Increase in AMPA receptor-mediated miniature EPSC amplitude after chronic NMDA receptor blockade in cultured hippocampal neurons

Synaptic scaling has been reported as scaling up of AMPA receptors (AMPAR)-mediated miniature excitatory postsynaptic currents (mEPSCs) induced by blockade of action potentials or AMPAR. Here, we show a novel type of synaptic scaling induced by N-methyl-D-aspartate receptors (NMDAR) blockade. In the present study, we analyzed AMPAR-mediated mEPSCs of D-(-)-2-amino-5-phosphonopentanoic acid (AP5)-treated hippocampal neurons (16 days in vitro) for 48 h in low-density cultures, using a whole-cell patch-clamp technique. The mEPSC amplitudes recorded from chronic AP5-treated neurons (25.5+/-0.3 pA; n=30 neurons) were significantly larger than that recorded from control neurons (21.6+/-0.2 pA; n=30 neurons, p<0.05), whereas the frequency of mEPSCs was not changed. Immunocytochemistry showed that the number of synapsin I clusters of AP5-treated neurons was not different from that of control neurons. Cumulative amplitude histograms revealed that the amplitude of mEPSCs was scaled multiplicatively after AP5 treatment. GluR2-lacking AMPAR were not involved in the scaling observed here. Together, our data indicate that NMDAR activity, as well as AMPAR activity, is involved in the negative feedback plasticity of AMPAR-mediated synaptic activity.     

Sodium influx blockade and hypoxic damage to CA1 pyramidal neurons in rat hippocampal slices.

We studied the effects of lidocaine and tetrodotoxin (TTX) on hypoxic changes in CA1 pyramidal neurons to examine the ionic basis of neuronal damage. Lidocaine (10 and 100 microM) and TTX (6 and 63 nM) delayed and attenuated the hypoxic depolarization and improved recovery of the resting and action potentials after 10 min of hypoxia. Lidocaine (10 and 100 microM) and TTX (63 nM) reduced the number of morphologically damaged CA1 cells and improved protein synthesis measured after 10 min hypoxia. Lidocaine (10 microM) attenuated the increase in intracellular sodium (181 vs. 218%) and the depolarization (-21 vs. -1 mV) during hypoxia but did not significantly attenuate the changes in ATP, potassium, or calcium measured at 10 min of hypoxia. Lidocaine (100 microM) attenuated the changes in membrane potential, sodium, potassium, ATP, and calcium during hypoxia. TTX (63 nM) attenuated the changes in membrane potential (-36 vs. -1 mV), sodium (179 vs. 226%), potassium (78 vs. 50%), and ATP (24 vs. 11%) but did not significantly attenuate the increase in calcium during hypoxia. These data indicate that the primary blockade of sodium channels can secondarily alter other cellular parameters. The hypoxic depolarization and the increase in intracellular sodium appear to be important triggers of hypoxic damage independent of their effect on cytosolic calcium; a treatment that selectively blocked sodium influx (lidocaine 10 microM) improved recovery. Our data indicate that selective blockade of sodium channels with a low concentration of lidocaine or TTX improves recovery after hypoxia by attenuating the rise in cellular sodium and the hypoxic depolarization. This blockade improves the resting and action potentials, histologic state, and protein synthesis of CA1 pyramidal neurons after 10 min of hypoxia to rat hippocampal slices. A higher concentration of lidocaine, which also improved ATP, potassium, and calcium concentrations during hypoxia was more potent. In conclusion, the depolarization and increased sodium concentration during hypoxia account for a portion of the neuronal damage after hypoxia independent of changes in calcium.     

Influence of agonist concentration on AMPA and kainate channels in CA1 pyramidal cells in rat hippocampal slices

We have determined the functional properties of single AMPA receptor (AMPAR) and kainate receptor channels present in CA1 cells in hippocampal slices, to shed light on the relationship between single-channel behaviour and synaptic currents in these cells. To derive basic properties of AMPA and kainate channels activated by their excitatory transmitter, we examined outside-out patches exposed to glutamate. The kainate agonist SYM 2081, was used to confirm the presence of kainate receptors. Channels activated by glutamate or SYM 2081 exhibited conductance levels of 2–20 pS. Properties of single channels depended on the glutamate or AMPA concentration used. We observed a marked increase in mean channel conductance (γ) from γ= 6.9, to 11.2 pS, when glutamate was increased from 10 μ m to 10 m m . The kinetic behaviour of AMPAR channels was also influenced by agonist concentration, with an increase in 'bursty' events at higher concentrations. In contrast, kainate channels were characterized by brief openings without bursts. Consistent with the view that 'bursty' events arose from AMPARs, these openings decreased in the presence of the AMPAR blocker GYKI 53655. Furthermore, our experiments revealed a concentration-dependent increase in the number of conductance states during an individual AMPAR opening; AMPAR channels displayed up to four distinct levels. Our results are consistent with the view that the AMPAR channel conductance depends on the number of transmitter molecules bound in CA1 cells. We consider the implications of these findings for the change in EPSC properties during long-term potentiation (LTP).     

Carbenoxolone depresses spontaneous epileptiform activity in the CA1 region of rat hippocampal slices

An important contributor to the generation of epileptiform activity is the synchronization of burst firing in a group of neurons. The aim of this study was to investigate whether gap junctions are involved in this synchrony using an in vitro model of epileptiform activity. Hippocampal slices (400 μm) were prepared from female Sprague–Dawley rats (120–170 g). The perfusion of slices with a medium containing no added magnesium and 4-aminopyridine (50 μM) resulted in the generation of spontaneous bursts of population spikes of a fast frequency along with less frequent negative-going bursts. The frequency of the bursts produced was consistent over a 3 h period. Carbenoxolone (100 μM), a gap junction blocker and mineralocorticoid agonist, perfused for 75 min, reduced the frequency of both types of spontaneous burst activity. Perfusion of spironolactone (1 μM), a mineralocorticosteroid antagonist, for 15 min prior to and during carbenoxolone perfusion did not alter the ability of carbenoxolone to depress the frequency of spontaneous activity. The incubation of hippocampal slices in carbenoxolone prior to recording increased the time taken for the spontaneous activity to start on change to the zero magnesium/4-aminopyridine medium and decreased the total number of spontaneous bursts over the first 60 min period.

The ability of carbenoxolone to delay induction of epileptiform activity and reduce established epileptiform activity suggests that gap junctions contribute to the synchronization of neuronal firing in this model.     

BK potassium channels control transmitter release at CA3–CA3 synapses in the rat hippocampus

Augmentation is a component of short-term synaptic plasticity with a gradual onset and duration in seconds. To investigate this component at the corticogeniculate synapse, whole cell patch-clamp recordings were obtained from principal cells in a slice preparation of the rat dorsal lateral geniculate nucleus. Trains with 10 stimuli at 25 Hz evoked excitatory postsynaptic currents (EPSCs) that grew in amplitude, primarily from facilitation. Such trains also induced augmentation that decayed exponentially with a time constant τ= 4.6 ± 2.6 s (mean ± standard deviation). When the trains were repeated at 1–10 s intervals, augmentation markedly increased the size of the first EPSCs, leaving late EPSCs unaffected. The magnitude of augmentation was dependent on the number of pulses, pulse rate and intervals between trains. Augmented EPSCs changed proportionally to basal EPSC amplitudes following alterations in extracellular calcium ion concentration. The results indicate that augmentation is determined by residual calcium remaining in the presynaptic terminal after repetitive spikes, competing with fast facilitation. We propose that augmentation serves to maintain a high synaptic strength in the corticogeniculate positive feedback system during attentive visual exploration.     

Chronic intermittent ethanol exposure alters CA1 synaptic transmission in rat hippocampal slices

We investigated the neuroadaptive changes in synaptic transmission in the CA1 region of the hippocampus as a result of chronic intermittent ethanol exposure. Male Wistar rats were exposed daily (14 h) to ethanol vapors (blood alcohol levels = 150-200 mg%) for 12-14 days, and synaptic field potentials elicited by Schaffer collateral stimulation were compared in hippocampal slices from control and chronic ethanol-treated rats. Excitatory postsynaptic responses of slices were recorded under three conditions: (i) normal physiological saline; (ii) continued presence of 33 mM (150 mg%) ethanol (chronic ethanol-treated rats only); (iii) acute exposure to 33 mM ethanol. When recorded in ethanol-free physiological saline, the mean amplitude of the dendritic synaptic potential and the somatic population spike were significantly smaller in slices from chronic ethanol-treated rats compared to slices from control rats. Under conditions of continuous ethanol exposure, somatic and dendritic synaptic responses of slices taken from chronic ethanol-treated rats were further depressed, suggesting that neural pathways in area CA1 remained sensitive to ethanol. Acute application of ethanol led to a more pronounced reduction of the mean somatic population spike amplitude in slices from chronic ethanol-treated rats than in slices from control rats. However, dendritic synaptic responses were unaffected by acute ethanol in slices from both control and chronic ethanol-treated rats. In addition, we examined the involvement of presynaptic mechanisms in the effects of chronic intermittent ethanol using paired-pulse protocols. When recorded in the continued presence of ethanol, slices from chronic ethanol-treated rats exhibited a significant reduction in paired-pulse facilitation of the dendritic synaptic response compared to slices from control rats, indicating a presynaptic component to the neuroadaptive effects of chronic intermittent ethanol exposure. Conversely, acute ethanol exposure resulted in an enhancement of paired-pulse facilitation of the dendritic synaptic response, an effect that was similar in slices from both control and chronic ethanol-treated rats. Paired-pulse facilitation of the somatic population spike amplitude was not altered by chronic ethanol treatment. However, acute ethanol exposure significantly enhanced paired-pulse facilitation of the somatic population spike in slices from chronic ethanol-treated rats. This effect of acute ethanol was not observed in slices from control rats. Paired-pulse inhibition was not significantly altered in slices from chronic ethanol-treated rats, suggesting that GABAergic inhibitory mechanisms were not involved in the neuroadaptive effects of chronic intermittent ethanol exposure. We suggest that chronic intermittent ethanol exposure can induce multiple neuroadaptive changes in synaptic transmission of CA1 pyramidal neurons that are detectable at both the pre- and postsynaptic levels. Alterations in paired-pulse facilitation indicate presynaptic changes involving the release of the excitatory neurotransmitter glutamate, whereas changes in dendritic synaptic responses suggest postsynaptic changes in the responsiveness of neurons to synaptic input. Moreover, differential effects of chronic ethanol treatment on synaptic responses recorded in the dendrites versus the somatic region implicate additional effects of ethanol on somatically located mechanisms of CA1 pyramidal neurons. Furthermore, we suggest that complete tolerance to ethanol does not occur in the CA1 region of the hippocampus following chronic intermittent ethanol exposure.     

Low-magnesium epilepsy in rat hippocampal slices: inhibitory postsynaptic potentials in the CA1 subfield

Spontaneous, synchronous epileptiform discharges were recorded in both CA3 and CA1 subfields of rat hippocampal slices perfused with Mg2+-free medium. Surgical separation of the two areas abolished the spontaneous discharges only in the CA1 subfield. However, epileptiform responses in the isolated CA1 subfield could still be evoked by orthodromic stimulation. Intracellularly these stimulus-induced responses were characterized by a depolarization associated with a burst of action potentials. Stimulation of the alveus still evoked a hyperpolarizing potential, presumably a recurrent inhibitory postsynaptic potential (IPSP) in CA1 pyramidal cells. Both spontaneous and stimulus-induced epileptiform discharges were blocked by the selective antagonist of N-methyl-D-aspartate (NMDA) receptors DL-2-amino-phosphonovalerate (APV). APV also reduced the amplitude and duration of the IPSP induced by alveus stimulation. Thus, epileptiform discharges evoked by lowering Mg2+ in the CA1 subfield are associated with a preservation of inhibitory mechanisms. Furthermore the effects exerted by APV upon the IPSP implicate that NMDA receptors might be involved in the neuronal circuit responsible for the hyperpolarizing IPSP generated by CA1 pyramidal neurons.     

Hyperexcitability of hippocampal CA1 region in brain slices after GABA withdrawal.

The interruption of GABA infusion in the cerebral cortex and in the hippocampus produces electrographic seizures in rats. Here, we have used the hippocampal slice preparation to induce a 'GABA withdrawal syndrome (GWS)'. With the stimulation parameters used (0.2 Hz, 200 microseconds), activation of the Schaffer afferents produced one population spike in the CA1 subfield, while multiple population spikes were observed in the slices previously incubated in GABA. Also, we recorded an increase in the amplitude of the population spike when compared to its control value. Paired pulse test showed absence of recurrent inhibition in these slices. These results suggest a dysfunction in GABAergic neurotransmission.     

Adenosine receptor blockade reveals N-methyl-D-aspartate receptor- and voltage-sensitive dendritic spikes in rat hippocampal CA1 pyramidal cells in vitro

The present study was done to determine the possible effects of endogenous adenosine, present in the extracellular fluid of the hippocampal slice, on pyramidal cells in the CA1 region using intracellular recording techniques. Administration of 5 microM of the adenosine receptor antagonist, 8-sulfophenyltheophylline (n=11), induced a depolarization (2.6+/-0.4 mV, mean+/-S.E.M.) with an increase in input resistance (6.7+/-2.1%) in pyramidal cells, and increased the amplitude of the excitatory postsynaptic potentials elicited by stimulation of Schaffer collateral afferents; 50 microM 8-sulfophenyltheophylline (n=68) produced a similar depolarization (3.4+/-1.7 mV) and an increase in input resistance (26+/-3.0%), but also produced spontaneous, synchronized giant excitatory postsynaptic potentials which could generate bursts of spikes. These effects lasted more than 10 min after washout. In the presence of 20 microM 6-cyano-7-nitro-quinoxaline-2,3-dione, a non-N-methyl-D-aspartate receptor antagonist, and 50 microM D-2-amino-5-phosphonovalerate, an N-methyl-D-aspartate receptor antagonist, 50 microM 8-sulfophenyltheophylline (n=4) induced only depolarization (3.1+/-1.3 mV) and an increase in input resistance (23+/-3.8%). In the presence of 20 microM 6-cyano-7-nitro-quinoxaline-2,3-dione only, 50 microM 8-sulfophenyltheophylline (n=7) induced not only the depolarization with an increase in input resistance, but also the occurrence of small-amplitude (11+/-5.6 mV), fast rising, all-or-none, voltage-sensitive spikes of 2-3 ms duration, which were attributed to a dendritic origin. The latency of these dendritic spikes in response to stimulation of Schaffer collateral afferents lasted up to 21 ms. These dendritic spikes could generate one or more action potentials, depending on the resting membrane potential and the frequency of the dendritic spikes. In the presence of 50 microM 8-sulfophenyltheophylline plus 20 microM 6-cyano-7-nitro-quinoxaline-2,3-dione, 50 microM D-2-amino-5-phosphonovalerate blocked the spontaneous dendritic spikes (n=4). In the presence of 5 microM 8-sulfophenyltheophylline, 200 microM N-methyl-D-aspartate (n=5) increased the occurrence of dendritic spikes.These data indicate that adenosine present in the extracellular fluid of the hippocampal slice tonically inhibits not only (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-mediated synaptic transmission, but also voltage- and N-methyl-D-aspartate receptor-sensitive dendritic spikes. Endogenous adenosine acting on adenosine A(1) receptors is thus visualized as a control to prevent the genesis of synchronized giant excitatory postsynaptic potentials. In our experiments, blockade of this tonic activation of adenosine receptors appears to have altered the origins of action potentials and led to epileptiform firing in CA1 pyramidal cells.     

Glycine antagonists block the induction of long-term potentiation in CA1 of rat hippocampal slices

The effects of the strychnine-insensitive glycine receptor antagonists, cycloleucine and 7-chlorokynurenic acid, on the induction of long-term potentiation (LTP) in CA1 of rat hippocampal slices were examined. A 5 min administration of cycloleucine (20-100 microM) or 7-chlorokynurenic acid (1-5 microM) during the delivery of high-frequency stimulation blocked the induction of LTP without affecting baseline synaptic transmission. Coapplication of 100 microM glycine with cycloleucine or 7-chlorokynurenic acid masked the inhibitory effect on the induction of LTP, supporting the hypothesis that these compounds act as glycine antagonists. These results indicate that glycine is a necessary factor for the induction of LTP in CA1 of the rat hippocampus.     

Nitric oxide inhibitors attenuate ischemic degeneration in the CA1 region of rat hippocampal slices

We examined the involvement of nitric oxide (NO) in ischemic brain damage using hippocampal slices prepared from 30 day old albino rats and exposed to 20 min of oxygen/glucose deprivation (ischemia) followed by 90 min postincubation in oxygen- and glucose-containing media. Damage in the CA1 region was rated on a 0 (intact) to 4 (severe neuronal damage) scale by a rater blind to the experimental condition. Control slices exposed to ischemia were rated as 2.8 ± 0.4 (N = 12). -N^G-Monomethylarginine (100 μM) and -N^G-nitroarginine (100 μm), non-selective NO synthase (NOS) inhibitors, diminished ischemic damage (0.6 ± 0.3, N = 8, and 1.0 ± 0.5, N = 4, respectively). An inhibitor of brain NOS, 7-nitroindazole (30 μM), was also effective against ischemic degeneration (0.7 ± 0.3, N = 5). These results suggest that activation of NOS is involved in ischemic degeneration in the CA1 region.     

Glucocorticoids promote localized activity of rat hippocampal CA1 pyramidal neurons in brain slices

The effects of glucocorticoids on rat hippocampal CA1 pyramidal neurons were studied using brain slice preparations. At 10 days after bilateral adrenalectomy, a localized region of CA1 showed a drastic reduction of excitability induced by CA3 stimulation as compared to control. The region of CA1 most effected was 1.4-2.0 mm from the most rostral side of the hippocampus. Upon perfusion of corticosterone, the response to synaptic activation was reduced in this region in slices from adrenalatomized animals increased rapidly toward control values, volatile responses in other regions were unaffected. These results suggest that glucocorticoid receptors are concentrated in restricted regions of hippocampus and that these receptors have important roles in regulation of synaptic excitability.     

Synaptic fatigue at the naive perforant path–dentate granule cell synapse in the rat

Synaptic activation at low frequency is often used to probe synaptic function and synaptic plasticity, but little is known about how such low-frequency activation itself affects synaptic transmission. In the present study, we have examined how the perforant path–dentate granule cell (PP–GC) synapse adapts to low-frequency activation from a previously non-activated (naive) state. Stimulation at 0.2 Hz in acute slices from developing rats (7–12 days old) caused a gradual depression of the AMPA EPSC (at −80 mV) to about half within 50 stimuli. This synaptic fatigue was unaffected by the NMDA and metabotropic glutamate (mGlu) receptor antagonists d -AP5 and LY-341495. A smaller component of this synaptic fatigue was readily reversible when switching to very low-frequency stimulation (0.033–0.017 Hz) and is attributed to a reversible decrease in release probability, which is probably due to depletion of readily releasable vesicles. Thus, it was expressed to the same extent by AMPA and NMDA EPSCs, and was associated with a decrease in quantal content (measured as 1/CV²) with no change in the paired-pulse ratio. The larger component of the synaptic fatigue was not readily reversible, was selective for AMPA EPSCs and was associated with a decrease in 1/CV², thus probably representing silencing of AMPA signalling in a subset of synapses. In adult rats (> 30 days old), the AMPA silencing had disappeared while the low-frequency depression remained unaltered. The present study has thus identified two forms of synaptic plasticity that contribute to fatigue of synaptic transmission at low frequencies at the developing PP–GC synapse; AMPA silencing and a low-frequency depression of release probability.     

Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons.

Dendritic spines serve as preferential sites of excitatory synaptic connections and are pleomorphic. To address the structure-function relationship of the dendritic spines, we used two-photon uncaging of glutamate to allow mapping of functional glutamate receptors at the level of the single synapse. Our analyses of the spines of CA1 pyramidal neurons reveal that AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-type glutamate receptors are abundant (up to 150/spine) in mushroom spines but sparsely distributed in thin spines and filopodia. The latter may be serving as the structural substrates of the silent synapses that have been proposed to play roles in development and plasticity of synaptic transmission. Our data indicate that distribution of functional AMPA receptors is tightly correlated with spine geometry and that receptor activity is independently regulated at the level of single spines.     

Selective blockade of Ca2+ permeable AMPA receptors in CA1 area of rat hippocampus

Using whole cell patch-clamp recording from pyramidal cells and interneurons in the CA1 area of hippocampal slices, the effect of IEM-1460, a selective channel blocker of Ca2+ permeable AMPA receptors (AMPARs), on postsynaptic currents (PSCs) was studied. Excitatory postsynaptic currents (EPSCs) were evoked by stimulation of Schaffer collaterals (SCs) in the presence of APV and bicuculline to pharmacologically isolate the EPSCs mediated by AMPAR activation. IEM-1460 (50 microM) did not affect the amplitude of EPSCs in CA1 pyramidal cells but reversibly decreased their amplitude in interneurons of pyramidal layer (15 cells), radiatum (37 cells) and border radiatum-lacunosum-moleculare (R-LM) (55 cells) layers. The ability of IEM-1460 to decrease EPSC amplitude correlated with EPSC rectification properties in CA1 interneurons, providing evidence for synaptic localization of Ca2+ permeable AMPARs at the SC synaptic input. Independent of their localization, the majority of interneurons studied exhibited only modest sensitivity to IEM-1460 (EPSC amplitude decreased by less than 30%), while in 15% of interneurons IEM-1460 induced more than 50% reduction in EPSC amplitude. To reveal possible afferent-specific localization of Ca2+ permeable AMPARs on R-LM interneurons, the effect of IEM-1460 on EPSCs evoked by stimulation of SC was compared with that of perforant path (PP). Although average sensitivities did not differ significantly, in 61% of R-LM layer interneurons, the SC-evoked EPSCs exhibited higher sensitivity to IEM-1460 than the PP-evoked EPSCs. Moreover, in 54% of R-LM layer interneurons the EPSCs evoked by SC stimulation were complex, having an initial peak followed by one or several late components. Kinetics, latency distribution and reversal potential of late components suggest di- and polysynaptic origin of the late components. Late EPSCs were strongly and reversibly inhibited by IEM-1460 indicating that Ca2+ permeable AMPARs are involved in the indirect excitation of R-LM layer interneurons. Despite the ability to decrease the excitatory synaptic input to interneurons, IEM-1460 did not affect interneuron-mediated inhibitory postsynaptic currents (IPSCs) evoked in pyramidal neurons by SC stimulation. These data suggest that interneurons with a synaptic input highly sensitive to IEM-1460 do not contribute specifically to the feed-forward inhibition of hippocampal pyramidal neurons.     

Rapid and slow swelling during hypoxia in the CA1 region of rat hippocampal slices.

The role of swelling in hypoxic/ischemic neuronal injury is incompletely understood. We investigated the extent and time course of cell swelling during hypoxia, and recovery of cell volume during reoxygenation, in the CA1 region of rat hippocampal slices in vitro. Cell swelling was measured optically and compared with simultaneous measurements of the extracellular DC potential, extracellular [K+], and synaptic transmission in the presence and absence of hypoxic depolarization. Hypoxia-induced swelling consisted of rapid and/or slow components. Rapid swelling was observed frequently and always occurred simultaneously with hypoxic depolarization. Additionally, rapid swelling was followed by a prolonged phase of swelling that was approximately 15 times slower. Less frequently, slow swelling occurred independently, without either hypoxic depolarization or a preceding rapid swelling. For slices initially swelling rapidly, recovery of both cell volume and the slope of field excitatory postsynaptic potentials were best correlated with the duration of hypoxia (r = 0.77 and 0.87, respectively). This was also the case for slices initially swelling slowly (r = 0.70 and 0.58, respectively). In contrast, the degree of recovery of cell volume was the same at 30 or 60 min of reoxygenation, indicating that prolonging the duration of reoxygenation within these limits was ineffective in improving recovery. Spectral measurements indicated that the hypoxia-induced changes in light transmittance were related to changes in cell volume and not changes in the oxidation state of mitochondrial cytochromes. The persistent impairment of synaptic transmission in slices swelling slowly (i.e., without hypoxic depolarization) indicates that swelling may play a role in this injury and that hypoxic depolarization is not required. Additionally, the correlation between the degree of recovery of cell volume and the degree of recovery of synaptic transmission during reoxygenation supports a role for swelling in hypoxic neuronal injury.     

Effects of noradrenaline on some potassium currents in CA1 neurones in rat hippocampal slices

Pyramidal (CA1) cells in rat hippocampal slices were voltage clamped using a single electrode voltage clamp. In the presence of tetrodotoxin (TTX), depolarizing pulses from holding potentials of −60 to −70 mV elicited a slow inward calcium (Ca²⁺) current and two outward potassium (K⁺) currents: an A current and a slower, Ca²⁺-dependent K⁺ current. Noradrenaline (NA) (20 μM) depressed the amplitude of the K⁺ currents without affecting the Ca²⁺ current. The effect of NA could be blocked with propranolol and was mimicked by isoprenaline, suggesting that NA depresses the K⁺ currents by binding to β-receptors.