Glial cell line-derived neurotrophic factor (GDNF) and a related protein, neurturin (NTN), require a GPI-linked coreceptor, either GFR alpha1 or GFR alpha2, for signaling via the transmembrane Ret tyrosine kinase. We show that mice lacking functional GFR alpha2 coreceptor (Gfra2-/-) are viable and fertile but have dry eyes and grow poorly after weaning, presumably due to malnutrition. While the sympathetic innervation appeared normal, the parasympathetic cholinergic innervation was almost absent in the lacrimal and salivary glands and severely reduced in the small bowel. Neurite outgrowth and trophic effects of NTN at low concentrations were lacking in Gfra2-/- trigeminal neurons in vitro, whereas responses to GDNF were similar between the genotypes. Thus, GFR alpha2 is a physiological NTN receptor, essential for the development of specific postganglionic parasympathetic neurons.

SUMMARY1. The ionic mechanism underlying the fast, GABAA receptor-mediated inhibitory postsynaptic potential (IPSPA) was examined in rat neocortical neurones using intracellular recording techniques. Synaptic responses were evoked by orthodromic stimulation applied to the subcortical white matter or to the pial surface. All experiments were carried out at a constant extracellular Cl-concentration.2. The resting membrane potential was -76-2 + 1I0 mV (mean+s. E. M., n = 32) and in most cells IPSPA was depolarizing. The reversal potential of IPSPA (EIPSP-A) was -70-2 + 0 9 mV (n = 32) and that of a more slowly developing hyperpolarizing response (IPSPB) was -91 4 + 1 3 mV (n = 28).3. An examination of the temporal relationships between excitatory postsynaptic potentials (EPSPs) and IPSPAS in different cells suggested that, despite partial overlap of these responses, EPSPs had little influence on the measured values of DEIPSP-A, 4. Application of 20 mm trimethylamine (TriMA), a membrane-permeant weak base which is expected to produce a rise in pHi (and hence in intracellular HC03-), induced a reversible positive shift in EIPSP-A of up to + 9-0 mV (mean + 4-2 mV) at an extracellular pH (pH.) of 7 4. In some experiments, the shift in reversal potential was associated with a change in the polarity of IPSPA from hyperpolarizing to depolarizing.5. Application of 20 mm lactate (a membrane-permeant weak acid which is expected to produce a fall in pHi and hence in intracellular HCO3 ) at pH. 7 0 produced a hyperpolarizing shift in EIPSP-A of up to -75 mV (mean -56 mV). In some experiments, exposure to lactate changed the polarity of IPSPA from depolarizing to hyperpolarizing.6. Changes in pH. from 7-4 to 7-0 reduced the effect of TriMA and augmented that of lactate on EIPSP-A' as could be expected on the basis of the pHo-dependent change in the fraction of membrane permeable non-charged weak base or acid.

SUMMARY1. The effects of y-aminobutyric acid (GABA) on membrane potential and conductance as well as on the intracellular Cl-activity (ai4k) and intracellular pH (pHi) were studied in crayfish muscle fibres using a three-microelectrode voltage clamp and ion-selective microelectrodes. In the presence of C02-HC03-, the intracellular HCO3-activity (a' o3) was estimated from pHi.2. In a nominally HCO3 -free solution, a near-saturating concentration of GABA (0-2 mM) produced a marked increase in membrane conductance but little change in potential. In a solution containing 30 mM-HCO3-(equilibrated with 5 % CO2 + 95 % air; pH 7-4), the GABA-induced increase in conductance was associated with a depolarization of about 15 mV, with an increase in ai1 and with a decrease in ah-o.All these effects were blocked by picrotoxin (PTX). The depolarizing action of GABA was augmented following depletion of extracellular and intracellular Cl-.3. The GABA-induced increase in a', which took place in the presence of HCO3 was blocked by clamping the membrane potential at its resting level. This indicates that the increase in a'1 was due to passive redistribution of Cl-. In both the presence and absence of HC03-, the GABA-activated transmembrane flux of Cl-showed reversal at the level of the resting potential, which indicates that under steady-state conditions the Cl-equilibrium potential (Ec1) is identical to the resting potential.4. In a Cl--free, 30 mM-HCO3--containing solution, 0-5 mM-GABA produced a PTX-sensitive increase in conductance which amounted to 15 % of the conductance activated in the presence of Cl-. In the absence of both Cl-and HC03-, the respective figure was 2-8 %. Assuming constant-field conditions, the conductance data yielded a permeability ratio PHCo3/PC1 of 0-42 for the GABA-activated channels.5. In a Cl--containing, HC03--free solution, the reversal potential of the GABAactivated current (EGABA) was, by about 1 mV, less negative than the resting membrane potential (RP). In a solution containing Cl-and 30 mM-HCO3-, EGABA -RP was 12 mV. Simultaneous measurements of EGABA, aci and allCO3 (pHi) gave a PHCO3/PC1 value of 0 33.6. In a Cl--free, HC03--containing solution EGABA was close to the HC03-* To whom correspondence should be addressed. 6 PHY 416K. KAILA AND OTHERS equilibrium potential (EHCO3) and an experimental acidosis which produced a negative shift in EHCO3 was associated with a similar shift in EGABA.7. The present results show that HCO3-permeability of GABA-gated channels can lead to a significant deviation of EGABA from Ecl, and, in particular, to a depolarizing effect of GABA in the absence of a postsynaptic, inwardly directed Clpump. The dependence of EGABA on HCO3-permeability may provide a novel link between postsynaptic regulation of pHi and the efficacy of synaptic inhibition.

The gamma-aminobutyric acid (GABA) receptor rho subunits recently cloned from rat and human retina are thought to form GABA receptor channels belonging to a pharmacologically distinct receptor class, termed GABA(C). In this work we have examined the distribution of rho1, rho2 and rho3 subunits, and found expression of all three transcripts in several regions of the rat nervous system. In situ hybridization revealed expression of rho2 in the adult rat retina and some other parts of the visual pathways. A high local rho2 expression was seen in the superficial grey layer of the superior colliculus, and in the dorsal lateral geniculate nucleus. Expression was also detected in the 6th layer of visual cortex and in the CA1 pyramidal cell layer of hippocampus. With reverse transcriptase-polymerase chain reaction, expression of rho1 was mainly seen in the adult rat retina and dorsal root ganglia, as well as, at a significantly lower level, in the superior colliculus, hippocampus, brain stem, thalamus, postnatal day 8 (P8) superior colliculus and P8 hippocampus. Expression pattern of rho3 mRNA was clearly different from that of rho1 and rho2, being strongest in the hippocampus, and significantly lower in the retina, dorsal root ganglia and cortex. No rho3 expression was observed in adult or P8 superior colliculus or in P8 hippocampus. The present results clearly demonstrate that expression of GABA receptor rho subunits is not restricted to the retina, but significant expression can also be detected in many other brain regions, especially in those belonging to the visual pathways. The expression pattern of the rho subunits may be helpful in solving the functional significance of the receptors formed from these subunits.