Vincenzo Crunelli

A T-type Ca2+ current underlies low-threshold Ca2+ potentials in cells of the cat and rat lateral geniculate nucleus.

1. The characteristics of a transient inward Ca2+ current (IT) underlying low‐threshold Ca2+ potentials were studied in projection cells of the cat and rat dorsal lateral geniculate nucleus (LGN) in vitro using the single‐electrode voltage‐clamp technique. 2. In cat LGN slices perfused at 25 degrees C with a solution which included 1 mM‐Ca2+ and 3 mM‐Mg2+, IT could be evoked by depolarizing voltage steps to ‐55 mV from a holding potential (Vh) of ‐95 mV and was abolished by reducing [Ca2+]o from 1 to 0.1 mM. IT was also blocked by 8 mM‐Mg2+ and 500 microM‐Ni2+, but 500 microM‐Cd2+ was a significantly less effective antagonist. 3. The inactivation of IT, which occurred at Vh positive to ‐65 mV, was removed as Vh approached ‐100 mV. The process of inactivation removal was also time dependent, with 800‐1000 ms needed for total removal. Activation curves for IT showed a threshold of ‐70 mV and illustrated that IT was extremely voltage sensitive over the voltage range from ‐65 to ‐55 mV. 4. The decay phase of IT followed a single‐exponential time course with a time constant of decay which was voltage sensitive and ranged from 20 to 100 ms. The mean peak conductance increase associated with IT was 8.4 nS (+/‐0.9, S.E.M.). 5. In more ‘physiological’ conditions (35 degrees C and 1.5 mM‐Ca2+, 1 mM‐Mg2+) the voltage dependence of activation and inactivation were unaffected. However, the development and decay of IT proceeded more rapidly and only 500‐600 ms were needed for total removal of inactivation. Under these conditions, the use of voltage ramps showed that depolarization rates of greater than 30 mV/s were necessary for IT activation. 6. The use of multiple voltage‐step protocols illustrated that the process of inactivation removal was rapidly reversed by brief returns to a Vh of ‐50 mV. Furthermore, any delay in IT activation, once the LGN cell membrane potential was in the IT activation range, resulted in a current of reduced amplitude. 7. Although IT in rat LGN cells was briefer and had a shorter latency to peak, it was otherwise similar to that seen in cat LGN cells. 8. The characteristics of IT are very similar to those of the T‐type Ca2+ currents of other excitable membranes. The properties of IT are discussed with respect to its role in generating the low‐threshold Ca2+ potentials which are central to the oscillatory behaviour of thalamic projection cells.

Two inward currents and the transformation of low-frequency oscillations of rat and cat thalamocortical cells.

1. The contribution of a slow, mixed Na(+)‐K+, inward rectifying current (Ih) and the T‐type Ca2+ current (IT) (that underlies low‐threshold Ca2+ potentials) to the low‐frequency oscillations observed in rat and cat thalamocortical (TC) cells in vitro was studied using current clamp and single‐electrode voltage clamp recordings. 2. From a holding potential of ‐50 mV, voltage steps negative to ‐60 mV showed the presence of a slow, non‐inactivating inward current, Ih. This current was unaffected by Ba2+ (1‐4 mM), tetrodotoxin (0.5‐1 microM) and TEA (20 mM, n = 6), reversibly blocked by Cs+ (1‐3 mM), and its reversal potential (‐33.0 +/‐ 1.2 mV) followed changes in the extracellular Na+ and K+, but not Cl‐, concentration. 3. Application of Cs+ (1‐3 mM) abolished the pacemaker oscillations (n = 9), while in six cells that did not show any oscillatory activity Cs+ first evoked the spindle‐like oscillations that, in the continuous presence of these ions, were then transformed into the pacemaker oscillations before all activities were finally blocked: all these effects were accompanied by a hyperpolarization and a progressive decrease and final blockade of Ih. Cs+ had no effect on the ‘N‐methyl‐D‐aspartate’ (NMDA) oscillations (n = 5) and Ba2+ (2 mM, n = 8) did not block the pacemaker, the spindle‐like and the ‘NMDA’ oscillations. 4. In ten cells that showed the pacemaker oscillations selective activation of beta‐adrenoceptors by 10‐50 microM‐noradrenaline (in the presence of alpha‐noradrenergic antagonists) or by 20 microM‐isoprenaline first transformed the pacemaker oscillations into the spindle‐like oscillations that, in the continuous activation of beta‐receptors, were finally abolished: all these effects were accompanied by a depolarization and a progressive increase of Ih. 5. In TC cells that showed the pacemaker oscillations application of 1‐octanol (50‐100 microM), an antagonist of T‐type Ca2+ currents, reversibly blocked this activity but concomitantly decreased (50%) the cell input resistance (n = 5). Application of Ni2+ (0.2‐0.5 mM, n = 13), another antagonist of IT reversibly blocked the pacemaker, the spindle‐like and the ‘NMDA’ oscillations. 7. In cells showing the pacemaker oscillations it was found that the current developing from the most hyperpolarized potential of an oscillation cycle was an inward relaxation whose time course differed from that of Ih evoked at the same potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Low‐frequency oscillatory activities intrinsic to rat and cat thalamocortical cells.

1. Low‐frequency membrane potential oscillations recorded intracellularly from thalamocortical (TC) cells of the rat and cat dorsal lateral geniculate nucleus (dLGN) and of the rat ventrobasal nucleus (VB) maintained in vitro were investigated. On the basis of their electrophysiological and pharmacological properties, four types of activity were distinguished and named: the pacemaker oscillations, the spindle‐like oscillations, the ‘very slow’ oscillations and the ‘N‐methyl‐D‐aspartate’ (NMDA) oscillations. 2. The pacemaker oscillations (95 out of 173 cells) consisted of rhythmic, large‐amplitude (10‐30 mV) depolarizations which occurred at a frequency of 1.8 +/‐ 0.3 Hz (range, 0.5‐2.9 Hz) and could often give rise to single or a burst of action potentials. Pacemaker oscillations were observed when the membrane potential was moved negative to ‐55 and positive to ‐80 mV, but in a given cell the upper and lower limits of this voltage range were separated by only 13.1 +/‐ 0.5 mV. Above ‐45 mV tonic firing consisting of single action potentials was seen in the cells showing this or the other types of low‐frequency oscillations. 3. The spindle‐like oscillations were observed in thirty‐nine (out of 173) TC cells and consisted of rhythmic (2.1 +/‐ 0.3 Hz), large‐amplitude depolarizations (and often associated burst firing) similar to the pacemaker oscillations but occurring in discrete periods every 5‐25 s and lasting for 1.5‐28 s. The spindle‐like oscillations were observed when the membrane potential was moved negative to ‐55 and positive to ‐80 mV and in two cells they were transformed into continuous pacemaker oscillations by depolarization of the membrane potential to ‐60 mV. 4. Pacemaker and spindle‐like oscillations were unaffected by tetrodotoxin (TTX) or by selective blockade of NMDA, non‐NMDA, GABAA, GABAB, nicotinic, muscarinic, alpha‐ and beta‐noradrenergic receptors. 5. The ‘very slow’ oscillations consisted of a TTX‐insensitive, slow hyperpolarization‐depolarization sequence (5‐15 mV in amplitude) which lasted up to 90 s and was observed in nine dLGN cells and in two VB cells. The pacemaker and the spindle‐like oscillations were recorded in one cell each which also showed the ‘very slow’ oscillations. 6. The ‘NMDA’ oscillations were observed only in a ‘Mg(2+)‐free’ medium (0 mM‐Mg2+, 2‐4 mM‐Ca2+; 64 out of 72 cells) and consisted of large‐amplitude (10‐25 mV) depolarizations that did not occur at regular intervals and were intermixed with smaller depolarizations present on the baseline and on the falling phase of the larger ones.(ABSTRACT TRUNCATED AT 400 WORDS)

Cl- - and K+-dependent inhibitory postsynaptic potentials evoked by interneurones of the rat lateral geniculate nucleus.

1. Hyperpolarizing potentials evoked by electrical stimulation of the optic tract were studied in projection cells of the rat dorsal lateral geniculate nucleus (LGN) in vitro. In the same cells the effects of gamma‐amino butyric acid (GABA), baclofen and acetylcholine (ACh) were also investigated. 2. In the majority of cells a short‐ (SHP) (34 ms) and a long‐lasting (LHP) (240 ms) hyperpolarizing potential could be recorded in the presence and in the absence of a preceding EPSP. They were blocked by tetrodotoxin (1 microM) and were more sensitive than the monosynaptic EPSP to a low‐Ca2+‐high‐Mg2+ solution. 3. The SHP was associated with a marked decrease (75%) in input resistance, was blocked by bicuculline (1‐100 microM) and its reversal potential (‐67 mV) was dependent on the extracellular Cl‐ concentration. 4. The LHP was associated with a smaller decrease (45%) in input resistance and its reversal potential (‐76 mV) was dependent on the extracellular K+ concentration. It was increased by bicuculline (100% at 50 microM) and nipecotic acid (30% at 10 microM), blocked by Ba2+ (1 mM), and unaffected by eserine (1‐10 microM), neostigmine (1‐10 microM) or by recording with EGTA‐filled electrodes. In the presence of bicuculline, a single LHP was able to evoke, as a rebound response, a low‐threshold Ca2+ spike that was, however, not followed by another LHP (or any other long‐lasting hyperpolarization). 5. Ionophoretic applications of GABA evoked in the same cell a Cl‐ ‐dependent hyperpolarization (reversal potential: ‐65 mV) and/or depolarization, both of which were associated with a marked decrease (91%) in input resistance and abolished by bicuculline. GABA was also able to evoke a bicuculline‐insensitive, K+‐dependent hyperpolarization that had a reversal potential of ‐75 mV and was associated with a smaller decrease (43%) in input resistance. 6. Baclofen, applied by ionophoresis, pressure ejection or in the perfusion medium (1‐100 microM), produced a hyperpolarization that had a reversal potential of ‐79 mV and was associated with a decrease (45%) in input resistance. 7. In the majority of cells (thirty‐seven out of forty) ACh evoked a slow depolarization and only in three cells a hyperpolarization which had a reversal potential of ‐80 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Blockade of amino acid-induced depolarizations and inhibition of excitatory post-synaptic potentials in rat dentate gyrus.

Excitatory post‐synaptic potentials (e.p.s.p.s) evoked by stimulation of the medial perforant path and depolarizations induced by excitatory amino acids were recorded from granule cells in the preparation of the hippocampal slice from the rat. The effects of (+/‐)‐2‐amino‐5‐phosphonovalerate (APV), gamma‐D‐glutamylglycine (gamma DGG) and cis‐2,3‐piperidinedicarboxylate (PDA), antagonists of excitatory amino acids on these phenomena were compared. gamma DGG was the most effective antagonist of the e.p.s.p. Its action was reversible and not associated with any change in the passive membrane properties of the granule cells or in the apparent reversal potential of the e.p.s.p. Quantal analysis showed that the reduction in the e.p.s.p. paralleled the decrease in quantal size rather than quantal content, confirming a post‐synaptic site of the action of gamma DGG. The potency of gamma DGG against the exogenous agonists was N‐methyl‐D‐aspartate greater than kainate greater than or equal to quisqualate. APV had very little effect on the e.p.s.p. but was a selective antagonist of N‐methyl‐D‐aspartate‐induced depolarizations. PDA depolarized granule cells and increased their membrane input resistance. Although gamma DGG was a potent antagonist of both glutamate‐ and aspartate‐induced depolarizations, no clear pattern of specificity could be found. The action of glutamate was unaffected by APV. These results indicate that the receptor for the transmitter at the synapses formed by the fibres of the perforant path with the granule cells is of the quisqualate and/or kainate type. The present data are consistent with the biochemical evidence that glutamate may be the endogenous transmitter at his synapse.

The GABAB antagonist phaclofen inhibits the late K+-dependent IPSP in cat and rat thalamic and hippocampal neurones

Phaclofen (0.5-1 mM) reversibly inhibited the late, bicuculline resistant, K+ dependent IPSP recorded in projection cells of the cat and rat dorsal lateral geniculate nucleus and in rat hippocampal CA1 pyramidal neurones. At the same concentrations, phaclofen reversibly blocked the K+ dependent, bicuculline insensitive hyperpolarization evoked by GABA and baclofen but had no effect on the GABAA IPSP. These results represent conclusive evidence that GABAB receptors mediate the late K+ dependent IPSP in cortical and subcortical neurones.

The ventral and dorsal lateral geniculate nucleus of the rat: intracellular recordings in vitro.

1. The membrane properties and the electrotonic structure of neurones in the ventral and dorsal lateral geniculate nucleus (l.g.n.) of the rat were studied using an in vitro slice preparation. 2. Following electrophysiological characterization, horseradish peroxidase (HRP) was injected intrasomatically and the morphological features of impaled cells were characteristic of principal neurones of the rat ventral and dorsal l.g.n. 3. Neurones in the ventral l.g.n. had a higher input resistance but similar membrane time constants (tau o) and resting potentials than cells in the dorsal l.g.n. 4. Using a simple neuronal model, the electrotonic length (L) and the dendritic to somatic conductance ratio (rho) were calculated and found to be similar for cells in both divisions of the l.g.n. The mean value of L (0.7) and rho (1.5) suggest that both groups of neurones are electrotonically compact. 5. The width and after‐hyperpolarization of directly evoked action potentials, but not their threshold or their amplitude, were different between cells of the ventral and dorsal l.g.n. 6. At potentials more negative than ‐55 mV, a slow rising and falling potential could be evoked in each neurone (n = 310) of the dorsal l.g.n. but only in three cells of the ventral l.g.n. (n = 94). The electrophysiological and pharmacological properties of this potential were identical with those of the low‐threshold Ca2+‐dependent potential observed in other thalamic nuclei. 7. These results indicate that some of the passive and active membrane properties of ventral and dorsal l.g.n. neurones are different. The implications of these findings for the control of the integrative capability and the response of l.g.n. neurones to visual stimulation are discussed.

Pacemaker-like and other types of spontaneous membrane potential oscillations of thalamocortical cells

During EEG-synchronized sleep, thalamic activity is characterized by rhythmic oscillations that till recently have been suggested to require the contribution of intra- and extra-thalamic inputs. The present experiments show that thalamocortical (TC) cells, mechanically and pharmacologically isolated from their intra-thalamic, cortical and brainstem inputs, are capable of different types of spontaneous membrane potential oscillations some of which resemble those observed in TC cells of the living animal during EEG-synchronization.

The reversal potential of excitatory amino acid action on granule cells of the rat dentate gyrus.

The responses of granule cells to glutamate, aspartate, N‐methyl‐D‐aspartate (NMDA), quisqualate and kainate applied by ionophoresis on to their dendrites in the middle molecular layer of the dentate gyrus were studied with intracellular electrodes using an in vitro hippocampal slice preparation. On passive depolarization 75% of the granule cells displayed anomalous rectification, which persisted in the presence of TTX and TEA but was eliminated by Co2+ or the intracellular injection of Cs+. Short ionophoretic applications of all the excitatory amino acids evoked dose‐dependent depolarizations that were highly localized: movement of the ionophoretic electrode by as little as 10 microns could substantially change the size of the response. The depolarizations evoked by glutamate, asparatate, quisqualate and kainate were unaffected by TTX and Co2+. The depolarization evoked by NMDA was unaffected by TTX but markedly reduced by Co2+. Following intracellular injection of Cs+, neurones could be depolarized to +30 mV and the depolarizations produced by glutamate, quisqualate, NMDA and kainate reversed. The reversal potentials (E) were Eglutamate: ‐5.6 +/‐ 0.4 mV; ENMDA: 1.8 +/‐ 1.9 mV; Equisqualate: ‐3.9 +/‐ 1.9 mV; Ekainate: ‐4.6 +/‐ 2.0 mV. The excitatory post‐synaptic potential (e.p.s.p.) evoked by stimulation of the medial perforant path could also be reversed and Ee.p.s.p. was ‐5.5 +/‐ 1.1 mV. The 6 mV difference between ENMDA and the equilibrium potential for the other exogenously applied excitatory amino acids and the statistically significant difference between ENMDA and Ee.p.s.p. (P less than 0.005; d.f.: 7) is consistent with our earlier hypothesis that both the transmitter released by the medial perforant path and exogenously applied glutamate are unlikely to interact with NMDA receptors.

Mg2+ dependence of membrane resistance increases evoked by NMDA in hippocampal neurones

The response of granule cells and CA1 pyramidal neurones to NMDA was studied in the presence and absence of Mg2+ using an in vitro slice preparation. In the absence of Mg2+ the depolarizing response of hippocampal neurones to NMDA is accompanied by a decrease in input resistance. In the presence of Mg2+ ions, however, the response to NMDA is always associated with an apparent increase in input resistance. These results indicate that the action of NMDA is by a classical mechanism of conductance increase and are in agreement with the suggestion that the apparent increase in input resistance associated with NMDA depolarizations is the result of voltage-dependent channel block by Mg2+ of the NMDA evoked current.