manner. True potentiation was defined as the current magnitude to simultaneous application of pentobarbital and propofol exceeding the additive responses to individual drug applications. A dose-dependent propofol potentiation of pentobarbital-induced current was observed in oocytes injected with alpha1beta3 or alpha1beta3gamma2 but not in beta3gamma2 or beta3 subunits, suggesting that the alpha1 subunit was necessary for this modulatory action of propofol. Further examination of the propofol potentiation in chimeras between the alpha1 and beta3 subunits showed that the extracellular amino-terminal half of the alpha1 subunit was sufficient to support propofol potentiation. The different requirements of the receptor structure for the agonistic (gating) and the potentiating actions suggest that these two actions of propofol are distinct processes mediated through its action at distinct sites.     

A dynamic and quantitative study of pattern visual evoked potentials and gamma-aminobutyric acid neurones in the lateral geniculate nucleus and the visual cortex of monocular deprivation cats

PURPOSE: To assess the effects of monocular lid closure during critical period on cortical activity. METHOD: Pattern visual evoked potentials (PVEP) of the normal and the monocular deprivation (MD) cats were dynamically measured and the number of gammaaminobutyric acid immunopositive (GABA-IP) neurones of the area 17 of the visual cortex and the lateral geniculate nucleus (LGN) was quantitatively compared by using immunohistochemical method (ABC). RESULTS: The amplitude of the N1-P1 attenuated in deprived eyes (DE), NE/DE at postnatal week (PNW) 7-8 (P < 0.05), NE/DE at PNW 15-16 (P < 0.01); while P1 latency delayed, NE/DE at PNW 7-8 (P > 0.05), NE/DE at PNW 15-16 (P< 0.05). The numbers of GABA-IP neurones in layer A1 of the ipsilateral LGN and in layer A of the contralateral LGN, compared to those in the corresponding normal laminae, were not significant at PNW 7-8 and PNW 11-12 (P > 0.05), while in the same cats a reduction in the number of GABA-IP neurones was found in layer IV of area 17 at PNW 11-12 (P < 0.05). However, with longer survival of 3-4 weeks in duration, the numbers of GABA-IP neurones in the deprived laminae of LGN were remarkably reduced (P < 0.05). CONCLUSIONS: The amplitude of N1-P1 components is sensitive to the effects of monocular deprivation. Monocular deprivation in cats during critical period leads to dramatic changes of the number of GABA-IP neurones in the LGN and cortical layer IV receiving inputs from the deprived eye in cats. The deprivation-induced reduction in GABA-IP neurones is delayed in the LGN compared with the visual cortex. PVEP of the MD cats is consistent with the damage of its GABA system in visual cortex.     

The distribution of calcium-binding proteins in the lateral geniculate nucleus and visual cortex of a New World monkey, the marmoset, Callithrix jacchus

Antibodies directed against the calcium-binding proteins, parvalbumin and calbindin, can be used to label distinct neuronal subgroups in the primate visual pathway. We analyzed parvalbumin immunoreactivity (P-IR) and calbindin immunoreactivity (C-IR) in the lateral geniculate nucleus (LGN) and visual cortex of the marmoset, Callithrix jacchus. We compared marmosets which were identified as having dichromatic or trichromatic color vision. Within the LGN, the density of P-IR neurones is highest in the parvocellular and magnocellular laminae, but C-IR neurones are found mainly in the koniocellular division of the LGN, that is, the interlaminar zones and S laminae. Not all interlaminar zone cells are C-IR. In the visual cortex, P-IR neurones are present in all laminae except lamina 1, in areas V1 and V2. Neurones which are strongly C-IR are mainly located in laminae 2 and 3 in V1 and V2. Lightly C-IR neurones are concentrated in lamina 4, and are more numerous in V1 than in V2. Quantitative analysis showed no differences in the density or distribution of IR neurones in either LGN or visual cortex when dichromat and trichromat animals were compared. We conclude that this functional difference is not associated with differences in the neurochemistry of calcium-binding proteins in the primary visual pathways.     

Characterization of human recombinant neuronal nicotinic acetylcholine receptor subunit combinations alpha2beta4, alpha3beta4 and alpha4beta4 stably expressed in HEK293 cells

Human embryonic kidney (HEK293) cells were transfected with cDNA encoding the human beta4 neuronal nicotinic acetylcholine (ACh) receptor subunit in pairwise combination with human alpha2, alpha3 or alpha4 subunits. Cell lines A2B4, A3B4.2 and A4B4 were identified that stably express mRNA and protein corresponding to alpha2 and beta4, to alpha3 and beta4 and to alpha4 and beta4 subunits, respectively. Specific binding of [3H]epibatidine was detected in A2B4, A3B4.2 and A4B4 cells with Kd (mean +/- S.D. in pM) values of 42 +/- 10, 230 +/- 12 and 187 +/- 29 and with Bmax (fmol/mg protein) values of 1104 +/- 338, 2010 +/- 184 and 3683 +/- 1450, respectively. Whole-cell patch-clamp recordings in each cell line demonstrated that (-)nicotine (Nic), ACh, cytisine (Cyt) and 1, 1-dimethyl-4-phenylpiperazinium iodide (DMPP) elicit transient inward currents. The current-voltage (I-V) relation of these currents showed strong inward rectification. Pharmacological characterization of agonist-induced elevations of intracellular free Ca++ concentration revealed a distinct rank order of agonist potency for each subunit combination as follows: alpha2beta4, (+)epibatidine (Epi) > Cyt > suberyldicholine (Sub) = Nic = DMPP; alpha3beta4, Epi > DMPP = Cyt = Nic = Sub; alpha4beta4, Epi > Cyt = Sub > Nic > DMPP. The noncompetitive antagonists mecamylamine and d-tubocurarine did not display subtype selectivity. In contrast, the Kb value for the competitive antagonist dihydro-beta-erythroidine (DHbetaE) was highest at alpha3beta4 compared with alpha2beta4 or alpha4beta4 receptors. These data illustrate that the A2B4, A3B4.2 and A4B4 stable cell lines are powerful tools for examining the functional and pharmacological properties of human alpha2beta4, alpha3beta4 and alpha4beta4 neuronal nicotinic receptors.     

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.     

Developmental changes of inhibitory synaptic currents in cerebellar granule neurons: role of GABA(A) receptor alpha 6 subunit

Eye opening and increased motor activity after the second postnatal week in rats imply an extensive development of motor control and coordination. We show a parallel development change in spontaneous IPSC (sIPSC) kinetics in cerebellar granule neurons. sIPSCs were studied by whole-cell recordings in cerebellar slices, prepared from 7-30 postnatal day old rats. Early in development, sIPSCs had slow decay kinetics whereas in older rats faster decaying sIPSCs were found in larger proportion. Currents elicited by 1 mM GABA pulses (GABACs) in nucleated patches excised from cerebellar granule neurons revealed that GABACs kinetics better approximate sIPSC decay in young but not in more developed rats. The expression of alpha 6 subunit of GABAA receptors, unique in cerebellar granule neurons, has been shown to increase during development. Therefore, we took advantage of the recently reported selective inhibition of GABAA receptors by furosemide to characterize the relative contribution of alpha 6 subunits to native receptors in inhibitory synapses of cerebellar granule neurons. Although furosemide inhibition of sIPSCs amplitude was highly variable among distinct granule cells, it increased during development. At the same time, furosemide failed to inhibit sIPSCs recorded from Purkinje neurons. From the comparison of furosemide inhibition and kinetics of sIPSCs with GABACs recorded from mammalian HEK293 cells transfected with combinations of alpha 1 and alpha 6 GABAA receptor subunits together with beta 2 gamma 2 subunits, we propose that an increased alpha 6 subunit contribution in the molecular assembly of postsynaptic receptors in cerebellar glomeruli is responsible for the developmental changes observed.     

Granule cells of the internal granule layer have increased expression of GABA(A) receptor beta 2/beta 3 subunits

We have examined the expression of GABA(A) receptor beta 2/beta 3 subunits from postnatal day 4 (P4) to P23. beta 2/beta 3 subunits are not detected in premigratory granule cells. Expression of beta 2/beta 3 was also low in granule cells found in the internal granule layers (IGLs) of P7 and P10 cerebella. However, between P10 and P16, the levels of beta 2/beta 3 increase substantially and reach high levels at P16-23. Because granule cells continue to migrate from the external granule layer (EGL) into the IGL after P10, this increase in the number of cells and the intensity of beta 2/beta 3 expression could be the result of a second wave of granule cells expressing beta 2/beta 3 that have migrated from the EGL. To test this hypothesis, migrating granule cells after P10 were eliminated by gamma-irradiation. Despite elimination of migrating granule cells, beta2/3 expression remained high in the IGLs of P16 irradiated animals similar to that observed in non-irradiated controls, suggesting that the increase of beta 2/beta 3 is not due to the arrival of a new population of granule cells expressing GABA(A) receptors. To explore the possibility that the increase of beta2/beta 3 is triggered by synaptic activity, we cultured P10 cerebellar sections, free of mossy fiber inputs, for 6 days in vitro. As observed in vivo, beta 2/3 expression in the IGL of cultured slices continued to increase, suggesting that beta 2/3 expression could be triggered without synaptic inputs from mossy fibers.     

GABA(A) receptor function in the cerebral cortex of alcohol-naive P and NP rats

In our previous paper (Biochim. Biophys. Acta 1379 (1998) 257-263), we demonstrated that bicarbonate promotes a cleavage of lactone ring of dehydroascorbate (DHA) on the basis of in vitro experiments. In the present study, we examined the degradation of DHA in blood circulation in vivo by using a high-performance liquid chromatographic method for the determination of ascorbate (AsA), DHA and 2,3-diketogulonate (2,3-DKG), which required no pretreatment of biological fluids. When DHA was intravenously administered to rats, a rapid disappearance of DHA (t1/2 < 1 min) and a concomitant appearance of 2,3-DKG in blood circulation were observed. Approximately 90% of the administered DHA were excreted into urine as resulting 2,3-DKG (55%) and AsA (31%), respectively. Furthermore, we elucidated that rat plasma lacks an enzyme having an aldonolactonase-like activity. The result of the present study suggests that this DHA disappearance is a function of both a chemical degradation to 2,3-DKG and a reduction to AsA.     

Differential changes in induced seizures after hippocampal treatment of rats with an antisense oligodeoxynucleotide to the GABA(A) receptor gamma2 subunit

Binding energy of DNA-Cro protein complexes is analyzed in terms of DNA elasticity, using a sequence-dependent anisotropic bendability (SDAB) model of DNA, developed recently [M.M. Gromiha, M.G. Munteanu, A. Gabrielian and S. Pongor, J. Biol. Phys. 22(1996) 227-243.]. The protein is considered to bind aspecifically to DNA that reduces the freedom of movement in the DNA molecule. In cognate DNA, the Cro protein moves on to form specific interactions and bends DNA. A comparison of the experimental data [Y. Takeda, A. Sarai and V.M. Rivera, Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 439-443.] with the calculated DNA stiffness data shows that delta G of the complex formation increases with stiffness of the ligand when the interactions are nonspecific ones, while an opposite trend is observed for specific binding. Both of these trends are in agreement with our approach using the SDAB model. A decomposition of the energy terms suggests that binding energy in the nonspecific case is used maily to compensate the free energy changes due to entropy lost by DNA, while the energy of specific interactions provide enough energy both to bend the DNA molecule and to change the conformation of the Cro protein upon ligand binding.     

The Ca2+ channel beta3 subunit differentially modulates G-protein sensitivity of alpha1A and alpha1B Ca2+ channels

We have shown previously that the Ca2+ channel beta3 subunit is capable of modulating tonic G-protein inhibition of alpha1A and alpha1B Ca2+ channels expressed in oocytes. Here we determine the modulatory effect of the Ca2+ channel beta3 subunit on M2 muscarinic receptor-activated G-protein inhibition and whether the beta3 subunit modulates the G-protein sensitivity of alpha1A and alpha1B currents equivalently. To compare the relative inhibition by muscarinic activation, we have used successive ACh applications to remove the large tonic inhibition of these channels. We show that the resulting rebound potentiation results entirely from the loss of tonic G-protein inhibition; although the currents are temporarily relieved of tonic inhibition, they are still capable of undergoing inhibition through the muscarinic pathway. Using this rebound protocol, we demonstrate that the inhibition of peak current amplitude produced by M2 receptor activation is similar for alpha1A and alpha1B calcium currents. However, the contribution of the voltage-dependent component of inhibition, characterized by reduced inhibition at very depolarized voltage steps and the relief of inhibition by depolarizing prepulses, was slightly greater for the alpha1B current than for the alpha1A current. After co-expression of the beta3 subunit, the sensitivity to M2 receptor-induced G-protein inhibition was reduced for both alpha1A and alpha1B currents; however, the reduction was significantly greater for alpha1A currents. Additionally, the difference in the voltage dependence of inhibition of alpha1A and alpha1B currents was heightened after co-expression of the Ca2+ channel beta3 subunit. Such differential modulation of sensitivity to G-protein modulation may be important for fine tuning release in neurons that contain both of these Ca2+ channels.