Clustering of delta glutamate receptors is regulated by the actin cytoskeleton in the dendritic spines of cultured rat Purkinje cells

The interaction of neurotransmitter receptors with the cytoskeleton is an important mechanism for the targeting of receptors to the postsynaptic membrane. Using cytoskeleton-perturbing agents, it was demonstrated that delta glutamate receptors, predominantly expressed on the dendritic spines of cerebellar Purkinje cells, are anchored to the actin cytoskeleton. The number of delta glutamate receptor-immunoreactive clusters was dramatically decreased following treatment of the Purkinje cells with the actin-disrupting agents, cytochalasin D or latrunculin A, without any significant effect on the number of presynaptic contacts of the granule cell axons. The clusters disrupted by latrunculin A were re-established 24 h after removal of the drug. These results suggest that morphological changes in the actin cytoskeleton regulate the delta glutamate receptor clustering on the dendritic spines, and may affect synaptic efficacy and plasticity.     

Synaptic amplification by active membrane in dendritic spines

The suspected functional role of dendritic spines as loci of neuronal plasticity (possibly memory and learning) is greatly enriched when active membrane properties are assumed at the spine head. Computations with reasonable electrical and structural parameter values (corresponding to an optimal range for spine stem resistance) show that an active spine head membrane can provide very significant synaptic amplification and also strongly non-linear properties that could modulate the integration of input from many afferent sources.     

Cellular and subcellular distribution of spinophilin, a PP1 regulatory protein that bundles F-actin in dendritic spines

Spinophilin is an actin binding protein that positions protein phosphatase 1 next to its substrates in dendritic spines. It contains a single PDZ domain and has the biochemical characteristics of a cytoskeletal scaffolding protein. Previous studies suggest that spinophilin is present in most spines, but the concentration of spinophilin varies from brain region to region in a manner that does not simply reflect differences in spine density. Here, we show that spinophilin is enriched in the great majority of dendritic spines in cerebral cortex, caudatoputamen, hippocampal formation, and cerebellum, irrespective of regional differences in spinophilin concentration. In addition, spinophilin is present postsynaptic to asymmetrical contacts on interneuronal dendritic shafts. We further show that, in hippocampus and ventral pallidum, spinophilin is occasionally present in dendritic shafts adjacent to gamma-aminobutyric acid-containing contacts. Thus, the functional role of spinophilin may not be exclusively restricted to excitatory synapses and may be significant at a small fraction of inhibitory contacts. These data also suggest that the concentration of spinophilin per spine is variable and is likely regulated by local physiological factors and/or regional influences.     

Loss of tumor necrosis factor (TNF)-receptor 1 and TNF-receptor 2 partially replicate effects of TNF deficiency on dendritic spines of granule cells in mouse dentate gyrus

The cytokine tumor necrosis factor (TNF) is involved in the regulation of physiological and pathophysiological processes in the central nervous system. In previous work, we showed that mice lacking constitutive levels of TNF exhibit a reduction in spine density and changes in spine head size distribution of dentate granule cells. Here, we investigated which TNF-receptor pathway is responsible for this phenotype and analyzed granule cell spine morphology in TNF-R1-, TNF-R2-, and TNF-R1/R2-deficient mice. Single granule cells were filled with Alexa568 in fixed hippocampal brain slices and immunostained for the actin-modulating protein synaptopodin (SP), a marker for strong and stable spines. An investigator blind to genotype investigated dendritic spines using deconvolved confocal image stacks. Similar to TNF-deficient mice, TNF-R1 and TNF-R2 mutants showed a decrease in the size of small spines (SP−negative) with TNF-R1/R2-KO mice exhibiting an additive effect. TNF-R1 mutants also showed an increase in the size of large spines (SP−positive), mirroring the situation in TNF-deficient mice. Unlike the TNF-deficient mouse, none of the TNF-R mutants exhibited a reduction in their granule cell spine densities. Since TNF tunes the excitability of networks, lack of constitutive TNF reduces network excitation. This may explain why we observed alterations in spine head size distributions in TNF- and TNF-R-deficient granule cells. The changes in spine density observed in the TNF-deficient mouse could not be linked to canonical TNF-R-signaling. Instead, noncanonical pathways or unknown developmental functions of TNF may cause this phenomenon.     

Plasticity of dendritic spines: Molecular function and dysfunction in neurodevelopmental disorders

Dendritic spines are tiny postsynaptic protrusions from a dendrite that receive most of the excitatory synaptic input in the brain. Functional and structural changes in dendritic spines are critical for synaptic plasticity, a cellular model of learning and memory. Conversely, altered spine morphology and plasticity are common hallmarks of human neurodevelopmental disorders, such as intellectual disability and autism. The advances in molecular and optical techniques have allowed for exploration of dynamic changes in structure and signal transduction at single‐spine resolution, providing significant insights into the molecular regulation underlying spine structural plasticity. Here, I review recent findings on: how synaptic stimulation leads to diverse forms of spine structural plasticity; how the associated biochemical signals are initiated and transmitted into neuronal compartments; and how disruption of single genes associated with neurodevelopmental disorders can lead to abnormal spine structure in human and mouse brains. In particular, I discuss the functions of the Ras superfamily of small GTPases in spatiotemporal regulation of the actin cytoskeleton and protein synthesis in dendritic spines. Multiple lines of evidence implicate disrupted Ras signaling pathways in the spine structural abnormalities observed in neurodevelopmental disorders. Both deficient and excessive Ras activities lead to disrupted spine structure and deficits in learning and memory. Dysregulation of spine Ras signaling, therefore, may play a key role in the pathogenesis of multiple neurodevelopmental disorders with distinct etiologies.     

Olfactory learning-induced morphological modifications in single dendritic spines of young rats

Learning-related morphological modifications in single dendritic spines were studied quantitatively in the brains of young Sprague-Dawley rats. We have previously shown that olfactory discrimination rule-learning results in transient physiological and morphological modifications in piriform cortex pyramidal neurons. In particular, spine density along the apical dendrites of neurons from trained rats is increased after learning. The aim of the present study was to identify and describe olfactory learning-induced modifications in the morphology of single spines along apical dendrites of the same type of neurons. By using laser-scanning confocal microscopy, we show that 3 days after training completion spines on neurons from olfactory discrimination trained rats are shorter as compared to spines on neurons from control rats. Further analysis revealed that spine shortening attributed to olfactory discrimination learning derives from shortening of spine head and not from shortening of spine neck. In addition, detailed analysis of spine head volume suggests that spines with large heads are absent after learning. As spine head size may be related to the efficacy of the synapse it bears, we suggest that modifications in spine head dimensions following olfactory rule-learning enhance the cortical network ability to enter into a 'learning mode', in which memories of new odours can be acquired rapidly and efficiently.     

Morphological development of dendritic spines on rat cerebellar Purkinje cells

The posterior cerebellum is strongly involved in motor coordination and its maturation parallels the development of motor control. Climbing and mossy fibers from the spinal cord and inferior olivary complex, respectively, provide excitatory afferents to cerebellar Purkinje neurons. From post-natal day 19 climbing fibers form synapses with thorn-like spines located on the lower primary and secondary dendrites of Purkinje cells. By contrast, mossy fibers transmit synaptic information to Purkinje cells trans-synaptically through granule cells. This communication occurs via excitatory synapses between the parallel fibers of granule cells and spines on the upper dendritic branchlets of Purkinje neurons that are first evident at post-natal day 21. Dendritic spines influence the transmission of synaptic information through plastic changes in their distribution, density and geometric shape, which may be related to cerebellar maturation. Thus, spine density and shape was studied in the upper dendritic branchlets of rat Purkinje cells, at post-natal days 21, 30 and 90. At 90 days the number of thin, mushroom and thorn-like spines was greater than at 21 and 30 days, while the filopodia, stubby and wide spines diminished. Thin and mushroom spines are associated with increased synaptic strength, suggesting more efficient transmission of synaptic impulses than stubby or wide spines. Hence, the changes found suggest that the development of motor control may be closely linked to the distinct developmental patterns of dendritic spines on Purkinje cells, which has important implications for future studies of cerebellar dysfunctions.     

Alterations in Purkinje cell spines of calbindin D-28 k and parvalbumin knock-out mice

The second messenger Ca2+ is known to act in a broad spectrum of fundamental cell processes, including modifications of cell shape and motility, through the intermediary of intracellular calcium-binding proteins. The possible impact of the lack of the intracellular soluble Ca2+-binding proteins parvalbumin (PV) and calbindin D-28 k (CB) was tested on spine morphology and topology in Purkinje cell dendrites of genetically modified mice. Three different genotypes were studied, i.e. PV or CB single knock-out (PV-/-, CB-/-) and PV and CB double knock-out mice (PV-/-CB-/-). Purkinje cells were microinjected with Lucifer Yellow and terminal dendrites scanned at high resolution with a confocal laser microscope followed by three-dimensional (3-D) reconstruction. The absence of PV had no significant effect on spine morphology, whereas the absence of CB resulted in a slight increase of various spine parameters, most notably spine length. In double knock-out mice, the absence of both PV and CB entailed a doubling of spine length, an increase in spine volume and spine surface, a higher spine density along the dendrites, as well as a more clustered spine distribution. In all three genotypes, a reduction in the number of stubby spines was observed compared with wild-type animals. These results suggest a morphological compensation for the lack of the soluble calcium buffers in the cytoplasm of Purkinje cell dendritic spines. The increase in various spine parameters, particularly volume, may counteract the lack of the calcium buffers, such as to adjust Ca2+-transients at the transitional zone between spines and dendrites.     

Regulation of afferent connectivity in the adult spinal cord by nerve growth factor

During development, nerve growth factor (NGF) regulates the density and character of peripheral target innervation (Barde, Neuron , 2 , 1525–1534, 1989; Ritter et al., Soc. Neurosci. Abstr. , 17 , 546.2, 1991); its role in adult animals is less well defined. Here we have asked if the availability of growth factors such as NGF in peripheral tissues can influence the pattern of primary afferent connections in the CNS. Using osmotic minipumps, we raised the levels of NGF in rat skeletal muscle in vivo , a tissue where the levels of this factor are normally very low (Korsching and Thoenen, Proc. Natl. Acad. Sci. USA , 80 , 3513–3516, 1983; Shelton and Reichardt, Proc. Natl. Acad. Sci. USA , 81 , 7951–7955, 1984; Goedert et al., Mol. Brain Res. , 1 , 85–92, 1986). After 2 weeks of treatment we asked if the sensory neurons innervating this tissue showed an altered strength and distribution of connections with dorsal horn neurons. The contralateral (vehicle-treated) muscle, and totally untreated animals, served as controls. In normal and vehicle-treated animals, electrical stimulation of muscle afferents excited relatively few neurons in the dorsal horn, and these generally showed only weak responses. In contrast, on the NGF-treated side many more dorsal horn neurons in the lumbar enlargement of the spinal cord were excited by muscle afferents. The increased responsiveness could not be explained by a generalized increase in dorsal horn excitability, since spontaneous activity was not enhanced, nor by a change in A-fibre-mediated inhibitions from the treated afferents. Thus, these afferents appeared to establish new synaptic connections or strengthened previously weak ones as a result of increased neurotrophic factor availability. The data suggest that, in the adult rat, the levels of growth factors in peripheral targets may be used to regulate an appropriate degree of afferent connectivity within the central nervous system.     

Ultrastructural plasticity of the dentate gyrus granule cells following recurrent limbic seizures: I. Increase in somatic spines

Various paradigms have been used to assess the capacity of the adult brain to undergo activity-dependent morphological plasticity. In this report we have employed recurrent limbic seizures as a means of studying the effects of this form of enhanced neuronal activity on cellular morphology and, in particular, on the incidence of somatic spines on the dentate gyrus granule cells. Seizure activity was induced by the placement of focal, unilateral electrolytic lesions in the dentate gyrus hilus of adult rats. At various intervals postlesion, rats with behaviorally verified seizures were sacrificed, and the hippocampi contralateral, to the lesions were removed and prepared for electron microscopy. Quantitative analysis showed that as early as 5 hours postlesion there was a dramatic increase in the density and morphological complexity of spines on the perikarya of the granule cells in rats that received seizure-producing hilus lesions when compared to granule cells from control rats. Many of the somatic spines received asymmetric synapses. The increase in somatic spines was dependent on seizure activity and persisted for at least 1 month following a single recurrent seizure episode. CA1 pyramidal neurons, which exhibit changes in gene expression in response to hilus lesion-induced seizures but do not normally possess somatic spines, did not exhibit an activity-dependent elaboration of somatic spines. Thus, the seizure-induced elaboration of somatic spines represents an amplification of an existing feature of the granule cells and not an effect occurring throughout hippocampus. These data provide evidence for very rapid and long-lasting structural plasticity in response to brief episodes of seizure activity in the adult brain. © 1994 Wiley-Liss, Inc.     

The diffusional properties of dendrites depend on the density of dendritic spines

We combined computational modeling and experimental measurements to determine the influence of dendritic structure on the diffusion of intracellular chemical signals in mouse cerebellar Purkinje cells and hippocamal CA1 pyramidal cells. Modeling predicts that molecular trapping by dendritic spines causes diffusion along spiny dendrites to be anomalous and that the value of the anomalous exponent (d(w) ) is proportional to spine density in both cell types. To test these predictions we combined the local photorelease of an inert dye, rhodamine dextran, with two-photon fluorescence imaging to track diffusion along dendrites. Our results show that anomalous diffusion is present in spiny dendrites of both cell types. Further, the anomalous exponent is linearly related to the density of spines in pyramidal cells and d(w) in Purkinje cells is consistent with such a relationship. We conclude that anomalous diffusion occurs in the dendrites of multiple types of neurons. Because spine density is dynamic and depends on neuronal activity, the degree of anomalous diffusion induced by spines can dynamically regulate the movement of molecules along dendrites.     

Developmental dynamics of Purkinje cells and dendritic spines in rat cerebellar cortex

Quantitative morphological changes of the developing Purkinje cells were studied from 6 to 90 postnatal (PN) days in the IVth lobule of vermis in the cerebellum of rats. The soma size (mean diameter) of Purkinje cells increased rapidly between 6 PN (on average 10 μm) and 18 PN (about 17 μm) days; it did not change between 18 and 25 PN days, but increased moderately again between 25 and 48 PN days (22–23 μm) and stabilized on the same value. In contrast, the number of Purkinje cells/100 μm (the “linear density”) decreased rapidly from 6 to 18 PN days. The molecular layer area belonging to 1 Purkinje cell increased rapidly from 6 to 25 PN days (from about 370 to 6,200 μm²) and less rapidly between PN days 30 to 48 (up to 9,300 μm²), followed by a moderate decrease at PN day 90 (about 6,600 μm²). The volume belonging to 1 Purkinje cell dendritic arbor was about 5,500 μm³ at PN day 6,93,000 μm³ at PN day 25, and 100,000 μm³ at PN day 90. The numerical density of dendritic spines in the molecular layer showed a biphasic curve: a rapid increase from PN days 6 to 21 followed by a significant but short decrease at PN day 25, moderate rise from PN days 25 to 48, and a subsequent decline between PN days 48 and 90. The number of spines belonging to 1 Purkinje cell showed two developmental “peaks”: the first peak at 21 PN days was moderate (5.6 × 10⁴ spines/Purkinje cell) while the second maximum at 48 PN days was more significant (1.2 × 10⁵ spines/Purkinje cell), which then declined to 6.3 × 10⁴ spines/Purkinje cell at PN day 90. It is suggested that the temporary overproduction and the following decline in the number of Purkinje dendritic spines during the development of the cerebellar cortex may be the morphological indicator of the dynamics of synaptogenetic and of synaptic stabilization processes. © 1994 Wiley-Liss, Inc.     

Neuroprotective actions of peripherally administered insulin-like growth factor I in the injured olivo-cerebellar pathway.

Exogenous administration of insulin-like growth factor I (IGF-I) restores motor function in rats with neurotoxin-induced cerebellar deafferentation. We first determined that endogenous IGFs are directly involved in the recovery process because infusion of an IGF-I receptor antagonist into the lateral ventricle blocks gradual recovery of limb coordination that spontaneously occurs after partial deafferentation of the olivo-cerebellar circuitry. We then analysed mechanisms whereby exogenous IGF-I restores motor function in rats with complete damage of the olivo-cerebellar pathway. Treatment with IGF-I normalized several markers of cell function in the cerebellum, including calbindin, glutamate receptor 1 (GluR1), gamma-aminobutyric acid (GABA) and glutamate, which are all depressed after 3-acetylpyridine (3AP)-induced deafferentation. IGF-I also promoted functional reinnervation of the cerebellar cortex by inferior olive (IO) axons. In the IO, increased expression of bax in neurons and bcl-X in astrocytes after 3AP was significantly reduced by IGF-I treatment. On the contrary, IGF-I prevented the decrease in poly-sialic-acid neural cell adhesion molecule (PSA-NCAM) and GAP-43 expression induced by 3AP in IO cells. IGF-I also significantly increased the number of neurons expressing bcl-2 in brainstem areas surrounding the IO. Altogether, these results indicate that subcutaneous IGF-I therapy promotes functional recovery of the olivo-cerebellar pathway by acting at two sites within this circuitry: (i) by modulating death- and plasticity-related proteins in IO neurons; and (ii) by impinging on homeostatic mechanisms leading to normalization of cell function in the cerebellum. These results provide insight into the neuroprotective actions of IGF-I and may be of practical consequence in the design of new therapeutic approaches for neurodegenerative diseases.     

Prolonged ampakine exposure prunes dendritic spines and increases presynaptic release probability for enhanced long‐term potentiation in the hippocampus

CX 546, an allosteric positive modulator of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid‐type ionotropic glutamate receptors (AMPARs), belongs to a drug class called ampakines. These compounds have been shown to enhance long‐term potentiation (LTP), a cellular model of learning and memory, and improve animal learning task performance, and have augmented cognition in neurodegenerative patients. However, the chronic effect of CX546 on synaptic structures has not been examined. The structure and integrity of dendritic spines are thought to play a role in learning and memory, and their abnormalities have been implicated in cognitive disorders. In addition, their structural plasticity has been shown to be important for cognitive function, such that dendritic spine remodeling has been proposed as the morphological correlate for LTP. Here, we tested the effect of CX546 on dendritic spine remodeling following long‐term treatment. We found that, with prolonged CX546 treatment, organotypic hippocampal slice cultures showed a significant reduction in CA3–CA1 excitatory synapse and spine density. Electrophysiological approaches revealed that the CA3–CA1 circuitry compensates for this synapse loss by increasing synaptic efficacy through enhancement of presynaptic release probability. CX546‐treated slices showed prolonged and enhanced potentiation upon LTP induction. Furthermore, structural plasticity, namely spine head enlargement, was also more pronounced after CX546 treatment. Our results suggest a concordance of functional and structural changes that is enhanced with prolonged CX546 exposure. Thus, the improved cognitive ability of patients receiving ampakine treatment may result from the priming of synapses through increases in the structural plasticity and functional reliability of hippocampal synapses.     

The roles of dendritic spine shapes in Purkinje cells

Shapes of dendritic spines are changed by various physiological or pathological states. The high degree of spine shape heterogeneity suggests that they would be the morphological basis for synaptic plasticity. An increasing number of proteins and signal transduction pathways have recently been shown to be associated with structural modifications of spines. Here, we review the possible functional roles of spine shapes in cerebellar Purkinje neurons. Several studies have suggested that spine shapes in Purkinje cells are regulated by both intrinsic and environmental factors, and different spine shapes could have significantly different consequences for brain function. Clearly constricted necks observed in thin, mushroom-shaped, and branched spines serve for compartmentalization of calcium and other second messenger molecules, influencing different signaling mechanisms and synaptic plasticity. Mushroom-shaped spines frequently have perforated postsynaptic density and the area of the spine head is much larger than simple spines, implying that membrane dynamics and receptor turnover are occurring. Branched spines might form additional synapses with afferent inputs resulting in the modification of neuronal circuits. Taken together, all these studies suggest that each spine shape is likely to have a distinct role in Purkinje cell function.     

Expression of insulin-like growth factor I by astrocytes in response to injury

Astrocytes are known to express several growth factors in response to injury and neurological disease. Insulin-like growth factor I (IGF-I) induces astrocytes to divide in vitro and is expressed by developing, but not adult astrocytes both in vivo and in vitro. We tested whether IGF-I is re-expressed by reactive astrocytes in response to injury. We found that astrocytes surrounding the lesioned parenchyma after introduction of a cannula through the cerebral cortex, hippocampus and midbrain contain high levels of immunoreactive IGF-I, as determined by immunocytochemistry using a highly sensitive and specific anti-IGF-I monoclonal antibody. Interestingly, the contralateral hippocampus also contained IGF-I positive astrocytes although in substantial lower numbers. Intact animals showed no detectable IGF-I immunoreactivity in astrocytes. IGF-I was detected at the first time point tested after the lesion was made, 1 week, and for at least 1 month thereafter. Reactive astrocytes expressing high levels of glial fibrillary acidic protein were found in a much wider distribution all along the lesioned area and beyond. We conclude that mechanical injury of the brain induces a specific pattern of expression of IGF-I by a subpopulation of astrocytes. These findings suggest that IGF-I is participating in the response of astrocytes to injury.     

Castration alters the number and structure of dendritic spines in the male posterodorsal medial amygdala

The posterodorsal medial amygdala (MePD) is responsive to androgens and participates in the integration of olfactory/vomeronasal stimuli for the display of sexual behavior in rats. Adult gonadectomy (GDX) affects the MePD structural integrity at the same time that impairs male mating behavior. At the cellular level, dendritic spines modulate excitatory synaptic transmission, strength, and plasticity. Here, we describe the effect of GDX on the number and shape of dendritic spines in the right and left MePD using confocal microscopy and 3D image reconstruction. Age‐matched adult rats were intact (n = 6), submitted to a sham procedure (n = 4) or castrated and studied 90 days after GDX (n = 5). The MePD neurons have a density of 1.1 spines/dendritic μm composed of thin, mushroom‐like, stubby/wide, and few ramified or atypical spines. Irrespective of brain hemisphere, GDX decreased the dendritic spine density in the MePD, but induced different effects on each spine type. That is, compared to control groups, GDX reduced (i) the number (up to 50%) of thin, mushroom‐like, and ramified spines, and (ii) the size and the neck length of thin spines as well as the head diameter of ramified spines. Besides, GDX increased the number of stubby/wide and atypical spines (up to 140% and 400%, respectively). These data show that GDX promotes a cellular and synaptic reorganization in a spine‐specific manner in the MePD. By altering the number and shape of these connectional elements, GDX can affect the neural transmission and hinder the function of integrated brain circuitries in the male brain.     

Morphologic changes of dendritic spines of striatal neurons in the levodopa‐induced dyskinesia model

Maladaptive plasticity at corticostriatal synapses plays an important role in the development of levodopa‐induced dyskinesia. Recently, it has been shown that synaptic plasticity is closely linked to morphologic changes of dendritic spines. To evaluate morphologic changes of dendritic spines of two types of striatal medium spiny neurons, which project to the internal segment of globus pallidus or the external segment of globus pallidus, in the levodopa‐induced dyskinesia model, we used 6‐hydroxydopamine‐lesioned rats chronically treated with levodopa. Dendritic spines were decreased and became enlarged in the direct pathway neurons of the model of levodopa‐induced dyskinesia. The same levodopa treatment to normal rats, in which no dyskinesia was observed, also induced enlargement of dendritic spines, but not a decrease in density of spines in the direct pathway neurons. These results suggest that a loss and enlargement of dendritic spines in the direct pathway neurons plays important roles in the development of levodopa‐induced dyskinesia. © 2014 International Parkinson and Movement Disorder Society     

Transsynaptic modulation of the synaptic vesicle cycle by cell-adhesion molecules

Delicate control of the synaptic vesicle cycle is required to meet the demands imposed on synaptic transmission by the brain's complex information processing. In addition to intensively analyzed intrinsic regulation, extrinsic modulation of the vesicle cycle by the postsynaptic target neuron has become evident. Recent studies have demonstrated that several families of synaptic cell-adhesion molecules play a significant role in transsynaptic retrograde signaling. Different adhesion systems appear to specifically target distinct steps of the synaptic vesicle cycle. Signaling via classical cadherins regulates the recruitment of synaptic vesicles to the active zone. The neurexin/neuroligin system has been shown to modulate presynaptic release probability. In addition, reverse signaling via the EphB/ephrinB system plays an important role in the activity-dependent induction of long-term potentiation of presynaptic transmitter release. Moreover, the first hints of involvement of cell-adhesion molecules in vesicle endocytosis have been published. A general hypothesis is that specific adhesion systems might use different but parallel transsynaptic signaling pathways able to selectively modulate each step of the synaptic vesicle cycle in a tightly coordinated manner.     

Protein kinase C activity modulates dendritic differentiation of rat Purkinje cells in cerebellar slice cultures

The molecular mechanisms underlying dendritic differentiation in neurons are currently poorly understood. We used slice cultures from rat cerebellum of postnatal day 8 to investigate the effect of protein kinase C (PKC) activity on dendritic development of Purkinje cells. After 12 days in culture under control conditions, Purkinje cells had developed a typical dendritic tree consisting of a few long primary dendrites with shorter side branches. Following treatment with the PKC agonist, phorbol-12-myristate-13-acetate (PMA), the dendritic tree area was strongly reduced to 32% of control and primary dendrites were short with only a few side branches. Delayed addition of PMA after 6 days resulted in a retraction of existing dendrites, whereas discontinuation of PMA treatment after 6 days resulted in a recovery of the dendritic tree to almost control values. In the presence of the PKC inhibitor, 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide (GF109203X), the dendritic tree area was increased to 158% of control with much more ramified branches after 12 days. The overall morphology of the cultures and the survival of Purkinje cells were unaffected by PKC modulators. Our data show that increased activity of PKC inhibits, and reduced activity of PKC promotes dendritic growth. This suggests that PKC activity is a critical regulator of dendritic growth and differentiation in cerebellar Purkinje cells.