Rosemarie Drake-Baumann

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.

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.

Effects of exposure to low-dose pyridostigmine on neuromuscular junctions in vitro

During the Persian Gulf War, pyridostigmine bromide (PB), a reversible inhibitor of acetylcholinesterase, was used as prophylaxis against exposure to nerve gas. Exposure to PB has been suggested as a potential cause of the persistent fatigue reported among Gulf War veterans. The aim of this study was to evaluate the effects of acute and continuous exposure to low doses of PB on the neuromuscular junction. Organotypic spinal cord‐muscle cocultures were used to examine in vitro the effects of PB under controlled conditions. Acute exposure to PB potentiated neuromuscular activity. Continuous exposure to PB produced a progressive decrease in the contractile activity of muscle fibers. Ultrastructural examination by electron microscopy revealed no abnormalities in the neuromuscular junctions after 1 week of exposure. Nerve terminal degeneration and atrophy of the postjunctional folds were evident after 2‐week exposure to low‐dose PB. The effects of PB were reversible following withdrawal. The reversibility of the PB‐induced changes in vitro suggests that such changes are causally unrelated to the fatigue reported by Persian Gulf War veterans years after exposure to PB.

Influence of functional glia on the electrophysiology of Purkinje cells in organotypic cerebellar cultures

Previous studies have shown that exposure of organotypic cerebellar explants to cytosine arabinoside (Sigma) for the first five days in vitro drastically reduced the granule cell population and severely affected glial function. Myelination was absent and astrocytes failed to ensheath Purkinje cells. In the absence of astrocytic ensheathment, Purkinje cell somata became hyperinnervated by Purkinje cell recurrent axon collaterals. Recurrent axon collaterals also projected to Purkinje cell dendritic spines. In later studies, exposure of cerebellar cultures to a different formulation of cytosine arabinoside (Pfanstiehl) also affected granule cells and oligodendrocytes but did not compromise astrocyte function. The different susceptibility of astrocytes to the two preparations of cytosine arabinoside (Sigma and Pfanstiehl) has provided the opportunity to examine the electrophysiological properties of Purkinje cells in the presence and absence of functional glia. Ensheathed Purkinje cells in granuloprival cultures exhibit within two weeks in vitro similar passive membrane properties as Purkinje cells in control cultures. Their input resistance is significantly higher and their spontaneous single-unit discharge is significantly lower than that of unensheathed Purkinje cells. This effect suggests that ensheathed Purkinje cells in cytosine arabinoside (Pfanstiehl)-treated cultures are more responsive to the profuse Purkinje cell recurrent axon collateral inhibitory projection to dendritic spines. These studies also show that the presence of functional glia and/or astrocytic ensheathment can be correlated with the development of complex spike activity by Purkinje cells in vitro. Purkinje cells in cultures treated with cytosine arabinoside (Pfanstiehl), which does not compromise astrocytic ensheathment, display membrane conductances and spike activity similar to mature Purkinje cells in control cultures. By contrast, Purkinje cells in cultures treated with cytosine arabinoside (Sigma), and devoid of astrocytic ensheathment, display mainly simple spike activity reminiscent of the type of activity seen in less mature neurons.

Electrophysiological differences between Purkinje cells in organotypic and granuloprival cerebellar cultures

Organotypic cerebellar cultures derived from newborn mice were exposed to cytosine arabinoside for the first five days in vitro to destroy granule cells and functionally compromise glia. Such granuloprival cultures undergo a circuit reorganization featured by Purkinje cells sprouting recurrent axon collaterals that hyperinnervate other Purkinje cells. Intracellular recordings were used to compare the electrophysiological properties of Purkinje cells in granuloprival cultures to those of Purkinje cells in standard cultures. Purkinje cells in granuloprival cultures have similar membrane potentials to those of Purkinje cells in standard cultures, but have a lower input resistance. A reduced input resistance could affect the effectiveness of inhibitory synaptic input. Intracellular recordings from Purkinje cells of standard cerebellar cultures between 13 and 21 days in vitro exhibit spike activity consisting of a mixture of complex and simple spikes. The complex spikes contain a fast rising action potential followed by a depolarizing potential on which a plateau and several spike-like components are superimposed. This type of activity has been observed in mature Purkinje cells in vivo and in vitro. By contrast, at resting membrane potential Purkinje cells in granuloprival cultures have simple spike activity reminiscent of the type of activity seen in immature Purkinje cells, while at hyperpolarized potentials they generate complex spikes. These observations indicate differences in the expression of intrinsic electrophysiological properties underlying complex spike generation between Purkinje cells of organotypic and granuloprival cerebellar cultures. Our results illustrate the considerable plasticity of Purkinje cells in the presence of altered neuronal circuitry. In the absence of normal excitatory input, their spontaneous activity is regulated by intrinsic membrane properties.

Dystrophic dendrites induced in cultured Purkinje cells by exposure to vinblastine.

Organotypic cerebellar cultures were exposed to the mitotic spindle inhibitor, vinblastine, and examined by light and electron microscopy, and electrophysiologically. Purkinje cells in exposed cultures had stunted, varicose dendrites when observed in preparations reacted with antibody to nonphosphorylated neurofilament protein. Ultrastructurally, microtubules were selectively reduced in Purkinje cell dendrites, which contained disarrayed excess smooth endoplasmic reticulum, but no large accumulations of densely packed neurofilaments. Spontaneous cortical discharges were initially increased in vinblastine treated cultures and then ceased, at which time the cultures became unresponsive to electrical stimulation. The vinblastine-induced changes were attributed to impaired intracellular transport secondary to disruption of Purkinje cell dendritic microtubules.

Neuromuscular responses to pyridostigmine bromide in organotypic spinal cord-muscle culture

Pyridostigmine bromide (PB) promotes and then silences cholinergic muscle activity, and disrupts the junctional regions of muscle fibers and associated nerve terminals, in organotypic mouse spinal cord-muscle cultures continuously treated with low concentrations of the drug for up to 14 days. Spontaneous muscle activity is restored within 1 week of drug removal.