Enrico Bracci

Cholinergic Interneurons Control the Excitatory Input to the Striatum

How the extent and time course of presynaptic inhibition depend on the action potentials of the neuron controlling the terminals is unknown. We investigated this issue in the striatum using paired recordings from cholinergic interneurons and projection neurons. Glutamatergic EPSCs were evoked in projection neurons and cholinergic interneurons by stimulation of afferent fibers in the cortex and the striatum, respectively. A single spike in a cholinergic interneuron caused significant depression of the evoked glutamatergic EPSC in 34% of projection neurons located within 100 μm and 41% of cholinergic interneurons located within 200 μm. The time course of these effects was similar in the two cases, with EPSC inhibition peaking 20–30 ms after the spike and disappearing after 40–80 ms. Maximal depression of EPSC amplitude was up to 27% in projection neurons and to 19% in cholinergic interneurons. These effects were reversibly blocked by muscarinic receptor antagonists (atropine or methoctramine), which also significantly increased baseline EPSC (evoked without a preceding spike in the cholinergic interneuron), suggesting that some tonic cholinergic presynaptic inhibition was present. This was confirmed by the fact that lowering extracellular potassium, which silenced spontaneously active cholinergic interneurons, also increased baseline EPSC amplitude, and these effects were occluded by previous application of muscarinic receptor antagonists. Collectively, these results show that a single spike in a cholinergic interneuron exerts a fast and powerful inhibitory control over the glutamatergic input to striatal neurons.

Prolonged epileptiform bursting induced by 0-Mg(2+) in rat hippocampal slices depends on gap junctional coupling.

The transition from brief interictal to prolonged seizure, or ictal, activity is a crucial event in epilepsy. In vitro slice models can mimic many phenomena observed in the electroencephalogram of patients, including transition from interictal to ictaform or seizure-like activity. In field potential recordings, three discharge types can be distinguished: (1) primary discharges making up the typical interictal burst, (2) secondary bursts, lasting several hundred milliseconds, and (3) tertiary discharges lasting for seconds, constituting the ictal series of bursts. The roles of chemical synapses in these classes of burst have been explored in detail. Here we test the hypothesis that gap junctions are necessary for the generation of secondary bursts. In rat hippocampal slices, epileptiform activity was induced by exposure to 0-Mg(2+). Epileptiform discharges started in the CA3 subfield, and generally consisted of primary discharges followed by 4-13 secondary bursts. Three drugs that block gap junctions, halothane (5-10 mM), carbenoxolone (100 microM) and octanol (0.2-1.0 mM), abolished the secondary discharges, but left the primary bursts intact. The gap junction opener trimethylamine (10 mM) reversibly induced secondary and tertiary discharges. None of these agents altered intrinsic or synaptic properties of CA3 pyramidal cells at the doses used. Surgically isolating the CA3 subfield made secondary discharges disappear, and trimethylamine under these conditions was able to restore them.We conclude that gap junctions can contribute to the prolongation of epileptiform discharges.

Differential contribution of dopamine D2S and D2L receptors in the modulation of glutamate and GABA transmission in the striatum.

Compelling evidence indicates that the long (D2L) and the short (D2S) isoform of dopamine (DA) D2 receptors serve distinct physiological functions in vivo. To address the involvement of these isoforms in the control of synaptic transmission in the striatum, we measured the sensitivity to D2 receptor stimulation of glutamate- and GABA-mediated currents recorded from striatal neurons of three mutant mice, in which the expression of D2L and D2S receptors was either ablated or variably altered. Our data indicate that both isoforms participate in the presynaptic inhibition of GABA transmission in the striatum, while the D2-receptor-dependent modulation of glutamate release preferentially involves the D2S receptor. Accordingly, the inhibitory effects of the DA D2 receptor agonist quinpirole (10 microM) on GABA(A)-mediated spontaneous inhibitory postsynaptic currents (IPSCs)correlate with the total number of D2 receptor sites in the striatum, irrespective of the specific receptor isoform expressed. In contrast, glutamate-mediated spontaneous excitatory postsynaptic currents (EPSCs) were significantly inhibited by quinpirole only when the total number of D2 receptor sites, normally composed by both D2L and D2S receptors in a ratio favoring the D2L isoform, was modified to express only the D2S isoform at higher than normal levels. Understanding the physiological roles of DA D2 receptors in the striatum is essential for the treatment of several neuropsychiatric conditions, such as Parkinsons disease, Tourettes syndrome, schizophrenia, and drug addiction.

Contribution of NMDA and non-NMDA glutamate receptors to locomotor pattern generation in the neonatal rat spinal cord.

The motor programme executed by the spinal cord to generate locomotion involves glutamate–mediated excitatory synaptic transmission. Using the neonatal rat spinal cord as an in vitro model in which the locomotor pattern was evoked by 5–hydroxytryptamine (5–HT), we investigated the role of N–methyl–D–aspartate (NMDA) and non–NMDA glutamate receptors in the generation of locomotor patterns recorded electrophysiologically from pairs of ventral roots. In a control solution, 5–HT (2.5–30 μM) elicited persistent alternating activity in left and right lumbar ventral roots. Increasing 5–HT concentration within this range resulted in increased cycle frequency (on average from 8 to 20 cycles min−1). In the presence of NMDA receptor antagonism, persistent alternating activity was still observed as long as 5–HT doses were increased (range 20–40 μM), even if locomotor pattern frequency was lower than in the control solution. In the presence of non–NMDA receptor antagonism, stable locomotor activity (with lower cycle frequency) was also elicited by 5–HT, albeit with doses larger than in the control solution (15–40 μM). When NMDA and non–NMDA receptors were simultaneously blocked, 5–HT (5–120 μM) always failed to elicit locomotor activity. These data show that the operation of one glutamate receptor class was sufficient to express locomotor activity. As locomotor activity developed at a lower frequency than in the control solution after pharmacological block of either NMDA or non–NMDA receptors, it is suggested that both receptor classes were involved in locomotor pattern generation.

Voltage‐dependent membrane potential oscillations of rat striatal fast‐spiking interneurons

We used whole‐cell recordings to investigate subthreshold membrane potential oscillations and their relationship with intermittent firing in striatal fast‐spiking interneurons. During current injections (100–500 pA, 1 s), these cells displayed a highly variable pattern of spike bursts (comprising 1–30 action potentials) interspersed with membrane potential oscillations. The oscillation threshold was −42 ± 10 mV, and coincided with that for action potentials. The oscillation frequency was voltage dependent and ranged between 20 and 100 Hz. Oscillations were unaffected by the calcium channel blockers cadmium and nickel and by blockers of ionotropic glutamate and GABA receptors. Conversely, the sodium channel blocker tetrodotoxin fully abolished the oscillations and the spike bursts. The first spike of a burst appeared to be triggered by an oscillation, since the timing and rate of rise of the membrane potential in the subthreshold voltage region was similar for the two events. Conversely, the second spike (and the subsequent ones) displayed much faster depolarisations in the subthreshold voltage range, indicating that they were generated by a different mechanism. Consistent with these notions, a small pulse of intracellular current delivered during the oscillation was effective in triggering a burst of action potentials that largely outlasted the pulse. We conclude that fast‐spiking interneuron oscillations are generated by an intrinsic membrane mechanism that does not require fast synaptic transmission, and which depends on sodium conductance but not calcium conductance, and that such oscillations are responsible for triggering the intermittent spike bursts that are typical of these neurons.

Activation of dopamine D1-like receptors excites LTS interneurons of the striatum

Dopamine (DA) has a crucial role in the modulation of striatal neuron activity. Along with projection cells, striatal interneurons receive dense dopaminergic innervation from midbrain neurons, thus, also suggesting that these intrinsic cells represent a synaptic target for DA action in the striatum. In the present study, we investigated the effects of DA on low‐threshold spike (LTS) interneurons of the rat striatum, by means of in vitro whole‐cell patch‐clamp electrophysiological recordings. Dopamine depolarized LTS cells, a pharmacological effect prevented by D1‐ but not D2‐like DA receptor antagonists. The membrane depolarization produced by DA was sufficient to trigger action potential discharge in the recorded cells and was insensitive to tetrodotoxin and glutamate receptor antagonists. In addition, this pharmacological effect was mimicked by D1‐ but not D2‐like DA receptor agonists, implying the selective involvement of D1‐like receptors in this action.

Excitatory effects of serotonin on rat striatal cholinergic interneurones

We investigated the effects of 5‐hydroxytryptamine (5‐HT, serotonin) in striatal cholinergic interneurones with gramicidin‐perforated whole‐cell patch recordings. Bath‐application of serotonin (30 μm) significantly and reversibly increased the spontaneous firing rate of 37/45 cholinergic interneurones tested. On average, in the presence of serotonin, firing rate was 273 ± 193% of control. Selective agonists of 5‐HT1A, 5‐HT3, 5‐HT4 and 5‐HT7 receptors did not affect cholinergic interneurone firing, while the 5‐HT2 receptor agonist α‐methyl‐5‐HT (30 μm) mimicked the excitatory effects of serotonin. Consistently, the 5‐HT2 receptor antagonist ketanserin (10 μm) fully blocked the excitatory effects of serotonin. Two prominent after‐hyperpolarizations (AHPs), one of medium duration that was apamin‐sensitive and followed individual spikes, and one that was slower and followed trains of spikes, were both strongly and reversibly reduced by serotonin; these effects were fully blocked by ketanserin. Conversely, the depolarizing sags observed during negative current injections and mediated by hyperpolarization‐activated cationic currents were not affected. In the presence of apamin and tetrodotoxin, the slow AHP was strongly reduced by 5‐HT, and fully abolished by the calcium channel blocker nickel. These results show that 5‐HT exerts a powerful excitatory control on cholinergic interneurones via 5‐HT2 receptors, by suppressing the AHPs associated with two distinct calcium‐activated potassium currents.

Serotonin excites fast-spiking interneurons in the striatum

Fast‐spiking interneurons (FSIs) control the output of the striatum by mediating feed‐forward GABAergic inhibition of projection neurons. Their neuromodulation can therefore critically affect the operation of the basal ganglia. We studied the effects of 5‐hydroxytryptamine (5‐HT, serotonin), a neurotransmitter released in the striatum by fibres originating in the raphe nuclei, on FSIs recorded with whole‐cell techniques in rat brain slices. Bath application of serotonin (30u2003μm) elicited slow, reversible depolarizations (9u2003±u20033u2003mV) in 37/46 FSIs. Similar effects were observed using conventional whole‐cell and gramicidin perforated‐patch techniques. The serotonin effects persisted in the presence of tetrodotoxin and were mediated by 5‐HT2C receptors, as they were reversed by the 5‐HT2 receptor antagonist ketanserin and by the selective 5‐HT2C receptor antagonist RS 102221. Serotonin‐induced depolarizations were not accompanied by a significant change in FSI input resistance. Serotonin caused the appearance of spontaneous firing in a minority (5/35) of responsive FSIs, whereas it strongly increased FSI excitability in each of the remaining responsive FSIs, significantly decreasing the latency of the first spike evoked by a current step and increasing spike frequency. Voltage‐clamp experiments revealed that serotonin suppressed a current that reversed around −100u2003mV and displayed a marked inward rectification, a finding that explains the lack of effects of serotonin on input resistance. Consistently, the effects of serotonin were completely occluded by low concentrations of extracellular barium, which selectively blocks Kir2 channels. We concluded that the excitatory effects of serotonin on FSIs were mediated by 5‐HT2C receptors and involved suppression of an inwardly rectifying K+ current.

Desensitization of AMPA Receptors Limits the Amplitude of EPSPs and the Excitability of Motoneurons of the Rat Isolated Spinal Cord

Intracellular recording was used to study the effect of cyclothiazide, a selective blocker of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptor desensitization, on lumbar motoneurons of the rat isolated spinal cord. Cyclothiazide (25 μM) enhanced the responses to AMPA in a tetrodotoxin‐insensitive fashion, without affecting those produced by N‐methyl‐D‐aspartate or γ‐aminobutyric acid. Excitatory postsynaptic potentials (EPSPs) evoked by dorsal root stimulation were strongly potentiated in amplitude while paired‐pulse depression (produced by applying pairs of pulses at 2 s interval) of the EPSP was decreased. In the presence of cyclothiazide the frequency of spontaneous synaptic events was greatly increased and network‐driven bursting activity developed with eventual loss of electrical excitability. The present results suggest that pharmacological block of AMPA receptor desensitization led to strong excitation of motoneurons and indicate a physiological role of desensitization in protecting these nerve cells from overactivity.

Layer-specific pyramidal cell oscillations evoked by tetanic stimulation in the rat hippocampal area CA1 in vitro and in vivo

Tetanic stimulation of axons terminating in the CA1 region of the hippocampus induces oscillations in the gamma‐to‐beta frequency band (13–100 Hz) and can induce long‐term potentiation (LTP). The rapid pyramidal cell discharge is driven by a mainly GABAA‐receptor‐mediated slow depolarization and entrained mainly through ephaptic interactions. This study tests whether cellular compartmentalization can explain how cells, despite severely reduced input resistance, can still fire briskly and have IPSPs superimposed on the slow GABAergic depolarization, and whether this behaviour occurs in vivo. Oscillations induced in CA1 in vitro by tetanic stimulation of the stratum radiatum or oriens were analysed using intracellular and multichannel field potentials along the cell axis. Layer‐specific effects of focal application of bicuculline indicate that the GABAergic depolarization is concentrated on tetanized dendrites. Current‐source density analysis and characteristics of partial spikes indicate that early action potentials are initiated in the proximal nontetanized dendrite but cannot invade the tetanized dendrite, where recurrent EPSPs and evoked IPSPs were largely suppressed. As the oscillation progresses, IPSPs recover and slow the neuronal firing to β frequencies, with a small subpopulation of neurons continuing to fire at γ frequency. Carbonic anhydrase dependence, threshold intensity, frequency, field strength and spike initiation/propagation of tetanus‐evoked oscillations in urethane‐anaesthetized rats, validate our observations in vitro, and show that these mechanisms operate in healthy tissue. However, the disrupted electrophysiology of the tetanized dendrites will disable normal information processing, has implications for LTP induction and is likely to play a role in pathological synchronization as found during epileptic discharges.