TrkB receptor signaling and activity-dependent inhibitory synaptogenesis.

When mouse organotypic cerebellar cultures were exposed to anti-GABA agents that increased neuronal activity early in development, there was a doubling of the ratio of inhibitory axosomatic synapse profiles to Purkinje cell somatic profiles after two weeks in vitro, which correlated with a decrease in spontaneous cortical discharges. When similar cultures were maintained in medium with activity blocking agents, Purkinje cell axosomatic synapses were reduced to approximately half of control values and, after recovery from activity blockade, the cultures discharged hyperactively. By contrast, the full complement of excitatory cortical synapses developed in the absence of neuronal activity. These results support the concept that neuronal activity is necessary for the complete development of inhibitory circuitry. When cerebellar cultures were simultaneously exposed to activity blocking agents and to neurotrophins BDNF or NT-4, both of which bound to the TrkB receptor, the numbers of inhibitory Purkinje cell axosomatic synapses were similar to those of untreated control cultures, and control rates of spontaneous cortical discharges were recorded. The TrkC receptor ligand, NT-3, did not promote inhibitory synapse development in the absence of neuronal activity, and such cultures exhibited hyperactive cortical discharges. These results are consistent with a role for TrkB receptor ligands in activity-dependent inhibitory synaptogenesis. Subsequent exposure of cerebellar cultures to antibody to the extracellular domain of TrkB induced an increased development of Purkinje cell axosomatic synapses, while similar antibody activation of TrkC had no effect on inhibitory synaptogenesis. The promotion of inhibitory synapse development by specific antibody activation of TrkB supports the concept that signaling for activity-dependent inhibitory synaptogenesis is via the TrkB receptor.

Signaling for activity-dependent inhibitory synaptogenesis via the TrkB receptor.

Organotypic cerebellar cultures derived from newborn mice were incubated for 2 weeks in vitro with antibodies that recognized the extracellular domains of the TrkB or TrkC receptor and were then examined by electron microscopy. Antibody activation of TrkB receptors resulted in development of an increased number of inhibitory Purkinje cell axosomatic synapses, while control numbers of axosomatic synapses were present in explants exposed to antibody to TrkC. Similar results had been obtained in previous studies when TrkB- and TrkC-specific ligands were applied to activity-blocked cerebellar cultures. These combined results are consistent with the concept that signaling for activity-dependent inhibitory synaptogenesis is via the TrkB receptor.

Interactions between cerebellar Purkinje cells and their associated astrocytes.

Some neurons, including cerebellar Purkinje cells, are completely ensheathed by astrocytes. When granule cell neurons and functional glia were eliminated from newborn mouse cerebellar cultures by initial exposure to a DNA synthesis inhibitor, Purkinje cells lacked glial sheaths and there was a tremendous sprouting of Purkinje cell recurrent axon collaterals, terminals of which hyperinnervated Purkinje cell somata, including persistent somatic spines, and formed heterotypical synapses with Purkinje cell dendritic spines, sites usually occupied by parallel fiber (granule cell axon) terminals. Purkinje cells in such preparations failed to develop complex spikes when recorded from intracellularly, and their membrane input resistances were low, making them less sensitive to inhibitory input. If granule cells and oligodendrocytes were eliminated, but astrocytes were not compromised, sprouting of recurrent axon collaterals occurred and their terminals projected to Purkinje cell dendritic spines, but the Purkinje cells had astrocytic sheaths, their somata were not hyperinnervated, the somatic spines had disappeared, complex spike discharges predominated, and membrane input resistance was like that of Purkinje cells in untreated control cultures. When cerebellar cultures without granule cells and glia were transplanted with granule cells and/or glia from another source, a series of changes occurred that included stripping of excess Purkinje cell axosomatic synapses by astrocytic processes, reduction of heterotypical axospinous synapses in the presence of astrocytes, disappearance of Purkinje cell somatic spines with astrocytic ensheathment, and proliferation of Purkinje cell dendritic spines after the introduction of astrocytes. Dendritic spine proliferation was followed by formation of homotypical axospinous synapses when granule cells were present or persistence as unattached spines in the absence of granule cells. The results of these studies indicate that astrocytes regulate the numbers of Purkinje cell axosomatic and axospinous synapses, induce Purkinje cell dendritic spine proliferation, and promote the structural and functional maturation of Purkinje cells.

TrkB receptor ligands promote activity-dependent inhibitory synaptogenesis.

Organotypic cerebellar cultures derived from newborn mice were simultaneously exposed to activity-blocking agents and neurotrophins for 2 weeks. Activity-blocked explants treated with the TrkB receptor ligands BDNF and neurotrophin-4 (NT-4) developed a full complement of Purkinje cell inhibitory axosomatic synapses, as defined ultrastructurally, and displayed control spontaneous cortical discharge rates after recovery from activity blockade. Otherwise untreated activity-blocked cultures and activity-blocked cultures exposed to the TrkC receptor ligand NT-3 had reduced inhibitory synapse development and persistent cortical hyperactivity after recovery. The added TrkB receptor ligands did not induce axonal sprouting to account for increased inhibitory synaptogenesis. Addition of neurotrophins to untreated cerebellar cultures did not increase the complement of Purkinje cell axosomatic synapses. Exposure of cerebellar cultures to a combination of antibodies to BDNF and NT-4 resulted in reduced inhibitory synapse formation, similar to the effects of activity blockade, indicating the necessity for endogenous neurotrophins for development of the full complement of inhibitory synapses in the presence of neuronal activity. Application of antibodies to BDNF and NT-4 to cerebellar explants exposed to picrotoxin to increase neuronal activity prevented the hyperinnervation of Purkinje cell somata by inhibitory terminals characteristic of cultures exposed to picrotoxin alone. These results are consistent with the concept that TrkB receptor ligands promote inhibitory synaptogenesis. The ability of neurotrophins to substitute for neuronal activity in encouraging development of inhibitory synapses may have therapeutic implications.

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.

BDNF and NT-4, but not NT-3, promote development of inhibitory synapses in the absence of neuronal activity.

Development of the full complement of inhibitory synapses in cerebellar cultures requires the presence of neuronal activity. The neurotrophins, BDNF, NT-3 and NT-4, were applied to cerebellar explants during activity blockade. Control numbers of inhibitory Purkinje cell axosomatic synapses developed in the presence of the TrkB receptor ligands, BDNF and NT-4, but not the TrkC receptor ligand, NT-3. The results suggest that BDNF and NT-4 have a role in the promotion of activity-dependent inhibitory synaptogenesis.

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.

The extracellular matrix molecule, laminin, induces purkinje cell dendritic spine proliferation in granule cell depleted cerebellar cultures.

Granule cells and glia were eliminated or reduced in organotypic cerebellar cultures exposed to cytosine arabinoside. Transplantation of such granuloprival cultures with glia or exposure to astrocyte conditioned medium in the absence of parallel fibers (granule cell axons) resulted in proliferation of Purkinje cell dendritic spines. The aim of the present study was to identify specific astrocyte secreted factors that induced dendritic spine proliferation. Known astrocyte secreted, neurite promoting factors were screened by application to granuloprival cultures and assayed for dendritic spine proliferation by electron microscopy. An extracellular matrix molecule, laminin, evoked sprouting of Purkinje cell dendritic spines. Dendritic spine proliferation was not associated with known neurite promoting parts of the laminin molecule, as two laminin-derived peptides with identified neurite promoting domains did not induce dendritic spine sprouting. The purpose of laminin-induced dendritic spine proliferation may be to elaborate postsynaptic membrane, thereby increasing the target area for arriving axon terminals during development or regeneration, both of which have been associated with the presence of laminin secreting astrocytes.

Serial changes in granuloprival cerebellar cultures after transplantation with granule cells and glia: a timed ultrastructural study.

Granuloprival cerebellar cultures derived from neonatal mice were transplanted at nine days in vitro with granule cells and glia, and the changes induced in the host explants were examined daily with the electron microscope from one to nine days post-transplantation. Granule cells and astrocytes had migrated into the host cultures within 24 h, and astrocytic processes began to ensheath Purkinje cells and to interpose themselves between axon terminals and Purkinje cell somata, reducing the number of axosomatic synapses. Occasional degenerating Purkinje cells were present. At two days post-transplantation, synapse formation between parallel fibre terminals and Purkinje cell dendritic spines was initially evident, and Purkinje cells began to proliferate dendritic spines near astrocytic processes. Degenerating Purkinje cells were more frequently encountered. Myelin was first observed in host cultures at three days after transplantation, and astrocytes continued to ensheath Purkinje cells and reduce the population of axosomatic synapses, a process that began to stabilize at four days post-transplantation. At this time astrocytic ensheathment had extended to Purkinje cell dendrites and dendritic spine synapses. Proliferation of Purkinje cell dendritic spines accelerated, and occasional synapses with presumptive parallel fibre terminals were present among clusters of proliferated spines. At five days after transplantation, contours of Purkinje cells were rounded, and there was a decrease of somatic spines and of synapses with somatic spines. Purkinje cells were fully ensheathed by astrocytic processes by six days post-transplantation and had assumed a mature appearance. Homotypical parallel fibre-Purkinje cell dendritic spine synapses were predominant in more developed areas of cortical neuropil as heterotypical recurrent axon collateral-Purkinje cell dendritic spine synapses were reduced. Increasing synapse formation was evident among clusters of proliferated spines, which continued at seven days post-transplantation, as the spine clusters became less frequent. At eight days after transplantation, space between Purkinje cells had increased and the cortical neuropil resembled that of comparably aged control cultures. Occasional degenerating Purkinje cells were still evident at nine days post-transplantation, at which time residual clusters of proliferated unattached dendritic spines were scarce. The sequence of changes after transplantation was consistent with the specific roles of the transplanted elements. Astrocytes were involved with the regulation of synapse density, including reduction of some heterotypical synapses, and induced proliferation of Purkinje cell dendritic spines. Granule cell axons synapsed with Purkinje cell dendritic spines, further reducing heterotypical synapses and restoring cortical circuitry to a near control state. The loss of heterotypical synapses was associated with programmed cell death of excess Purkinje cells, reducing the Purkinje cell population to control levels.

Recovery and repair issues after stroke from the scientific perspective.

Animal and human studies have shown that motor and somatosensory cortex are capable of reorganization throughout adult life. Cortical reorganization has been associated with functional recovery in experimental and human stroke. Retraining after experimental cortical infarction has been shown to extend cortical representation of the infarcted area and improve skilled motor performance.

Cerebellar culture models of dendritic spine proliferation after transplantation of glia.

Studies of Purkinje cell dendritic spine proliferation after transplantation of cytosine arabinoside (Ara C) treated organotypic cerebellar cultures with glia and granule cells, either separately and in combination, were reviewed. Exposure of cerebellar explants to Ara C for the first 5 days in vitro results in the destruction of granule cells, the only excitatory cortical neurons, and oligodendroglia, and functionally compromises surviving astrocytes so that they do not appose neuronal membranes. In the absence of granule cells, there is a sprouting of Purkinje cell recurrent axon collaterals, the terminals of which project to and form heterotypical synapses with Purkinje cell dendritic spines, which are usually occupied by terminals of granule cell axons (parallel fibers). After this reorganization has been achieved, the explants can be transplanted with the missing elements to induce a second round of reorganization, with approximate restoration of the usual interneuronal relationships. Addition of both granule cells and glia resulted in a proliferation of clusters of Purkinje cell dendritic spines, which formed synapses with axon terminals of transplanted granule cells, and as synapse formation progressed, the spine clusters became reduced. Transplantation of Ara C-treated cultures with glia alone resulted in a proliferation of clusters of Purkinje cell dendritic spines, but in the absence of granule cells the spines remained unattached, and the clusters persisted throughout the period of observation. Purkinje cell dendritic spine proliferation was induced by exposure of Ara C- treated cultures to astrocyte-conditioned medium. When Ara C-treated cerebellar cultures were transplanted with granule cells in the absence of functional glia, parallel fiber- Purkinje cell dendritic spine synapses formed, but no clusters of Purkinje cell dendritic spines were observed. These findings suggest that Purkinje cell dendritic spine proliferation is induced by an astrocyte-secreted factor, resulting in an expansion of postsynaptic sites available for synaptogenesis.

Neural plasticity in cerebellar cultures.

Cerebellar granule cells and oligodendrocytes are destroyed and astrocytes are functionally compromised by exposure of organotypic cerebellar cultures derived from newborn mice to cytosine arabinoside for the first 5 days in vitro. Consequently, myelin does not form and Purkinje cells survive in increased numbers, but without astrocytic ensheathment. In the absence of glial sheaths, Purkinje cells have altered membrane properties and reduced input resistance. Their inhibitory recurrent axon collaterals sprout enormously and hyperinnervate the unensheathed somata of other Purkinje cells and form heterotypical synapses with Purkinje cell dendritic spines normally occupied by homotypical excitatory parallel fiber (granule cell axon) terminals. This reorganization of the cortical circuitry, in which recurrent axon collaterals are the dominant inhibitory elements, allows retention of some inhibition in the absence of parallel fiber excitation of the inhibitory interneurons. In the absence of neuronal activity, the full complement of inhibitory synapses is not developed and the cultures exhibit sustained cortical hyperactivity after recovery from the blockade. If granule cells and glia are replaced, a second round of reorganization ensues, in the direction of restoration of the normal cortical circuitry. The cultures are myelinated and the number of recurrent axon collaterals is reduced. Astrocytes ensheath Purkinje cell somata and strip excess axosomatic synapses, as well as eliminate some of the heterotypical synapses in the cortical neuropil. Parallel fibers synapse with already present Purkinje cell dendritic spines and with newly proliferated spines, the latter induced by an astrocyte secreted factor. As homotypical synapses develop and heterotypical synapses decline, Purkinje cells undergo apoptosis and their population is reduced to control levels. With the restoration of parallel fiber excitation, recurrent axon collaterals are no longer the dominant cortical inhibitory elements. If neuronal activity is blocked as the granule cells and glia are replaced, there is incomplete formation of inhibitory synapses, and cortical discharges are hyperactive after recovery from activity blockade.

Activity-dependent changes in "transplanted" cerebellar cultures.

Organotypic cerebellar cultures were used to assess the effects of increasing or blocking neuronal activity on circuit reconstruction in an in vitro transplantation model. Granule cells and oligodendrocytes were destroyed and astrocytes were functionally compromised by exposing newborn mouse-derived cerebellar explants to cytosine arabinoside for the first 5 days in vitro. Such cultures were "transplanted" at 9 days in vitro with granule cells and glia and maintained in standard nutrient medium; in medium with the GABA antagonist, picrotoxin, to increase neuronal activity; or with tetrodotoxin and elevated levels of magnesium to block neuronal activity. Transplanted cultures exposed to picrotoxin were not significantly different from control transplanted cultures. Transplanted cultures deprived of neuronal activity had reduced inhibitory synaptogenesis, greater persistence of heterotypical axospinous synapses, and hyperactive cortical spontaneous discharges after recovery from the blockade. Transplantation-induced changes that were not affected included myelination, reduction of sprouted Purkinje recurrent axon collaterals, astrocytic ensheathment of Purkinje cells, reduction of excess Purkinje cell axosomatic synapses, and formation of excitatory parallel fiber-Purkinje cell dendritic spine synapses. The results were consistent with previous studies indicating the necessity of neuronal activity for the full development of inhibitory circuitry, and suggested that neuronal activity is also necessary for the reconstruction of inhibitory circuitry after transplantation.

Circuit reorganization in Ara-C-treated cerebellar cultures chronically exposed to picrotoxin.

Organotypic cerebellar cultures derived from neonatal mice were exposed to the DNA synthesis inhibitor, cytosine arabinoside, or to cytosine arabinoside plus picrotoxin, an anti-GABA agent that increased neuronal activity, for the first five days in vitro. The group treated with cytosine arabinoside alone was subsequently maintained in standard nutrient medium, while the group exposed to both cytosine arabinoside and picrotoxin was continuously maintained in medium with incorporated picrotoxin. Granule cells were destroyed and astrocytes were functionally compromised in both culture groups, and both groups exhibited Purkinje cell axon collateral sprouting, with projection of sprouted inhibitory terminals to unensheathed Purkinje cell somata and to Purkinje cell dendritic spines in equal numbers. Spontaneous cortical discharge rates were the same in both groups, and antidromic stimulation of Purkinje cell axons induced inhibition of cortical activity. These results differed from those of a previous study in which chronic exposure of otherwise untreated cerebellar cultures to anti-GABA agents increased the complement of inhibitory terminals on glially ensheathed Purkinje cell somata and resulted in a reduction of spontaneous cortical discharge rates. These differences were attributed to the failure of picrotoxin (1) to alter the plastic changes consequent to exposure to cytosine arabinoside, in which Purkinje cells had excess inhibitory projections, and (2) to extend inhibitory synaptogenesis in a system in which inhibitory synapse development was already enhanced.

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.

Circuit reorganization in granuloprival cerebellar cultures in the absence of neuronal activity.

Neonatal mouse cerebellar cultures exposed to cytosine arabinoside for the first 5 days in vitro to destroy granule cells and compromise glia undergo a circuit reorganization featured by profuse sprouting of Purkinje cell recurrent axon collaterals, which hyperinnervate the somata of other Purkinje cells and project to Purkinje cell dendritic spines. Such granuloprival cultures were exposed continuously from explanation to tetrodotoxin and elevated levels of magnesium to block neuronal activity. A similar circuit reorganization occurred, except that there was a reduction in the number of axospinous synapses and Purkinje cell axosomatic synapses, which in this case were all inhibitory. Functionally, after recovery from the blockade, granuloprival cultures developed sustained cortical hyperactivity, which was consistent with the reduction of inhibitory synapses. While the absence of neuronal activity did not prevent reorganizational changes following granule cell loss, the full development of the inhibitory circuitry was not attained. These results further support the concept that spontaneous neuronal activity is necessary for the complete development of inhibitory synapses.

Molecular compartmentation expressed in cerebellar cultures in the absence of neuronal activity and neuron-glia interactions.

The purpose of the study was to determine if zebrin compartmentation developed in permanently isolated cerebellar cultures, in the presence of agents that block neuronal activity and in the absence of myelination and astrocytic ensheathment of Purkinje cells. Parasagittally oriented organotypic cultures derived from newborn mice and carefully undercut at explantation to exclude extracerebellar afferents were subjected to three conditions: 1) Some were maintained in standard nutrient medium; 2) some were chronically exposed to tetrodotoxin and elevated levels of magnesium to block neuronal activity; and 3) some were exposed to cytosine arabinoside for the first 5 days in vitro (DIV) to destroy granule cells and oligodendrocytes and functionally compromise astrocytes, so that the astrocytic survivors did not ensheath Purkinje cells. Cultures fixed as whole-mount preparations were reacted with antibody to zebrin II. Cultures that were cryostat sectioned were dually reacted with antibody to zebrin II and calbindin. Groups of zebrin+ and zebrin- Purkinje cells were evident after 14 DIV in all of the experimental conditions, indicating that zebrin compartmentation developed 1) in isolated cerebellar explants, 2) in the absence of neuronal activity, and 3) in the absence of neuron-glia interactions such as myelination and glial ensheathment of Purkinje cell somata and dendrites. These results are consistent with the concept that expression of the zebrin+ and zebrin- phenotypes is an intrinsic property of Purkinje cells. The fact that zebrin expression seems to depend on an intrinsic program of differentiation in Purkinje cells suggests some role for zebrin compartmentation in cerebellar function.