Dean E. Hillman

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.

Distribution and functional significance of the P-type, voltage-dependent Ca2+ channels in the mammalian central nervous system

In addition to the three types of voltage-dependent calcium channels presently recognized in the CNS, the L-, the T- and the N-types, a fourth distinct type known as the P-type channel has recently been described. This channel, initially recognized in Purkinje cells (and thus the name), is not blocked by dihydropyridines or by omega-conotoxin (GVIA), but is blocked by native funnel-web spider venom and by a polyamine (FTX) extracted from such venom. In addition, a synthetic polyamine (sFTX) has been produced that also specifically blocks P-channels in brain slices and at the neuromuscular junction, and blocks presynaptic Ca2+ currents in other vertebrate and invertebrate forms, as well as channels expressed in Xenopus oocytes following CNS mRNA injections. Using sFTX to form an affinity gel, a protein was isolated and reconstituted into lipid bilayers where it manifests single-channel properties that are electrophysiologically and pharmacologically similar to those of the native P-channels. Rabbits immunized with the isolated protein produced a polyclonal antibody that gave a positive western blot with the purified P-channel protein and generated a reaction product at specific sites in the CNS that agree with the physiological distribution of P-channel activity.

The potassium channel subunit KV3.1b is localized to somatic and axonal membranes of specific populations of CNS neurons

Potassium channels play major roles in the regulation of many aspects of neuronal excitability. These channels are particularly well suited for such multiplicity of roles since there is a large diversity of channel types. This diversity contributes to the ability of specific neurons (and possibly different regions of the same neuron) to respond uniquely to a given input. Neuronal integration depends on the local response of spatially segregated inputs to the cell and the communication of these integration centers with the axon. Therefore, the functional implications of a given set of K+ channels varies depending on their precise location on the neuronal surface. Site- specific antibodies were utilized to characterize the distribution of KV3.1b, a subunit of voltage-gated K+ channels in CNS neurons. KV3.1b subunits are expressed in specific neuronal populations of the rat brain, such as cerebellar granule cells, projecting neurons of deep cerebellar nuclei, the substantia nigra pars-reticulata, the globus pallidus, and the ventral thalamus (reticular thalamic nucleus, ventral lateral geniculate and zona incerta). The KV3.1b protein is also present in various neuronal populations involved in the processing of auditory signals, including the inferior colliculus, the nuclei of the lateral lemniscus, the superior olive, and some parts of the cochlear nuclei; as well as in several other neuronal groups in the brainstem (e.g., in the oculomotor nucleus, the pontine nuclei, the reticulotegmental nucleus of the pons, trigeminal and vestibular nuclei, and the reticular formation) and subsets of neurons in the neocortex, the hippocampus and the caudate-putamen shown by double staining to correspond to neurons containing parvalbumin. KV3.1b subunits are localized predominantly in somatic and axonal membranes (particularly in axonal terminal fields) but are much less prominent in dendritic arborizations. This distribution is different than that of other subunits of voltage gated K+ channels and is consistent with a role in the modulation of action potentials. KV3.1b proteins have a cellular and subcellular distribution different than the related KV3.2 subunits which express in Xenopus oocytes currents similar to those expressed by KV3.1b.

Dendritic Spikes and Their Inhibition in Alligator Purkinje Cells

Alligator Purkinje cells generate action potentials in the peripheral dendritic tree, after synaptic depolarization via superficial parallel fibers. These action potentials are inhibited at the dendrite level by preceding parallel-fiber volleys at close intervals. We conclude that this inhibition is produced by the activation of the inhibitory interneurons of the molecular layer, the stellate cells, which establish synaptic contacts with the dendrites of the Purkinje cells.

Colocalization of neurotransmitters in the deep cerebellar nuclei.

SummaryAn abundance of glycine and glycine receptor immunoreactivities was found in all three parts of the deep cerebellar nuclei. Glycine immunoreactivity was restricted to small neurons throughout most of the deep cerebellar nuclei except for a few large positive neurons in the ventral part of the fastigial nuclei. In addition, glycine immunoreactivity was found in boutons outlining somata of large glycine negative neurons. Complementary to the glycine positive boutons was an intense glycine receptor immunoreactivity on large deep cerebellar nuclei neurons. Comparisons of immunoreactivities for glycine, GABA and aspartate in consecutive one micron sections revealed that many small neurons colocalized glycine and GABA, while some large neurons in the fastigal region colocalized glycine and aspartate.Ultrastructural investigations revealed glycine receptors on postsynaptic sites of dendrites and somata. Most boutons, which were presynaptic to glycine receptor sites, were filled with small flattened vesicles; however, a small percentage of boutons had round clear or dense core vesicles. Frequently, each bouton apposed multiple active zones on the dendrite or soma. One of these active zones was positive for glycine receptor and another was negative.This study supports: (1) glycine as a neurotransmitter in deep cerebellar nuclei, and (2) glycine and GABA colocalization in the same cell and bouton, but releasing to different receptor sites on the target neuron. Furthermore, the coexistence of glycine with GABA in the same deep cerebellar neuron may play an important role in controlling the conset and duration of signal transmission.

Chronic Neurological Deficits and Nesacaine-CE-An Effect of the Anesthetic, 2-Chloroprocaine, or the Antioxidant, Sodium Bisulfite?

Chronic neurological deficits have been described in patients after presumed accidental subarachnoid injection of 2-chloroprocaine-CE (Nesacaine-CE; N-CE) intended for epidural block. This study investigated the possible role of pure 2-chloroprocaine (2-CP) and sodium bisulfite, two components of Nesacaine-CE, in causing these complications when injected separately into the lumbar subarachnoid space of neurologically intact awake rabbits. Repeated 2–4-mg spinal anesthetic doses of pure 2-CP in lactated Ringers solution did not produce chronic hindlimb paralysis even though accumulated doses reached 50 mg. However, 1.2–2.4 mg of sodium bisulfite, the antioxidant in N-CE added to prolong shelf-life, resulted in irreversible hindlimb paralysis in 12 out of 14 animals. This amount of bisulfite is contained in 12–24 mg of 2% N-CE. The demonstration that persistent paralysis resulted from low dosages of sodium bisulfite contained in commercially available 2-CP requires revaluation of the suitability of this antioxidant for products prepared for intrathecal use.

Plasticity of the parallel Fiber-Purkinje cell synapse by spine takeover and new synapse formation in the adult rat

Alteration in synaptic connectivity between Purkinje cell spines and parallel fibers of the cerebellum were studied following partial deafferentation of Purkinje cells in the the adult rat. Transection of parallel fibers by two lesions placed at a 1 mm interval on the folial crest were used to produce degeneration of these afferents. Ultrastructural analysis of synapses on Purkinje cell spines revealed degeneration with vacating of postsynaptic sites within 6 h. Reactive synaptogenesis as takeover of Purkinje cell spines by formation of new synapses from remaining parallel fibers occurred even before degenerating parallel fibers had vacated postsynaptic sites. This was accompanied by a marked increase in the number of dual innervations by reactive parallel fibers within one day. Some vacated postsynaptic sites were lost as indicated by a reduction in the number of synapses and others may have been taken over by newly formed synapses on spines. In addition, new synapses formed between the shafts of Purkinje cell branchlets and parallel fibers. Sprouting of parallel fibers occurred as small extensions without tubules while Purkinje cell spines reacted by forming elongated and multiple heads which contacted different parallel fibers. After 5 days degenerating boutons were rarely found. Enlarged spine heads were each capped by a proportionally enlarged parallel fiber bouton and joined by an elongated synaptic junction to parallel fibers. Some parallel fiber boutons were greatly enlarged and capped numerous profiles of spines. This study shows that formation of new pre- and postsynaptic sites takes precedence over reoccupation of original contacts and that multiple synapses on individual spines are being eliminated to give rise to single contacts with boutons. This elimination resulted in enlargement of synaptic contact areas between Purkinje cell spines and parallel fibers by taking over postsynaptic sites from some vacated and eliminated boutons.

Selective ablation of neurons by methylazoxymethanol during pre- and postnatal brain development

Neonatal or pregnant albino rats were injected with either single or double doses of methylazoxymethanol (20 mg/kg) to test the temporal specificity of its effect on clearly definable regions of the brain. A single dose, to dams from gestational day 11 to 21 (G11-G21) and to neonatal rats from birth to postnatal day 5 (P0-P5), produced differential weight reductions among various brain regions. Two prominent peaks of reduction were found: one occurring between G13 and G15 for the cerebrum and hippocampus and one occurring between P0 and P1 for the cerebellum and olfactory bulbs. Dual injections of the drug on G14 and G15 produced 60% weight reduction in the cerebrum, and slightly earlier injections on G13 and G14 reduced the weight of the cerebellum by about 23%. This weight reduction was accompanied by narrowing of the cerebellar width, which we believe was due to fewer Purkinje cells. Dual injections of methylazoxymethanol at P0 and P1 reduced the weight of the olfactory bulb by 65%, the cerebellum by 62%, and the hippocampus by 18%. These results show that its short action is within the window of cell division for various neurons and becomes additive on two successive days. This precise toxic effect on brain development can be used to disproportionally reduce the number of neuroblasts in specific regions of the brain. A differential ablation allows analysis of plasticity on pyramidal and nonpyramidal cells of the neocortex and hippocampus, Purkinje and granule cells of the cerebellum, and the granule and mitral cells of the olfactory bulbs.

Vulnerability of cerebellar development in malnutrition—I. Quantitation of layer volume and neuron numbers

Abstract The volumes of the cerebellar layers and numerical relationships, between granule cells and Purkinje cells, were analyzed following developmental malnutrition for differences in cerebellar changes between male and female rats. Control and protein-deficient diets were administered during pregestation, gestation, lactation and to offspring from weaning until death at 60 days of age. Volume of the granular, molecular and myelinated layers and the area of the Purkinje cell layer were determined from area and length measurements on serial sections. Density of granule and Purkinje cells was quantitated and used with granular layer volume and area of the Purkinje cell layer, respectively, for defining total cell numbers of each cell type. Although the wet weight of the cerebellum was not different between male or female deficient groups, the volume of the entire cerebellum, as well as its layers, was reduced more in the deficient female group than the deficient males. This difference between the two types of preparations was attributed to larger interfolial spaces in the deficient female group, presumably due to overall brain volume displacement within a cranial cavity space having the same size. The most striking difference between sexes was seen in the volume of the molecular layer where deprived females had a nearly 30% reduction as compared to their controls. The granular layer was decreased twice as much in the deficient females as in the deficient males. The largest volume change but least difference between sexes was in the myelinated layer. The reduction in granule cell number was reflected in a decreased granular layer volume rather than granular cell density. Granule cells were reduced by 20% in deficient females and 10% in deficient males but the number of Purkinje cells was constant between all groups. The result was a major shift in the ratio of granule cells to Purkinje cells for the deficient female group and a small change in ratio for the deficient male group. The reason for the more marked effect of malnutrition on the female could not be determined from this experimental paradigm, although much of the difference may be the result of behavior in competitive feeding during lactation.

Regulation of granule cell number by a predetermined number of Purkinje cells in development

Development dysgenesis of Purkinje cells or granule cells was analyzed for the reciprocal effect of reduced number of each cell type on the other. A single pre- or postnatal injection of methylazoxymethanol acetate (MAM) in the rat reduces either the number of Purkinje cells or the number of granule cells when administered at the time of their respective genesis. The total number of these two types of neurons was obtained from cell density values of each layer and the total volume of the granular layer and the area of the Purkinje cell layer. The results show that Purkinje cells (targets) strictly determine the maximum number of granule cells (afferent neurons) following deficits in the number of Purkinje cells produced by prenatal MAM administration. Deficits in Purkinje cells were accompanied by a proportionally smaller number of granule cells so that the ratio remained constant. On the other hand, the reduction in the number of granule cells of the postnatal MAM model did not affect the number of Purkinje cells. These results indicate that the maximum number of these afferent neurons is constrained unidirectionally through a property defined by the number of their target neurons which develop earlier. Furthermore the number of afferent cells had no effect on the number of target cells.