B

Fig. 1 Identification of the neuronal population as Purkinje neurons. Cells cultured from ED8 chick cerebellum after 14 DIV (equivalent to hatch) are positive for both the Purkinje neuron-specific marker calbindin D-28 (A) linked to an FITC-labeled secondary antibody and the pan-neuronal marker NSE (B) linked to an RITC-liked secondary antibody. The majority of cells were double labeled. Inserts contain the negative control for each primary antibody. Scale bar 10 ^m. Reprinted from Jeffrey et al. (1996), with permission.

overnight at 4° C (Fig. 1). Coverslips were washed three times in PBS and incubated for 1 h in the dark with antimouse FITC (1:100) or antirabbit RITC (1:100), made up in 1% goat serum in PBS. Coverslips were mounted in FluorSave Reagent (Calbiochem Corp) and viewed under a confocal laser-scanning microscope (Wild Leitz Instrument, Heidelberg, Germany). Incubations were performed in a humidified chamber.

D. Morphological Analysis

1. Materials

Ethanol/acetic acid Ethanol Polyester wax

2. Morphological Types

The stages of Purkinje neuron development were first described by Armengol and Sotelo (1991) in the rat. This system of classification was applied to the development of avian Purkinje neurons in the in vitro culture and for comparison to the in vivo situation.

The basic features of the neuronal types described for the rat have been modified to formulate five morphological categories of avian Purkinje neurons in the developing avian cerebellum: type I, bipolar cells with smooth oval cell body and processes at opposite ends; type II, cells with less than four processes growing from all parts of the cell body; type III, cells with processes that are being withdrawn or regressing; and type IV, stellate cells characterized by the progressive outgrowth of processes. Another category has been added for avian analysis: type V, cells with a primary dendrite and secondary dendritic branching.

a. In Vivo Purkinje Neuron Morphology

Cerebellar tissue from ED10, ED13, ED15, ED17, and hatch chicks was fixed in ethanol/acetic acid (95/5, v/v) on ice and embedded in soft polyester wax as described previously by Sheppard et al. (1988). Sections (4 ym) were blocked in 10% normal goat serum for 15 min and were stained with antibodies parvalbumin (1:100) or calbindin (1:150) for 1 h, followed by the secondary antibody of rabbit antimouse-conjugated alkaline phosphatase (1:1000) and NBT/BCIP. Only nucleated Purkinje neurons in sections were counted. Counts were determined on 20 sections of each age between ED10 and ED17. The morphological types can be recognized in the developing chick cerebellum (type 1, bipolar with two processes, Fig. 2A; type II, greater than four processes emanating from the cell body, Fig. 2B; type III, processes are regressive, Fig. 2C; type IV, stellates form where new processes grow out of the cell body, Fig. 2D; and type V, formation of the primary dendrite, Fig. 2E). The quantitation of this conversion from type I at ED10 to type V at ED17 is presented in Fig. 3B.

b. In Vitro Purkinje Neuron Morphology

In vitro morphological types can be quantitated in simple cultures with different aged astrocytes or in coculture with isochronic granule cell addition. Counts were carried out at four time points—2, 8, 16, and 21 DIV—using coverslips stained with either calbindin D-28 or NF200. Only single Purkinje neurons were counted, with data expressed as a percentage of total counts at each particular time point (Figs. 2F-2I; Fig. 3A).

E. Glutamate Uptake in Purkinje Neurons

1. Materials

Phosphate-buffered Krebs-Ringer solution (PBKR) L-[3H]glutamate, 28 Ci/mmol d- and L-Threo-3-hydroxyaspartic acids (D- and L-t-3OHA) l-trans-Pyrrolidine-2,4-dicarboxylic acid (L-t-PDC) Antibody to GLT-1, a generous gift from Dr. J. D. Rothstein

Fig. 2 Identification of the morphological phenotypes of avian Purkinje neurons according to the modified criteria of tt

Armengol and Sotelo (1991) in vivo (A-E) and in vitro (F-I). (A) Type I with process at opposite ends of the cell body. (B) S

Type II, with less than four processes emanating from the cell body. (C) The regressive form, when the processes are .

retracted. The emergence of new fibers, and also the beginning of the primary dendrite, is seen in vivo (D and E) and in vitro J?

(F-I) from coculturing with granule cells. In vitro neurons are stained for NF200. In vivo sections of ED10 Purkinje ?

neurons are stained for parvalbumin, but all other embryonic ages are stained for calbindin-D (Fig. 3B). Scale bar: 10 ^m. y Reprinted from Jeffrey et at. (1996), with permission.

Fig. 2 Identification of the morphological phenotypes of avian Purkinje neurons according to the modified criteria of tt

Armengol and Sotelo (1991) in vivo (A-E) and in vitro (F-I). (A) Type I with process at opposite ends of the cell body. (B) S

Type II, with less than four processes emanating from the cell body. (C) The regressive form, when the processes are .

retracted. The emergence of new fibers, and also the beginning of the primary dendrite, is seen in vivo (D and E) and in vitro J?

(F-I) from coculturing with granule cells. In vitro neurons are stained for NF200. In vivo sections of ED10 Purkinje ?

neurons are stained for parvalbumin, but all other embryonic ages are stained for calbindin-D (Fig. 3B). Scale bar: 10 ^m. y Reprinted from Jeffrey et at. (1996), with permission.

Fig. 3 Quantitation of Purkinje neuron morphological types under various experimental conditions. Purkinje neuron types were quantitated as described in the text and graphed as a percentage of the total count at each time point. Black bars refer to type I, type II is represented by dark striped bars, crosshatched bars show type III, and type IV is represented as a light striped diagonal. The type V type, which is mature with prominent primary dendrites, is shown by open bars. (A) Temporal distribution of the different types of immature Purkinje neurons over a period of 21 DIV in the presence of ED16 astrocytes. Error bars are the mean ± SD from four individual experiments. (B) Temporal distribution of the different types of Purkinje neurons present in the developing avian cerebellum from ED10 to ED17. Purkinje were identified in sections by calbindin-D28 staining, except ED10, which were stained for parvalbumin. (C) The effect of the addition of granule cells from ED13 (ED13GN) and ED15 (ED15GN) on Purkinje neuron morphology after 21 DIV. Purkinje neurons from ED8 cerebellum were cultured in the presence of ED16 astrocytes for 5 and 7 DIV before the addition of ED13 and ED15 granule cells, respectively. Results are given as mean ± SD for 3 individual experiments for each age of granule cells. For comparison, the distribution after 21 DIV in the presence of ED16 astrocytes and in the absence of granule cells and the in vivo avian ED17 data are given. Reprinted from Jeffrey et al. (1996), with permission.

Fig. 3 Quantitation of Purkinje neuron morphological types under various experimental conditions. Purkinje neuron types were quantitated as described in the text and graphed as a percentage of the total count at each time point. Black bars refer to type I, type II is represented by dark striped bars, crosshatched bars show type III, and type IV is represented as a light striped diagonal. The type V type, which is mature with prominent primary dendrites, is shown by open bars. (A) Temporal distribution of the different types of immature Purkinje neurons over a period of 21 DIV in the presence of ED16 astrocytes. Error bars are the mean ± SD from four individual experiments. (B) Temporal distribution of the different types of Purkinje neurons present in the developing avian cerebellum from ED10 to ED17. Purkinje were identified in sections by calbindin-D28 staining, except ED10, which were stained for parvalbumin. (C) The effect of the addition of granule cells from ED13 (ED13GN) and ED15 (ED15GN) on Purkinje neuron morphology after 21 DIV. Purkinje neurons from ED8 cerebellum were cultured in the presence of ED16 astrocytes for 5 and 7 DIV before the addition of ED13 and ED15 granule cells, respectively. Results are given as mean ± SD for 3 individual experiments for each age of granule cells. For comparison, the distribution after 21 DIV in the presence of ED16 astrocytes and in the absence of granule cells and the in vivo avian ED17 data are given. Reprinted from Jeffrey et al. (1996), with permission.

2. Uptake of L-[3H]Glutamate

1. Place three small drops mounting medium (Euparal, GBI Laboratories, Manchester, UK) on the bottom of petri dishes and allow to harden for a few days. This prevents coverslips containing Purkinje neurons from adhering too closely to the bottom of the petri dish and makes the removal of coverslips at the end of incubation much simpler.

2. Remove coverslips containing 7 DIV and 14 DIV Purkinje neurons from neuronal culture medium.

3. Wash by dipping into 80ml of PBKR (Balcar et al., 1994) (PBKR: 120mM NaC1, 4.5mM KC1, 1.8mM MgCl2, 1.8mM CaCl2, 5.5mM glucose, pH 7.4) at room temperature (23-25° C).

4. Store in same PBKR medium for 5-15 min before use.

5. Transfer coverslips to 35-mm petri dishes containing 1 yM L-[3H]glutamate in 2 ml incubation medium (PBKR), with or without inhibitors. For best results, it is suggested that the specific activity be set at 0.5 to 1 yCi/ml if 1 yM L-glutamate uptake is used or higher if L-[3H]glutamate uptake is studied at higher concentrations.

7. After the incubation period, wash coverslips by rapidly dipping twice into three 100-ml beakers filled with PBKR and finally place in 1 ml water.

8. For best results, the washing procedure should be completed within 5 s.

9. After 24 h, determine tritium radioactivity in a 1-ml aqueous sample by liquid scintillation counting.

10. Determine protein on coverslips by solubilizing protein from parallel coverslips in 0.25 M NaOH and using the Lowry protein assay (Lowry et al., 1951). Alternatively, it is possible to determine water-soluble protein in the aqueous extract. It is possible to extract the cultures with 0.1-0.5 M NaOH to solubilize the proteins and then use the Lowry protein assay.

11. Process data with Prism software (Graphpad, San Diego) or any other suitable high-power statistical package.

III. Applications

The method presented here has allowed the study of the development of avian Purkinje neurons in a controlled coculture system. The system is rapidly manipulated, and two approaches already published from this laboratory are presented as examples of the suitability of the system for investigating the morphological, immunocytochemical, and electrophysiological differentiation of Purkinje neurons (Jeffrey et al., 1996; Meaney et al., 1998; Jeffrey et al., 1998).

A. Astrocyte Effects on Purkinje Neurons

1. Survival and Limited Morphological Development Requires the Presence of Astrocytes

Purkinje neurons are identified by markers and morphological characteristics following days in vitro culture. Following 14 DIV, which is roughly equivalent to hatch, Purkinje neurons are positive for the pan-neuronal marker, neuronal-specific enolase, and the specific Purkinje neuron marker, calbindin D-28 (Fig. 1), and other markers, cyclic GMP kinase, MAP 2, NF68, NF200, and Thy-1. The size of neuronal soma increases from 8 ^mat7DIV to 19.0 ± 1.7 ^mat21 DIV.

Astrocyte monolayers present on Millicells were necessary to promote neuronal survival by providing essential factors, as Purkinje neuron survival was extremely low in the absence of Millicell astrocyte monolayers at even a high plating density (6-8000 cells/mm2).

Astrocytes derived from two embryonic ages caused different effects on the Purkinje neurons. Purkinje neurons in the presence of ED8 astrocytes exhibited increased fasciculation of neurites and the number of neurons in aggregates. With ED16 astrocytes, Purkinje neurons remain predominately as single cells with minimal aggregation and with a slightly more mature phenotype and are hence a better source for morphological studies. These findings implicate temporally regulated astrocyte factors affecting neuronal aggregation and fasciculation. Pur-kinje neurons grown for 5 DIV with ED16 astrocyte layers on Millicells form long simple neurites. Removal of Millicells at 5 DIV and further culturing for a further 9 DIV resulted in a dramatic retraction of the processes. Cell bodies remained normal and no cell death was noted. Thus following an initial period of culture in the presence of astrocytes, their removal does not affect survival but causes a retraction of processes, indicating different factors being present with increasing time in culture.

2. Limited Development of Purkinje Neuron Morphology by Astrocyte-Conditioned Media

Coculture with ED16 astrocytes over a period of 21 DIV results in a maturation block in Purkinje neuron development at the type II phenotype. This limited differentiation clearly evident at 21 DIV was seen routinely up to 28 DIV (2 weeks posthatch in vivo) in our culture system and is quantitated in Fig. 3A. The limited Purkinje neuron development was independent of the age of the cerebellar astro-cytes present on Millicells.

B. Granule Cell Effects on Purkinje Neurons

1. Formation of Mature Purkinje Neuron Phenotype Requires Isochronic Addition of

Granule Cells

In order to keep the in vitro coculture system as close a possible to the in vivo situation with regard to the avian developmental timetable (Feirabend, 1990), isochronic granule cells purified from cerebellum were added to the cultures.

Adding ED13-purified granule cells (ratio 5/1 to Purkinje neurons) to Purkinje neuron cultures from ED8 cerebellum following 5 DIV produced a dramatic effect on the morphology, inducing a more mature phenotype where types III - V predominate (Fig. 3C), and was less dramatic with ED15 granule cells. The effect was dependent on the number of granule cells added; ratios of granule cells to Purkinje neurons less than 1/1 had no effect on maturation, whereas ratios of 10/1 showed no increased maturation over the 5/1 ratio but analysis became-difficult. Representative profiles of mature Purkinje neurons are shown (Figs. 2F-2I) and quantitated in Fig. 3C.

The importance of granule neuron interactions with developing Purkinje neurons has been reported in mouse where plating of immature Purkinje neurons onto networks of parallel fibers enhances Purkinje neuron maturation (Baptista et al., 1994).

The shift to the mature phenotype is not complete, reflecting the role of other neuronal cell types in the cerebellum on Purkinje dendritic tree development and the two-dimensional nature of this culture system.

Clearly, two phases of in vitro avian Purkinje neuron development can be defined by this system.

1. Survival and limited morphological development requires astrocytes.

2. Attainment of mature phenotypes is dependent on the presence of granule cells added in an isochronic manner.

C. Glutamate Transport in Developing Purkinje Neurons

The coculture of cerebellar Purkinje cells and glial monolayers provides an excellent model system for studying the effects of epigenetic factors on the differentiation of neurons.

Excitatory amino acids, specifically L-glutamate, have been proposed as cofac-tors essential for the development and differentiation of Purkinje neurons, particularly in relation to the morphology of the dendrites (Cohen-Cory et al., 1991; Mount et al., 1993). The actions of L-glutamate could be mediated by ionotropic or metabotropic receptors (iGluR, mGluR) that are usually positioned on the outer surface of the cytoplasmic membrane. Interaction of L-glutamate with its receptors (GluR) can, however, contribute not only to the mechanisms of neuronal signalling or morphogenesis, but, in the case of uncontrolled GluR activity, could trigger a cascade of events resulting in the death of the neurons. Extracellular concentrations of L-glutamate therefore have to be regulated precisely.

The actions of L-glutamate released at excitatory glutamatergic synapses in the adult brain are limited temporally and spatially by sodium-dependent transport (GluT) that is located usually, but not always, in the cytoplasmic membrane of neighboring astrocytes (Danbolt, 2001). Transporters mediating GluT are expressed in brain tissue at very high densities both in the forebrain and in the cerebellar cortex (Danbolt, 2001). GluT can thus very efficiently "sweep" the synaptic cleft, as well as the perisynaptic extracellular space, remove any excess l-glutamate, transport it against very steep concentration gradients (Danbolt, 2001), and include it in normal glial metabolism (Rae et al., 2000; Moussa et al., 2002). At least five distinct transporters (not counting numerous splice variants present in brain; Robinson, 1998) can handle L-glutamate in a Na+-dependent manner: GLAST (EAAT1), GLT (EAAT2), EAAC1 (EAAT3), EAAT4, and EAAT5 [see Danbolt (2001) or Balcar (2002) for the explanation of nomenclature]. While GLT is the principal glutamate transporter in astrocytes of the forebrain, GLAST is found at very high densities in glial cells of the cerebellar cortex (Danbolt, 2001). EAAC1 is found in neurons throughout the CNS, but EAAT4 and EAAT5 have very specific locations: EAAT4 exists in significant amounts only in cerebellar Purkinje cells, whereas EAAT5 is expressed appreciably only in the retina (Pow, 2001; Rauen, 2000).

1. Glutamate Uptake by Developing Purkinje Neurons

There seems to be a period of time during neuronal differentiation when glutamate transporters, such as GLT and GLAST, which are, in the adult brain, found only in glial cells, appear transiently in developing neurons. Sutherland et al. (1996) reported increased densities of glutamate transporter mRNA in regions with high synaptogenesis and, subsequently, the expression of GLT was demonstrated both in developing hippocampal neurons (Mennerick et al., 1998) and in Purkinje cells of the chick cerebellum (Meaney et al., 1998). It has also been shown that expression of the transporter protein is associated with the appearance of a vigorous uptake of L-[3H]glutamate (Meaney et al., 1998; Gaillet et al., 2001). The 7 DIV culture of ED8 chick Purkinje neurons accumulates 1 yML-[3H]-glutamate by a high-affinity transport system that could be inhibited in order of potency L-t-DOC~L-t-3-OHA>D-t-3OHA. The IC50 decreased for D-t-3OHA between 7 and 14 DIV, indicating the appearance of heterogeneity for transport sites at later times. Furthermore, hippocampal neurons were shown to coexpress GLAST together with GLT (Plachez et al., 2000).

2. Inhibition of High-Affinity Glutamate Transport Results in Changes in Purkinje Neuron Morphology

The hypothesis that L-glutamate acts as an epigenetic factor during neuronal morphogenesis can be tested using cultured Purkinje cells, and we have done so by inhibiting either GluT or GluR of the NMDA type. Typical results of such experiments are displayed in Fig. 4. While the extracellular levels of L-glutamate would be expected to rise as a consequence of GluT inhibition, thus increasing the stimulation of GluR, MK-801 would have an exactly opposite effect, causing a potent inhibition of NMDA receptors. Inhibition of GluT by L-trans-pyrrolidine-2,4-dicarboxylate reduces the number of neurites (Fig. 4), whereas MK-801 has, in fact, an opposite effect. Therefore, it appears that L-glutamate, acting on GluR of the NMDA type, actually tends to downregulate the growth and branching of the

Fig. 4 Effects of L-t-PDC on Purkinje neuron morphology. The addition of L-t-PDC, a potent inhibitor of glutamate transport (IC50 116 ^m at 7 DIV) to Purkinje neuron cultures, causes a massive regression of neurites. L-t-PDC was added at 1 DIV (1-250 ^M) and grown for 9 DIV and was fixed and stained for NF200. Neuronal coverslips are presented in B and D, where D is a higher power magnification showing extensive neurite outgrowth and fasciculation. Contrast to A and C, following the addition of L-t-PDC over an 8 DIV period, results in almost complete neurite retraction. Scale bars: 50 ^m (A and B) and 10 ^m (C and D). Reprinted from Meaney et al. (1998), with permission.

Fig. 4 Effects of L-t-PDC on Purkinje neuron morphology. The addition of L-t-PDC, a potent inhibitor of glutamate transport (IC50 116 ^m at 7 DIV) to Purkinje neuron cultures, causes a massive regression of neurites. L-t-PDC was added at 1 DIV (1-250 ^M) and grown for 9 DIV and was fixed and stained for NF200. Neuronal coverslips are presented in B and D, where D is a higher power magnification showing extensive neurite outgrowth and fasciculation. Contrast to A and C, following the addition of L-t-PDC over an 8 DIV period, results in almost complete neurite retraction. Scale bars: 50 ^m (A and B) and 10 ^m (C and D). Reprinted from Meaney et al. (1998), with permission.

neurites of Purkinje cells. Stimulation of NMDA receptors would increase the concentration of Ca2+ in the cytosol, thus activating numerous enzymes such as Ca2+-dependent proteases and kinases that could affect either synthesis or post-translational processing of proteins important for the development of the neuritic tree. Whether such mechanisms are also active in vivo and contribute to the process of "sculpting" the developing dendrites to develop the large, complex, and precisely oriented tree-like structure so characteristic of Purkinje neurons in the adult cerebellar cortex is not known, but a strong transient expression of GLT has since been reported in the ovine cerebellum in utero (Northington et al., 1999).

GLT and GLAST are, however, not the only glutamate transporters found on or in the vicinity of Purkinje neurons, and there are at least two other glutamate transporters that could be important in the differentiation of Purkinje cells: EAAT3 and EAAT4. Little is known of the expression of EAAT3 either by the adult or developing Purkinje neurons, but findings from hippocampal neurons suggest that EAAT3 could move rapidly between the cytoplasm and the cyto-plasmic membrane in response to changing neuronal activity, and one might conjecture that such intracellular movements are part of the mechanism involved in synaptic plasticity (Danbolt, 2001; Robinson, 2002). As such phenomena have also been studied in Purkinje neurons (in the form of long-term depression, LTD), the presently discussed coculture system would seem to represent an excellent experimental model for studies involving both morphological and molecular aspects of the hypothesis.

Probably the most enigmatic glutamate transporter expressed by Purkinje neurons is EAAT4. In the adult brain it is found mainly in a precise arrangement close to the dendritic spines (Danbolt, 2001). Like most other transporters, EAAT4, when transporting L-glutamate, also permits passive passage of chloride ions across the neuronal membrane (Fairman et al., 1995). This could influence depolarization of the neuronal membrane caused by ionotropic glutamate receptors and thus further modify the activity of glutamatergic synapses on Purkinje neurons (Auger and Attwell, 2000; Otis et al., 1997). EAAT4 is distributed heterogeneously not only within cells, but also across the cerebellar cortex; it has been reported that it codistributes with zebrin II, thus forming characteristic parasagittal "stripes" (Dehnes et al., 1998).

Several potent GluT inhibitors have been identified and/or synthesized over the past three decades (review Balcar et al., 2001), and the structural specificity of GluT has been studied in great detail (review Balcar, 2002). Moreover, clear differences exist between the pharmacological characteristics of GluT in the forebrain and the cerebellar cortex, respectively (Takamoto et al., 2002; Balcar, 2002), thus leaving hope that substrates and inhibitors specific for GluT in Purkinje neurons could be identified and tested in systems such as the glial/ neuronal coculture discussed here.

Acknowledgments

Work in this laboratory is supported by the NH&MRC. The authors thank Dr. Christine Smythe for excellent work on the confocal microscope.

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