Arnold L. Leiman

Toxic effects of kainic acid on mouse cerebellum in tissue culture

Cultures of mouse cerebellum were exposed for various intervals after explantation to kainic acid, a glutamic acid analog. Purkinje cells and intracerebellar nucleus neurons were destroyed and cortical laminar formation was inhibited by exposure to kainic acid, while granule cells were relatively spared. Prolonged kainate treatment also reduced the granule cell population. The destructive effects of kainic acid were evident upon exposure of Purkinje cells prior to the development of parallel fiber-Purkinje cell synapses, the neurotransmitter for which is believed to be glutamic acid. Glutamate application to intracerebellar nucleus neurons in vitro did not evoke extracellularly recorded excitatory effects, suggesting that these kainate-sensitive neurons do not have significant numbers of glutamate receptors. The combination of these observations suggests that neuronal toxic effects of kainic acid are not exclusively mediated by action on glutamate receptors, but involve other, less specific mechanisms as well.

Cytosine arabinoside effects on developing cerebellum in tissue culture

Cerebellar explants derived from neonatal mice were exposed to cytosine arabinoside, an inhibitor of DNA synthesis. Following such exposure, the cortical regions of the explants contained numerous closely packed large neurons with few intervening elements and without lamination. The surviving large cortical neurons included Golgi cells and Purkinje cells, the latter with persistent dendritic spines in the absence of granule cells. A marked increase in density of subcortical and intracortical neurites was evident in fiber stains. The neurites were identified as Purkinje cell axons and axon collaterals by fiber tracing. Myelin failed to form around the axonal elements. Both regular and phasic spontaneous discharges were recorded electrophysiologically. Trains of cortical stimuli elicited both excitatory and inhibitory responses in the absence of parallel fibers. Antidromic stimulation of Purkinje cell axons evoked inhibition of spontaneous cortical discharges. By contrast, antidromic activation of Purkinje cell axons in control cultures had no effect on spontaneous cortical discharges, or provoked a transient increase in discharge rate. These responses were interpreted as consistent with a cortical remodeling in granuloprival cerebellar cultures in which basket-stellate cell inhibition of Purkinje cells was preempted by Purkinje cell recurrent axon collateral inhibition.

Spontaneous and evoked bioelectric activity in organized cerebellar tissue cultures

Abstract Electrophysiological studies were performed on cultures of neonatal mouse cerebellum which had retained a high degree of anatomical organization after 19–27 days. Both spontaneous neuronal discharges and stimulus-elicited activity were recorded over a temperature range of 25–38 C. Surface cortical stimulation provoked short- and long-latency responses, inhibitory pauses and decremental responsiveness to repetitive stimulation. These effects were attributed to the presence of functional synapses in the cultures.

Morphological correlates of altered neuronal activity in organotypic cerebellar cultures chronically exposed to anti-GABA agents

Organotypic cerebellar cultures derived from newborn mice were chronically exposed to medium containing picrotoxin or bicuculline from explanation until they were recorded from extracellularly or fixed for morphological studies. Cultures exposed to anti-GABA agents for 13-18 days in vitro had decreased spontaneous cortical discharge rates when compared with sister control cultures and prolonged inhibitory responses to cortical stimulation. Electron microscopic examination of exposed cultures after 14-16 days in vitro revealed a hyperinnervation of Purkinje cell somata by inhibitory terminals predominantly of basket cell origin. The sprouted terminals penetrated otherwise intact Purkinje cell astrocytic sheaths. These changes represent a departure from the usual developmental pattern, a departure induced by exposure to anti-GABA agents that increased neuronal activity early in the development of the cerebellar circuitry in vitro. The precise signals that initiated the changes are unknown, but the altered development is most likely in response to increased Purkinje cell activity.

Responses of inferior colliculus neurons to free field auditory stimuli

Abstract The cues for auditory localization depend, in the natural state, on the size and shape of an animals head and pinnae and on the frequency of the acoustic stimulus. This study examined the responses of neurons in the inferior colliculus to sounds presented from various locations in a free field. A group of loudspeakers in an anechoic chamber was arranged in a semicircle around the cats head with a minimum speaker resolution of 20°. The frequency characteristics of the speakers were compensated for by a computer controlled attnuator. The relation of firing rate to auditory direction was quite varied among inferior colliculus neurons. In many cells the form of functions relating number of stimulus elicited spikes to position in the horizontal plane varied with frequency. These observations suggested that neurons of inferior colliculus did not have the property of acoustic space maps. Many cells could define a median directiton as suggested by marked slope changes. The ease with which the median plane is represented has suggested that the control of various acoustically driven movements of the eyes, pinnae and head may be a primary functional output of neurons at this level. From this perspective, the neurons of the inferior colliculus may drive a center-null device which restores the organism to a region of maximum sensitivity, i.e., the median plane.

Development of spontaneous and evoked electrical activity of cerebellum in tissue culture.

Abstract The development of spontaneous and evoked electrical activity of the cerebellar cortex was studied in tissue cultures derived from newborn mice. Occasional spontaneous spikes were observed after 1 day in vitro . Sustained, large-amplitude cortical spikes were initially evident at 8 days in vitro , and by 15 days in vitro irregular, frequently occurring, spontaneous discharges like those of mature cultures had become established. Single cortical cellular spikes were first evoked by parallel fiber stimulation at 8 days in vitro , and barrages of evoked cortical spikes were initially observed at 12 days in vitro . Inhibitory evoked responses also first appeared at 8 days in vitro , and developed subsequently with increasing age of the explants. Sequences of excitation-inhibition-excitation in response to cortical stimulation were initially observed at 12 days in vitro and had assumed a mature form by 15 days after explantation. Evoked summated fiber responses were elicted in deep portions of the cortex prior to their appearance in superficial regions. The development of increasingly complex electrical activity in concert with the structural maturation of synapses in vitro and the sequence of functional maturation in cultures in parallel with that of development in situ suggest that both overall synaptogenesis and the specific sequence of synaptic development of cerebellar cortex are intrinsically programmed, as both are expressed in the cerebellum maintained in total isolation.

Cytosine arabinoside effects in mouse cerebellar cultures in the presence of astrocytes

Organotypic cerebellar cultures derived from neonatal mice were exposed to recent preparations of cytosine arabinoside that destroyed oligodendrocytes and drastically reduced granule cells, but did not reduce the astrocyte population. The cultures were analysed by light and electron microscopy, and by extracellular electrophysiological recording. Purkinje cells survived in greater numbers than in untreated explants and sprouted excess recurrent axon collaterals that formed heterotypical synapses with Purkinje cell dendritic spines. These changes were similar to those found in earlier studies with a cytosine arabinoside preparation that did reduce the astrocyte population, in addition to destroying oligodendrocytes and granule cells. Results with recent cytosine arabinoside preparations that differed from those obtained previously included astrocytic ensheathment of Purkinje cells and apposition of many unattached dendritic spines, encasement of heterotypical synapses by astroglial processes, a loss of Purkinje cell somatic spines, and a lack of somatic hyperinnervation of Purkinje cells by sprouted recurrent axon collateral terminals. All of these differences were attributed to the presence of adequate numbers of competent astrocytes. Heterotypical synapses formed by sprouted recurrent axon collateral terminals and Purkinje cell dendritic spines were functional, as indicated by cortical inhibition in response to antidromic Purkinje cell activation in the absence of somatic hyperinnervation. These results give further definition to the role of astrocytes in cerebellar development and plasticity.

Spontaneous versus driven activity in intracerebellar nuclei: A tissue culture study

Abstract Intracerebellar nuclei in cerebellar explants derived from newborn mice were recognized as focal points of converging Purkinje cell axons. Morphological studies demonstrated characteristic architectonic features and abundant typical large and small intracerebellar nucleus neurons. Spontaneous electrical activity was recorded extracellularly from cortical regions of cerebellar cultures, but no endogenous activity was evident in recording from intracerebellar nuclei. These neurons were pharmacologically isolated from the inhibitory influence of Purkinje cells by application of bicuculline and lidocaine-HCl, without the resultant emergence of endogenous activity. Intracerebellar nucleus neurons in deafferented cerebellar cultures proved incapable of generating spontaneous electrical discharge, suggesting that background excitatory activity in intracerebellar nuclei in situ may be driven by collaterals of cerebellar afferent fibers.

State-dependent choice behavior in the Rhesus monkey.

Abstract Rhesus monkeys given a series of successive brightness reversals while either drugged with pentobarbital or given saline on alternate reversals showed no evidence of interference from one reversal to the next. We suggest that a change of drug state permits independent (dissociated) learning to occur in each state. Retention of an appropriate cue within a state was remarkably good even when an animals immediately preceding experience was training to another cue while in the alternate state. Retention appears state-dependent.

Maturation of electrical activity of cerebral neocortex in tissue culture

The neurophysiological development of cerebral neocortex cultures derived from 2 or 3 day old mice was studied using extracellular microelectrode recording techniques. The explants were examined during an in vitro growth period extending from 1 to 33 days. Spontaneous activity was rarely noted, although drugs such as penicillin and strychnine could provoke prolonged discharges. Electrical stimuli delivered to the dorsal cortical surfaces of the cultures provoked short-duration spike potentials during the initial 1 to 5 days of in vitro growth. These responses were attributed to axonal activity. After this early period, dorsal surface stimulation additionally elicited labile large-amplitude slow-wave activity which was of maximal amplitude in the middle third of the explants, a region often characterized by aggregates of pyramidal cell bodies and basal dendrites. Within the age range of 5 to 33 days of in vitro growth, the properties of the elicited slow waves failed to show any regular, progressive modifications such as increments in complexity of waveforms or prominence of oscillatory activity. These data were interpreted as reflecting the constraints on maximal functional development imposed by the isolation of a portion of an emerging nervous system. Although characteristic intrinsic circuitry may show extensive anatomical development in this state, more elaborate sequential functional development may depend upon the capacity of customary input pathways to provide triggers for additional structural and functional modifications.