Rodolfo R. Llinás

ELECTROPHYSIOLOGICAL PROPERTIES OF IN VITRO PURKINJE CELL DENDRITES IN MAMMALIAN CEREBELLAR SLICES

1. Intradendritic recordings from Purkinje cells in vitro indicate that white matter stimulation produces large synaptic responses by the activation of the climbing fibre afferent, but antidromic potentials do not actively invade the dendritic tree. 2. Climbing fibre responses may be reversed in a manner similar to that observed at the somatic level. However, the reversal does not show the biphasicity often seen at somatic level. 3. Input resistance of these dendrites was found to range from 15 to 30 M omega. The non‐linear properties seen at the somatic level for depolarizing currents are also encountered here. However, there seems to be less anomalous rectification. 4. Detailed analysis of repetitive firing of Purkinje cells elicited by outward DC current shows that, as in the case of the antidromic invasion, the fast somatic potentials (s.s.) do not invade the dendrite actively. However, the dendritic spike bursts (d.s.b.s) interposed between the s.s. potentials are most prominent at dendritic level. 5. Two types of voltage‐dependent Ca responses were observed. At low stimulus level a plateau‐like depolarization is accompanied by a prominent conductance change; further depolarization produces large dendritic action potentials. These two classes of response are TTX‐resistant but are blocked by Cd, Co, Mn or D600, or by the removal of extracellular Ca. 6. Following blockage of the Ca conductance, plateau potentials produced by a non‐inactivating Na conductance are observed mainly near the soma indicating that this voltage‐dependent conductance is probably associated with the somatic membrane. 7. Spontaneous firing in Purkinje cell dendrites is very similar to that observed at the soma. However, the amplitude of these bursts is larger at dendritic level. It is further concluded that these TTX‐insensitive spikes are generated at multiple sites along the dendritic tree. 8. Six ionic conductances seem to be involved in Purkinje cell electroresponsiveness: (a) an inactivating and (b) a non‐inactivating Na conductance at or near the soma, (c) a spike‐ and (d) a plateau‐generating Ca conductance, and (e) voltage‐dependent and (f) Ca‐dependent K currents. 9. The possible role of these conductances in Purkinje cell integration is discussed.

The excitatory synaptic action of climbing fibres on the Purkinje cells of the cerebellum

1. A single climbing fibre makes an extraordinarily extensive synaptic contact with the dendrites of a Purkinje cell. Investigation of this synaptic mechanism in the cerebellum of the cat has been based on the discovery by Szentagothai & Rajkovits (1959) that the climbing fibres have their cells of origin in the contralateral inferior olive.

Electrophysiology of mammalian inferior olivary neurones in vitro. Different types of voltage-dependent ionic conductances.

The electrophysiological properties of guinea‐pig inferior olivary (I.O.) cells have been studied in an in vitro brain stem slice preparation. 1. Intracellular recordings from 185 neurones in this nucleus reveal that antidromic, orthodromic or direct stimulation generates action potentials consisting of a fast spike followed by an after‐depolarizing potential (ADP). The ADP had an amplitude of 49 +/‐ 8 mV (mean +/‐ S.D.) and a duration which varied over a wide range with the level of depolarization. This ADP is followed by an after‐hyperpolarizing potential (AHP) having an amplitude of 12 +/‐ 3 mV (mean +/‐ S.D.) from rest and lasting up to 250 msec. The AHP shows a rebound depolarization wave. 2. Synaptic activation may be obtained by peri‐olivary stimulation with a bipolar electrode located in the immediate vicinity of the I.O. nucleus. These potentials are a mixture of depolarizing and hyperpolarizing synaptic events which can be reversed by direct membrane polarization. 3. Addition of tetrodotoxin (TTX) to the bath, or removal of extracellular Na, abolishes the fast initial action potential but does not modify the ADP or the AHP. Blockage of Ca conductance by Co, Mn, Cd or D600, or replacement of Ca by Mg, abolishes the ADP‐‐AHP sequence. 4. Hyperpolarization of the neurone uncovers a low‐threshold Ca conductance which is inactivated at rest and has similar pharmacological properties to the ADP. This low‐threshold spike plays a central role in the rebound potential following the AHP. 5. Simultaneous impalement of I.O. neurone pairs demonstrated the presence of electrotonic coupling between neurones, which is especially prominent in the medial accessory olive.

Relationship between presynaptic calcium current and postsynaptic potential in squid giant synapse

The relationship between calcium current and transmitter release was studied in squid giant synapse. It was found that the voltage-dependent calcium current triggers the release of synaptic transmitter in direct proportion to its magnitude and duration. Transmitter release occurs with a delay of approximately 200 mus after the influx of calcium. A model is presented which describes these relations formally.

Compartmentalization of the submembrane calcium activity during calcium influx and its significance in transmitter release

Quantitative modeling indicates that, in presynaptic terminals, the intracellular calcium concentration profile during inward calcium current is characterized by discrete peaks of calcium immediately adjacent to the calcium channels. This restriction of intracellular calcium concentration suggests a remarkably well specified intracellular architecture such that calcium, as a second messenger, may regulate particular intracellular domains with a great degree of specificity.

Of dreaming and wakefulness

Following a set of studies concerning the intrinsic electrophysiology of mammalian central neurons in relation to global brain function, we reach the following conclusions: (i) the main difference between wakefulness and paradoxical sleep lies in the weight given to sensory afferents in cognitive images; (ii) otherwise, wakefulness and paradoxical sleep are fundamentally equivalent brain states probably subserved by an intrinsic thalamo-cortical loop. From this assumption, we conclude that wakefulness is an intrinsic functional realm, modulated by sensory parameters. In support of this hypothesis, we review morphological studies of the thalamocortical system, which indicate that only a minor part of its connectivity is devoted to the transfer of direct sensory input. Rather, most of the connectivity is geared to the generation of internal functional modes, which may, in principle, operate in the presence or absence of sensory activation. These considerations lead us to challenge the traditional Jamesian view of brain function according to which consciousness is generated as an exclusive by-product of sensory input. Instead, we argue that consciousness is fundamentally a closed-loop property, in which the ability of cells to be intrinsically active plays a central role. We further discuss the importance of spatial and temporal mapping in the elaboration of cognitive and perceptual constructs.

The Functional Organization of the Olivo-Cerebellar System as Examined by Multiple Purkinje Cell Recordings.

Multiple recordings from Purkinje cells in the rat cerebellum allowed the mechanism responsible for the activation of rows of synchronous complex spikes to be investigated. By determining the spatial distribution of the climbing fibre reflex that follows electrical microstimulation of the cerebellar cortex, it was shown that the mechanism for the simultaneity of firing was the electrotonic interactions between neurons in the inferior olive (IO). The spatial organization of the complex spike activity was shown to be regulated by GABAergic inhibitory input into the IO, probably arising from the cerebellar nuclear neurons. The rostro‐caudal organizion of the complex spike activity following physiological stimulation (tactile stimulation of the upper and lower lip) demonstrated the same spatial distribution of synchronous activity in the cerebellar cortex as did the spontaneous activity and this was also disrupted by GABA blockers. Finally, complex spike responses to physiological stimulation indicate that the IO is capable of gating sensory inputs in accordance with its intrinsic autorhythmicity and that strong peripheral stimuli reset the oscillatory properties of the IO. The functional implications of the synchronicity and of the temporo‐spatial organizion of complex spikes in the cerebellar cortex are discussed in the context of motor coordination and timing.

Brain modeling by tensor network theory and computer simulation. The cerebellum: Distributed processor for predictive coordination

Abstract A fundamental problem regarding the functional interpretation of neural networks in the central nervous system is that of establishing the principles of their parallel, distributed organization. Available morphological and physiological knowledge concerning the cerebellum suggests that the central nervous system may use organizational principles other than the traditionally assumed ‘random connectivity’, ‘reflex loops’ or ‘redundancy’. We propose formally, and demonstrate by computer modeling, that the firing frequencies of individual cells over a cerebellar cortical area may be interpreted as a spatially distributed, finite, series expansion of a time function, which is reconstructed, by summation, in the nucleus where the cortical cells project. Thus, for example, the firing of Purkinje cells, when considered as representing a Taylor expansion, yield a prediction in the cerebellar nuclei of the frequency-time function of the input arriving at the cortex. This ‘lookahead’ (Δ) is an emergent property of the inherently parallel, distributed network. In order to analyze how a Taylor expansion-like process is used by the cerebellum on a system level, the linear algebraic matrix- and vector-representation of a distributed network was generalized in such a way as to regard the neuronal networks as tensors. Thus, the brain is envisioned as a set of tensorial systems which communicate with each other through vectorial channels (the pathways). These pathways carry multidimensional frequency vectors which are transformed by the tensors. In these terms the function of a particular subsystem of the central nervous system, in the present case the cerebellum, is represented in a multidimensional space. The frequency-hyperspace is characterized by the matrix of the cerebellar tensor which specifies a curved set of trajectories: a cerebellar vector field. Dynamic posture and balance are interpreted as displacement or stabilization of the functional status vector of the motor system along these curved trajectories. Cerebellar coordination of ballistic movements can be described as guiding the movement onto the ‘wired in’ trajectories of the vector field, by virtue of the coordination and inhibition vectors provided by the cerebellum. A proposal is also introduced that the climbing fiber system operates by momentary perturbations of the vector field, leading to deformations of the trajectories of the cerebellar frequency hyperspace. To bridge the gap between an attempt to treat parallel, distributed, neuronal networks, such as the cerebellum, as geometrical objects (leading, at the first approximation, to a linear mathematical formulation) and the simultaneous task of incorporating and interpreting experimental data, computer simulation methods are required. This combined approach of mathematical treatment (which provides an abstract language) and computer simulation (which, by accommodating data into the model, explores the significance of deviations from linear character) appears at present to be the most adequate technique for dealing with central nervous system function.

The olivo-cerebellar system: Functional properties as revealed by harmaline-induced tremor

SummaryIntracellular recording from Purkinje cells in cat cerebellar cortex demonstrated an 8–10/sec burst activity following intravenous administration of harmaline (10 mg/kg), a drug known to produce tremor at the same frequency. The burst activation of Purkinje cells was generated by large all-or-none depolarizations similar to climbing fiber (CF) excitatory postsynaptic potentials (EPSPs). Polarization of the cell membrane through the recording electrode (via a Wheatstone bridge) revealed that the all-or-none depolarization had an equilibrium potential and time course identical to the electrically evoked CF-EPSP, demonstrating directly that tremor is associated with specific activation of the CF afferent system.Interspike frequency histograms of the burst responses of Purkinje cells show that the rhythmic CF activity may continue for several hours with approximately 10% frequency scatter, the actual frequency depending on the level of anesthesia. Simultaneous extracellular recordings from Purkinje cells near the midline vermis indicated that CFs projecting to this area fire in a synchronous manner, while simultaneous recording from three Purkinje cells at different lateralities from the midline showed that the rhythmic activity is reduced in the lateral vermis and may be absent in the cerebellar hemispheres.Intra- and extracellular recordings from cerebellar nuclear cells (fastigial) disclosed a bursting type of activation following harmaline; a similar type of activity could be recorded in the reticular formation neurons and at inferior olive level. At spinal cord level, harmaline induced a repetitive and rhythmic activation of motoneurons which was not modified by dorsal root section. Cooling of the cerebellar cortex produced a definite desynchronization of the rhythmic motoneuronal firing. However, the basic 10/sec firing of the spinal cord motoneurons could still be observed. Following lesion of the inferior peduncles which interrupted the olivo-cerebellar pathway, the rhythmic activation of Purkinje cells, nuclear cells, vestibular and reticular cells and motoneurons disappeared. However, the rhythmic activity was maintained at inferior olivary level. It is suggested that harmaline acts directly on the inferior olive since in animals with low decerebration, cerebellectomy and spinal transection, rhythmic activity of the inferior olive could still be observed.The results of these experiments strongly suggest that the inferior olive is able to generate the activation of motoneurons and that such influence can only take place through the activation of the cerebellar nuclei. Possible functions of the inferior olive as a generator of fast muscular transients are discussed.

Inferior olive: its role in motor learing.

Specific chemical lesion of the rat inferior olive by intraperitoneal administration of 3-acetylpyridine prevents recuperation from motor abnormalities generated by unilateral labyrinthine lesion. Moreover, in animals that have recuperated from the balyrinthine lesion, 3-acetylpyridine produces a reversal of the symptoms within 2 hours of administration. These results indicate that the integrity of the olivo-cerebellar system is necessary for the acquisition and retention of this form of motor learning, but that the cerebellum itself is not the seat of such learning.