The Cerebellum: Brain for an Implicit Self (14 page)

IO neurons receive GABAergic synapses, most of which arise from cerebellar nuclear neurons (
De Zeeuw et al., 1988
). In rats, an antibody against a GABA-synthesizing enzyme (GAD) labeled axon terminals impinging onto dendrites (94% of the sample) or with somata (6%). These terminals were intermingled with dendrodendritic gap junctions within the complex structure of glomeruli (
Sotelo et al., 2004
). The functional association between IO neurons and axon terminals of cerebellar nuclear neurons was indicated by the systematic presence of GAD-labeled cerebellar nuclear neurons’ axon terminals directly apposed to the IO cells’ dendritic appendages linked by gap junctions, and by the frequent presence of type II synapses (allegedly inhibitory) straddling both elements. Gap junctions were located mostly in glomeruli at the distal dendrites of IO neurons (
Sotelo et al., 1974
; De Zeeuw et al., 1990a,b). One glomerulus contained a core of five to six
dendritic and axonal spiny appendages, derived from different IO neurons, coupled by gap junctions, and surrounded by both excitatory and inhibitory synaptic terminals of extrinsic origin (De Zeeuw et al., 1990a). These arrangements appear to serve for the modulation of electrical synapses by the release of GABA, which increases nonjunctional membrane conductance and thereby shunts the electric coupling between inferior olive neurons.

Under certain conditions, the electrical synapses mediate regular sinusoidal oscillations of the membrane potential of IO neurons. About 10% of these neurons tested in guinea pig brainstem slices exhibited spontaneous subthreshold oscillations in their membrane potential at a frequency of 4–6 Hz and an amplitude of 5–10 mV. This oscillation was synchronized in all cells tested within the slice, persisted in the presence of tetrodotoxin, and was blocked by Ca
²⁺
conductance blockers or by removal of Ca
²⁺
from the bathing solution. The oscillation was not affected by the intracellular activation of any given neuron. Therefore, the oscillation seemed to reflect the ensemble properties of a large number of IO neurons coupled in a network. A similar ensemble oscillation was induced by adding harmaline and serotonin to the bathing solution and it was blocked by adding noradrenaline to the bathing solution (
Llinás and Yarom, 1986
).

Given that IO neurons discharge with a highly regular rhythm under selected conditions, a hypothesis has been proposed that climbing fibers provide a periodic clock for coordinating movements or motor timing (
Kazantsev et al., 2004
). In alert monkeys, however, climbing fiber signals have been reported to occur randomly at a characteristically low rate (~1 Hz) (
Keating et al., 1995
). It appears that IO neurons recode the high-frequency information carried by its synaptic inputs into stochastic, low-rate discharge in their climbing fiber outputs. This possibility has been simulated by a computational model (Schweighofer et al., 2004).

The characteristic multiple spike discharges induced in Purkinje cells by IO stimulation partly reflect (1) Ca
²⁺
spikes in the Purkinje cell membrane (see
Figure 15B
) and (2) the long-lasting after-depolarizing potential (ADP) that follows a single spike in IO neurons. The number of spikes in the burst discharge of IO axons was shown to depend on the phase of their subthreshold oscillations, which suggested that the burst stage encoded the state of the IO network (
Mathy et al., 2009
).

Preolivary neurons projecting ascending pathways from the spinal cord to the IO complex can be labeled by the anterograde transport of WGA-HRP from spinal segments (
Matsushita et al., 1992
). It was thus shown that C1-T2 and also L6-S1 segments project to the caudal half of the MAO, C1-C4 segments to the most
medial part of the DAO, and C5-T1 segments more laterally. Even more lateral projections to the DAO were shown to arise from T2-L5 and L6-S1 spinal segments. These findings imply distinct somatotopic projections in the mediolateral order to the caudal MAO and the DAO. On the other hand, certain midbrain neurons project descending axons to the IO complex. They have been labeled by the retrograde transport of HRP in cats. Such midbrain neurons were shown to be located in the ipsilateral mesencephalon, from the rostral pole of the red nucleus to the caudomedial border of the thalamus. Heavily labeled nuclear groups included the parvocellular red nucleus, the interstitial nucleus of Cajal, the nucleus of Darkschewitsch, and the caudomedial extremity of the subparafascicular nucleus (
Saint-Cyr and Courville, 2004
). A few cells were also labeled in the reticular formation lateral to the interstitial nucleus of Cajal, in the caudomedial parafascicular nucleus, in the nucleus of the fields of Forel, and in the central gray. Elsewhere in the midbrain, there were HRP-labeled cells in the deep layers (IV, VI) of the superior colliculus, predominantly on the side contralateral to the injection site, in the nucleus of the optic tract, and in the ipsilateral anterior and posterior pretectal nuclei. These latter groups were best labeled after caudally located injections into the olive. No labeled cells were found by Saint-Cyr and Courville (
2004
) in the basal ganglia, rostral raphe nuclei, and the nucleus of Edinger-Westphal, despite a previous report to the contrary.

Among beaded fibers so far identified, serotonergic fibers originate from the raphe nucleus, and noradrenergic fibers from the locus coeruleus. These aminergic fibers exert a unique facilitatory action on inhibitory neurons in the granular layer (
Chapter 5
, “Inhibitary Interneurons and Glial Cells in the Cerebellar Cortex”). Histaminergic fibers originate from a group of hypothalamic neurons. Histamine was shown to excite rat Purkinje cells via H2 receptors (
Tian et al., 2000
).

Twenty-four different neuropeptides have been identified in the cerebellum (
Ito, 2009
), with many expressed in mossy fibers, climbing fibers, and several cellular structures. It is also known that angiotensin II and orexin are located in beaded fibers that originate in the hypothalamus. The stimulation of orexinergic neurons in the hypothalamus was shown to excite Purkinje cells in a small area (folium-p) of the flocculus. Hence, it was suggested that orexinergic fibers facilitate folium-p Purkinje cells together with brainstem structures involved in defense reactions (
Nisimaru et al., 2010
).

Four types of neurons are packed into cerebellar nuclei. First are excitatory projection neurons, which release glutamate as the transmitter and project to the medulla and midbrain structures. These neurons have the largest somata and dendritic trees among cerebellar nuclear cells. Second, smaller GABAergic neurons project to the IO (
De Zeeuw et al., 1989
). In glutamate decarboxylase (GAD) 67-green fluorescent protein (GFP) knock-in mice, GABAergic neurons were identified as GAD-positive neurons. Compared with GAD-negative neurons, GAD-positive neurons generated broader action potentials, displayed stronger frequency accommodation, and did not reach high firing frequencies during depolarizing current injections. GAD-positive cells also displayed slower spontaneous firing rates but exhibited a longer-lasting rebound depolarization and associated spiking after a transient membrane hyperpolarization (
Uusisaari et al., 2007
). Third are glycinergic neurons recently found in the medial cerebellar (fastigial) nuclei of mice (
Bagnall et al., 2009
). Glycinergic fastigial neurons form projections to vestibular and reticular neurons in the ipsilateral brainstem, whereas their glutamatergic counterparts project contralaterally. These three types of projection neurons receive inhibitory inputs from Purkinje cells and excitatory inputs via collaterals of mossy fibers and climbing fibers. Fourth are the smallest cells, which extend their axons within the cerebellar nuclei (
Chan-Palay, 1977
). Both GABA and glycine transmitters were shown to co-localize in these cells (
Chen and Hillman, 1993
). Their input and output connections remain unknown, however. Among rat cerebellar nuclear cells, projection neurons (first type) show cyclic burst firing with underlying Na
⁺
and Ca
²⁺
plateau potentials, low-threshold Ca
²⁺
spikes, and a slow Ca
²⁺
-dependent afterhyperpolarization. A small subset of cerebellar nuclear neurons lacks slow plateau potentials and low threshold spikes, and they have a lower rate of spontaneous discharge. These neurons were conjectured to be among the fourth type of cerebellar nuclear interneurons (
Czubayko et al., 2001
).

Aizenman and Linden (
1999
) provided evidence that a characteristic feature of cerebellar nuclear neurons was their prominent rebound depolarization that occurred at the end of injected hyperpolarizing current pulses leading to a Na
⁺
spike burst. This rebound depolarization depends on low-threshold T-type voltage-gated Ca
²⁺
channels and may involve also the activation of a hyperpolarization-activated cation current. Similar rebound was observed also in vestibular neurons receiving inhibition from flocculus Purkinje cells (
Sekimjak et al., 2003
). Such rebound has been interpreted as playing several functional roles, including timing, encoding information (pause coding;
Steuber et al., 2007
), and mediating synaptic
plasticity (see
Chapters 8
and
9
). However, it has been claimed that such a rebound does not occur unless the test cell is slightly depolarized by microelectrode penetration (
Alviña et al., 2008
;
Walter and Khodakhah, 2009
). As such, the physiological meaning of this rebound is still a matter of discussion.

In the flocculus, Purkinje cells project directly to certain brainstem neurons rather than cerebellar nuclear cells. There are four studied cases. (1) Purkinje cells that project to the superior and medial vestibular nuclei (
Fukuda et al., 1972
;
Kawaguchi, 1985
;
Sekimjak et al., 2003
). Recall that these nuclei process the VOR. Such VOR neurons are either excitatory (glutamatergic) or inhibitory (glycinergic) in their action on oculomotor neurons (
Chapter 10
). (2) Purkinje cells in zone B of the vermis that inhibit the dorsal part of the lateral vestibular nucleus of Deiters (
Ito et al., 1964
), which in turn exerts excitatory actions on segmental motoneurons and interneurons (
Wilson and Yoshida, 1969
). (3) Purkinje cells located in the lateral edge of the nodulus that project directly to the medial portion of the ipsilateral parabrachial nucleus, which is located immediately rostral to the superior vestibular nucleus (
Nisimaru et al., 1998
). The parabrachial nucleus has been shown to mediate the vestibulo-sympathetic reflex and thereby maintain mean blood pressure constant during head movement. (4) Purkinje cells that have been shown to project directly to the lateral portion of the ipsilateral parabrachial nucleus (
Nisimaru et al., 2010
). This nucleus mediates the somatosympathetic reflex, which redistributes arterial blood flow in defense reactions and locomotion (
Chapter 11
).

Morphological, immunocytochemical, and genetic analyses have advanced remarkably to reveal new properties of neurons in the cerebellum and its related structures. We now need more information about the activity of these neurons in behaving animals. It is hoped that this advance should be forthcoming in the near future through the development of new technologies for improved telemetry, optogenetics mapping, and molecular imaging.

Among electrical and chemical signaling capabilities of neurons, synaptic plasticity is a unique process that induces persistent changes in synaptic transmission efficacy in an activity-dependent manner. The best-known examples are long-term potentiation (LTP) and depression (LTD). A cascade of cellular/molecular events, which are initiated by an experience, generates these changes. They are considered to culminate in a durable form of altered synaptic transmission efficacy. “Conjunctive LTD,” which is induced postsynaptically by the combined activation of parallel fibers and a climbing fiber converging onto a Purkinje cell, is a unique type of synaptic plasticity in the cerebellum because of the involvement of climbing fibers that are unique to the cerebellum. The characteristic physiological properties of such conjunctive LTD and its underlying mechanisms are discussed in the following sections.

Conjunctive LTD occurs in parallel fiber-Purkinje cell synapses when parallel fibers are activated simultaneously with a climbing fiber that converges onto the same Purkinje cell. The whole story of its discovery in the cerebellum was introduced in
Chapter 3
, “
The Cerebellum as a Neuronal Machine
.” In rat and mouse
in vitro
cerebellar slices, conjunctive stimulation at 1 Hz for 5 minutes (300 pulses) is optimal (
Karachot et al., 1994
) for reducing by ~30% the EPSPs (their rising slope) and EPSCs of Purkinje cells, as activated by parallel fiber input. A similar result is obtained when brief stimulation of parallel fibers by 8 pulses (100) precedes stimulation of a climbing fiber with a low frequency train of 3 pulses (20 Hz) (
Schreurs et al., 1996
).

Conjunctive LTD involves postsynaptic glutamate receptors in parallel fiber-Purkinje cell synapses. This finding is based on the following observations. When parallel fiber stimulation is replaced by the iontophoretic application of glutamate to directly activate the postsynaptic membrane of a Purkinje cell, the conjunction of this and climbing fiber stimulation depresses the glutamate sensitivity of the cell (
Ito et al., 1982
). In a cultured Purkinje cell devoid of dendritic spines, a combination of glutamate (or quisqualate) pulses and membrane depolarization, the latter causing Ca
²⁺
entry, effectively depresses glutamate and AMPA sensitivity (
Linden et al., 1991
,
1995
). In mice deficient in mGluR4, which is normally expressed in parallel fibers, parallel-fiber-induced paired-pulse facilitation and post-tetanic potentiation are impaired, but conjunctive LTD remains fully operative (
Pekhletski et al., 1996
). This showed that LTD does not require the mGluR4-regulated presynaptic mechanism to maintain synaptic efficacy during repetitive activation.