Donald J. Woodward

The ontogenetic development of synaptic junctions, synaptic activation and responsiveness to neurotransmitter substances in rat cerebellar purkinje cells

Ultrastructural quantitation of synaptogenesis in cerebellum showed the onset of contacts stained by ethanolic phosphotungstic acid at days 3 and 4 with a progressive increase in density of such contacts at later ages. Electrophysiological studies revealed that both responses to parallel fiber stimulation and the occurence of climbing fiber burst responses appear late in the third postnatal day. Studies of drug sensitivity employing microiontophoretic techniques showed that spontaneous activity of Purkinje cells between 0 and 4 postnatal days is accelerated by glutamate, and inhibited by gamma-aminobutyric acid, norepinephrine, adenosine cyclic monophosphate (cyclic AMP) and dinitrophenol. Prostaglandin E1 antagonized the action of norepinephrine. These studies indicate that the membrane chemosensitivity to the adult spectrum of putative transmitters precedes the onset of synaptogenesis.

Postnatal development of electrical and enzyme histochemical activity in Purkinje cells

Abstract Extracellular action potentials were recorded from Purkinje cells of rats, from several hours after birth to adult in age, anesthetized with Nembutal and paralyzed with curare. Lactate and succinate dehydrogenase and γ-amino butyrate-α-ketoglutarate transaminase activities were histochemically determined using tetrazolium reduction techniques. Action potentials were observed as early as several hours after birth with regular sustained spontaneous activity beginning at 1–2 days. There is a sharp increase in activity of all three enzymes between 0 and 3 days restricted in the cerebellar cortex to Purkinje-cell soma. Maximum activities of these enzymes were found at 7 days. Spontaneous climbing-fiber bursts were observed by 7 days. Between 10 and 15 days after birth there occurs an increase in spontaneous firing rates, a decrease in spike duration, and a shift from regular to irregular discharge patterns. There is a simultaneous appearance of histochemical activity in the neuropil of the molecular layer, the glomeruli, and the cerebellar interneurons at this time.

Inhibition of Purkinje cells in the frog cerebellum. II. Evidence for GABA as the inhibitory transmitter

Abstract Microiontophoresis of gamma-aminobutyric acid (GABA) from multi-barreled micropipettes mimics the effects of stellate cell inhibition in frog cerebellar Purkinje (P) cells, producing cessation of P cell discharge accompanied by hyperpolarization and decreased membrane resistance. Microiontophoresis of picrotoxin or bicuculline, but not strychnine, antagonized P cell responses to GABA, and reproducibly blocked the inhibitory effects of local (LOC) stimulation on P cell spontaneous discharge. These experiments suggest that LOC-induced inhibition does exist in frog cerebellum, and that GABA may be the inhibitory neurotransmitter released by stellate cells.

NORADRENALINE MODULATION OF THE RESPONSES OF THE CEREBELLAR PURKINJE CELL TO AFFERENT SYNAPTIC ACTIVITY

Noradrenaline, applied by microiontophoresis to rat cerebellar Purkinje neurones, selectively depressed spontaneous neuronal discharge. Simple spike and complex spike excitations, evoked by stimulation of the mossy and climbing fibres, were relatively preserved during the inhibition of spontaneous activity, and the number of full‐sized action potentials in the complex spike increased. Inhibition mediated by the basket and stellate cells was augmented. Thus, relative to the change in spontaneous activity, noradrenaline increased the responsiveness of the Purkinje cell to afferent input.

In Vivo Extracellular Recording of Striatal Neurons in the Awake Rat Following Unilateral 6-Hydroxydopamine Lesions

The purpose of this study was to further understand the functional effects of dopaminergic input to the dorsal striatum and to compare the effects of dopaminergic lesions in awake and anesthetized animals. We examined the effects of unilateral 6-hydroxydopamine (6-OHDA) lesions of the ascending dopaminergic bundle on the firing properties of dorsal striatal neurons in the awake freely moving rat using chronically implanted microwire electrode arrays. We recorded extracellular activity of striatal neurons under baseline conditions and following the systemic injection of apomorphine in awake and anesthetized subjects. Firing rates were higher in the hemisphere ipsilateral to the 6-OHDA lesion compared to rates of neurons from the contralateral unlesioned hemisphere. Striatal firing rates from sham and no-surgery control rats were, in general, higher than those from the contralateral unlesioned striatum of experimental subjects. Apomorphine (0.05 mg/kg, sc) normalized the differences in firing rates in lesioned animals by increasing firing of neurons within the contralateral unlesioned side, while simultaneously decreasing firing of neurons within the ipsilateral lesioned side. Mean firing rates were substantially higher in awake animals than in subjects anesthetized with chloral hydrate, perhaps reflecting anesthesia-induced decreases in excitatory input to striatal neurons. Chloral hydrate anesthesia decreased firing rates of neurons in the lesioned, unlesioned, and control striata to a similar degree, although absolute firing rates of neurons from the 6-OHDA-lesioned striata remained elevated over all other groups. Unilateral 6-OHDA lesions also altered the pattern of spike output in the awake animal as indicated by an increase in the number of bursts per minute following dopaminergic deafferentation. This and other burst parameters were altered by apomorphine. Our findings show that effects of dopaminergic deafferentation can be measured in the awake behaving animal; this model should prove useful for testing the behavioral and functional effects of experimental manipulations designed to reduce or reverse the effects of dopaminergic cell loss. In addition, these results suggest that the contralateral changes in striatal function which occur in the unilateral dopaminergic lesion model should be considered when evaluating experimental results.

An improved constant current source for micro-iontophoretic drug application studies ☆

Abstract A simple circuit is described for injecting current into micropipettes for electrophoresing and retaining chemical substances. Due to the high voltage output amplifier employed, it will deliver a high (500 nA) current through a very high resistance (200 MΩ) drug micropipette. Provision is made for injecting a balancing current equal in magnitude and opposite in polarity to the sum of the injection currents, for recording a signal proportional to output current and for controlling current injection with external devices.

Responses of cultured cerebellar neurons to iontophoretically applied amino acids.

Electrical activity of spontaneously firing neurons in explant cultures of neonatal rat cerebellum was recorded with extracellularly placed microelectrodes. Several different normal patterns of firing were noted, common ones being single spikes at regular intervals, irregularly spaced single spikes and rapid bursts of up to 10 spikes separated by periods of silence. The firing rate could be modified by the local application of drugs through iontophoresis onto the soma and adjacent processes. Firing rates were increased by glutamate and homocysteate, and decreased by gamma-aminobutyric acid (GABA) and glycine. The action of GABA was blocked by bicuculline and picrotoxin. GABA was relatively more effective than glycine. Acetylcholine was generally ineffective, but rarely had weak excitatory actions. We conclude that the sensitivities of the cultured Purkinje cell with respect to these drugs is the same as the adult Purkinje cellin vivo and similar to the immature cell at the time of explantation. We suggest that pharmacological and physiological criteria be used to identify cell types and evaluate the extent of differentiation of neurons in culture.

Deep brain stimulation of the substantia nigra pars reticulata exerts long lasting suppression of amygdala-kindled seizures

Deep brain stimulation (DBS) has been used to treat a variety of neurological disorders including epilepsy. However, we have limited knowledge about effective target areas, optimal stimulation parameters, and long-term effect of DBS on epileptic seizures. Here we examined the effects of DBS of the substantia nigra pars reticulata (SNr) on amygdala-kindled seizures. Microwire electrodes were implanted into the SNr and amygdala of adult male rats. When stage 5-kindled seizures were achieved by daily amygdala kindling, high frequency stimulation was delivered to the SNr bilaterally 1 s after cessation of kindling. Our DBS protocol completely blocked kindled seizures in 10 out of 23 (43.5%) rats studied. Furthermore, when the same amygdala kindling procedure was performed 24 h later without DBS, the kindling failed to elicit any seizure signs in 6 of these 10 rats. Some of the post-DBS period of seizure suppression lasted for up to 4 days. In other 3 rats, only mild stage 1 to 2 seizures appeared following amygdala kindling. Only 1 of the 10 rats for which DBS had blocked kindled seizures exhibited full-scale 5 stage-kindled seizures 24 h after DBS. These results suggest that highly plastic neural networks are involved in amygdala-kindled seizures and that DBS, if well timed with the onset of amygdala kindling, may exert long lasting effects on the networks that may prevent the recurrence of kindled seizures.

CEREBELLAR DEVELOPMENT IN THE RAT AFTER EARLY POSTNATAL DAMAGE BY METHYLAZOXY-METHANOL: DNA, RNA AND PROTEIN DURING RECOVERY

—Total cerebellar DNA, RNA, protein and wet weight were determined for normal cerebellum and for cerebellum damaged early in postnatal development by the neurotoxic action of the drug methylazoxymethanol (MAM) acetate. Injections of a 10 mg/kg dose of the drug were given on postnatal days 1, 2, 3 and 4 as a means of inducing a transient insult to the proliferating stem cell population of the external granular layer. Cerebellar DNA and other quantitative measures were reduced to 50 per cent of normal at day 7. Growth in the MAM‐treated cerebellum was relatively more rapid than in normals so that the eventual deficit in the adult was 80 per cent of normal. We concluded that early postnatal transient injury of the developing cerebellum is followed by a regenerative phase of repair but that quantitatively significant mitosis in the injured cerebellum ceases normally at about post‐natal day 17. The conditions evaluated in this investigation may serve as a model for other types of injury induced by drugs, irradiation, viruses or nutritional factors.

β-Adrenergic-control of cyclic AMP-generating systems in cerebellum: Pharmacological heterogeneity confirmed by destruction of interneurons

Abstract Rats subjected to neonatal x-irradiation selectively lose the vast majority of late-maturing granule, basket, and stellate cells, while the earlier-maturing Purkinje cells remain intact. These animals were used as a model to examine the electrophysiological and neurochemical interaction of neuroleptic compounds with the β-adrenergically linked cyclic AMP-generating systems of cerebellum. Intracisternal injection of 6-hydroxydopamine in x-irradiated animals resulted in an 80% increase in Purkinje cell mean discharge rate, suggesting an inhibitory adrenergic input comparable to that seen in normal animals. Iontophoresis of fluphenazine reversed the inhibitory effects of iontophoretically applied norepinephrine on Purkinje cell discharge rate in both x-irradiated animals and x-irradiated animals treated with 6-hydroxydopamine. The norepinephrine-stimulated formation of cyclic AMP was significantly reduced in cerebellar slices prepared from x-irradiated rats as compared with controls. Furthermore, while fluphenazine (10 μ m ) reduced the norepinephrine-stimulated formation of cyclic AMP by 50 to 60% in cerebellar slices from control rats, the same concentration of fluphenazine reduced norepinephrine-elicited accumulation of cyclic AMP to values which were not significantly different from basal values in slices from x-irradiated rats. These results indicate that at least two populations of cells containing catecholamine-sensitive cyclic AMP-generating systems exist in the cerebellum. One population, including the Purkinje cell, has β-receptors sensitive to the inhibitory effects of the clinically-active neuroleptic, fluphenazine. The other population of cells, destroyed by x-irradiation, has β-receptors which are resistant to the inhibitory effects of this neuroleptic. Moreover, the locus of the neuroleptic inhibition of catecholamine-sensitive cyclic AMP-generating systems in the cerebellum appears to be postsynaptic, since the electrophysiological effects of fluphenazine were qualitatively similar in both x-irradiated rats and x-irradiated rats treated with 6-hydroxydopamine.