The different roles of Na + /Ca 2+ (NCX) exchangers and Na + / Ca 2+ /K + (NCKX) exchangers in regulation of the ionic homeostasis in neurones are poorly understood. We have previously shown that serotonin excites histaminergic tuberomamillary (TM) neurones by activation of 5-HT 2C -receptors and Na + /Ca 2+ exchange. With the help of single-cell RT-PCR (sc-RT-PCR) we have now determined the coexpression pattern of different subtypes of NCX and NCKX with serotonin receptors. The majority of TM neurones express NCX1, NCX2 and NCKX3. Serotonin 2C receptor-mRNA was detected in 70% while 5-HT 2A mRNA was found in only 10% of TM neurones. In all neurones expressing the 5-HT 2C receptor NCX1-mRNA was present. Double immunostaining revealed the presence of the NCX1 protein in histidine decarboxylasepositive neurones. In the majority of TM neurones one or two out of five isoforms, NCX1.4, NCX1.5, NCX1.7, NCX1.14, NCX1.15, were detected by cDNA sequencing and/or by restriction analysis. The alternative splicing region is important for the Ca 2+ sensitivity and presumably for the modulation of NCX1 function by second messengers. We conclude that several exchanger-subtypes can be coexpressed in single neurones and that TM cells are heterogeneous with respect to their calcium homeostasis regulation. Calcium plays a central role in neuronal signalling and after a quick rise in response to physiological stimuli free calcium must be immediately removed and finally extruded from the cell in order to maintain its low resting level. Two systems, the plasma membrane Ca 2+ ATPase (Carafoli 1994) and the sodium-calcium exchangers (Philipson and Nicoll 1992) are responsible for the efflux of intracellular calcium. These exchangers come from two gene families: Na + /Ca 2+ (NCX) and Na + /Ca 2+ /K + (NCKX) exchangers (Schulze et al. 2002b). In vitro expression of each known gene results in the formation of a functional protein which imports across the cellular membrane 3Na + for every Ca 2+ that is pumped out (NCX; Kimura et al. 1987) or 4Na + for 1K + plus 1Ca 2+ (NCKX; Blaustein and Lederer 1999). Function of these exchangers in native neurones remains elusive mainly because of the lack of selective antagonists for NCX versus NCKX. Nevertheless, recent studies attribute an important role to NCKX and NCX for Ca 2+ extrusion from living neurones (Ranciat-McComb et al. 2000;Lee et al. 2002).We have recently shown that serotonin depolarizes histaminergic neurones and increases their firing rate (Eriksson et al. 2001b. This action was suppressed by the two Na + /Ca 2+ exchange inhibitors KB R7943 (80 lM, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate) and DCB (100 lM, 3¢,4¢-dichlorobenzamil). Moreover, other characteristic features of serotonin-induced depolarization, such as dependence on external Na + and temperature suggested involvement of an exchanger. However, the available pharmacological tools were inadequate for an unambiguous conclusion on the type of Na + /Ca 2+ exchanger involved in the observed p...

(1) Pre-mRNA editing of serotonin 2C (5-HT2c) and glutamate (Glu) receptors (R) influences higher brain functions and pathological states such as epilepsy, amyotrophic lateral sclerosis, and depression. Adenosine deaminases acting on RNA (ADAR1-3) convert adenosine to inosine on synthetic RNAs, analogous to 5-HT2cR and GluR. The order of editing as well as mechanisms controlling editing in native neurons is unknown. (2) With single-cell RT-PCR we investigated the co-expression of ADAR genes with GluR and 5-HT2cR and determined the editing status at known sites in the hypothalamic tuberomamillary nucleus, a major center for wakefulness and arousal. (3) The most frequently expressed enzymes were ADAR1, followed by ADAR2. The Q/R site of GluR2 was always fully edited. Editing at the R/G site in the GluR2 (but not GluR4) subunit was co-ordinated with ADAR expression: maximal editing was found in neurons expressing both ADAR2 splice variants of the deaminase domain and lacking ADAR3. (4) Editing of the 5-HT2cR did not correlate with ADAR expression. The 5-HT2cR mRNA was always edited at A, in the majority of cells at B sites and variably edited at E, C and D sites. A negative correlation was found between editing of C and D sites. The GluR4 R/G site editing was homogeneous within individuals: it was fully edited in all neurons obtained from 12 rats and under-edited in six neurons obtained from three rats. (5) We conclude that GluR2 R/G editing is controlled at the level of ADAR2 and therefore this enzyme may be a target for pharmacotherapy. On the other hand, further factors/enzymes besides ADAR must control or influence 5-HT2cR and GluR pre-mRNA editing in native neurons; our data indicate that these factors vary between individuals and could be predictors of psychiatric disease.

The histaminergic tuberomamillary (TM) nucleus, a center for the regulation of wakefulness, is excited by glutamatergic, aminergic and peptidergic inputs. AMPA receptor properties in relation to their expression were investigated in acutely isolated TM neurons with the help of whole-cell patch-clamp recordings combined with single-cell RT-PCR. The mRNAs encoding for the AMPA receptor GluR2 (100% of the neurons) and GluR1 (75%) were the most frequently detected, followed by the mRNA for GluR4 (56%), whereas GluR3 cDNA amplification did not yield a PCR product in any neuron. Flip splice variants prevailed over flop, in keeping with a strong glutamate-response potentiation by cyclothiazide. The expression pattern of AMPA subunits in their two splice variants was correlated with the different subtypes of Na+/Ca2+ (NCX) and Na+/Ca2+/K+ (NCKX) exchangers: glutamate receptor subunits GluR1-4 displayed no coordinated pattern with NCX. However, NCKX2 mRNA occurred only in TM cells with a fast desensitizing glutamate response, where it was coexpressed with the GluR4 subunit in the flop splice variant. NCKX3 mRNA was detected in neurons with fast or slow desensitization of glutamate responses. AMPA receptors in TM neurons were Ca2+-impermeable. As reverse Na+/Ca2+ exchange contributes to the immediate rise in intracellular calcium resulting from glutamate receptor activation, we suggest that the coordinated expression of NCKX2 with the fast desensitizing AMPA receptor-type reflects either a receptor-exchanger coupling or separate mechanisms for maintaining calcium homeostasis in neurons with fast or slow glutamate responses.