University of Groningen

Ultrastructural localisation and functional implications of Corticotropin releasing factor,Urocortin and their receptors in cerebellar neuronal developmentSwinny, Jerome Dominic

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115

CHAPTER 6

Corticotropin releasing factor and Urocortin modulatePurkinje cell dendritic outgrowth and differentiation in vitro

J. D. Swinny1, F. Metzger2 , J. IJkema-Paassen2, A. Gramsbergen2,

J.J.L. van der Want1

1Laboratory for Cell Biology and Electron Microscopy,2 Department of Medical Physiology, Graduate School of Behavioural and

Cognitive, Neuroscience, University of Groningen,

The Netherlands

Submitted: Mol. Cell. Neuroscience

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ABSTRACT

The precise outgrowth and arborisation of dendrites is crucial for their function as

integrators of signals relayed from axons and hence, the functioning of the brain.

Proper dendritic differentiation is particularly resonant for Purkinje cells since the

intrinsic activity of this cell-type is governed by functionally distinct regions of its

dendritic tree. The initial phase of dendritic development is governed by intrinsic

patterns. However, activity dependent mechanisms, under the influence of electrical

signalling and trophic factors account for the most active period of dendritogenesis.

A yet unexplored trophic modulator of Purkinje cell dendritic development is

corticotropin releasing factor (CRF) and family member, urocortin, both localised in

climbing fibres. Here we use organotypic cerebellar slice cultures to investigate the

roles of CRF and urocortin on Purkinje cell dendritic development. Intermittent

exposure (12 hours per day for 10 days in vitro) of CRF and urocortin induced

significantly more dendritic outgrowth (45 % and 70 % respectively) and elongation

(25 % and 15 % respectively) compared with untreated cells. Conversely, constant

exposure to CRF and urocortin significantly inhibited dendritic outgrowth. Using

specific antagonists against the CRF receptors, it could be demonstrated that both

receptors are involved in the induction of dendritic outgrowth whereas CRF receptor

1 modulates CRF-induced elongation of dendrites. Furthermore, the trophic effects

of CRF and urocortin are mediated by the protein kinase A and the mitogen activating

protein kinase pathways. The study shows unequivocally that CRF and urocortin are

important regulators of dendritic development. However, their effects are dependent

upon their degree of expression that in turn, influences the functional status of their

receptors. The current data offer insights, and possible therapeutic possibilities for

growing number of pathologies that show perturbations between CRF peptide

expression and their receptors.

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corticotropin releasing factor and Urocortin

INTRODUCTION

Dendrites play a crucial role in the integration and computation of signals relayed

from axons and consequently, the activity of neurons (Magee, 2000; Reyes, 2001).

The diversity of dendritic arbor types within the CNS underscores the significance

of dendritic morphology to the specialised functioning of neurons (Vetter et al., 2001).

Since the spatial patterning of dendritic arborisation is integral to neuronal function,

and hence proper brain functioning, an understanding of the processes and factors

involved in the precise growth and differentiation of dendrites is crucial. This will

also facilitate more directed interventions of malformations and pathologies of a

developmental origin such as spinocerebellar ataxia (Yang et al., 2000; Jeong et al.,

2000).

The precise orientation and differentiation of the most elaborate dendritic trees in the

brain, namely those of Purkinje cells is of particular importance. This is highlighted

by the fact that the functionally distinct regions of its dendritic tree (Bravin et al.,

1999) serve specific roles in the intrinsic activity of Purkinje cells (Womack and

Khodakhah, 2002). Also, reciprocal trophic interactions between the Purkinje cell

dendrites and their afferents, climbing fibres and parallel fibres (Strata et al., 1997)

ensure that the afferents selectively innervate distinct regions on the dendritic tree.

Indeed, parallel fibres which convey sensory information from the mossy fibre system

form synapses exclusively on the distal thorny dendritic spines while axons of the

inferior olivary complex, namely climbing fibres, which convey signals from higher

brain centres, selectively innervate the more proximal stubby dendritic spines

(Sugihara et al., 2000). Since the Purkinje cell is considered the organising centre of

the cerebellar cortex, the formation and differentiation of Purkinje cell dendrites is

central for the integration and computation of its diverse inputs, allowing the

cerebellum to function as the centre for motor coordination.

During development, intrinsic programs contribute to the initial acquisition of a

polarised form, with distinct axons and dendrites (Dotti et al., 1988; Zhang et al.,

2002) and also allowing for the distinctive morphologies of different neuronal types

(Cannon et al., 1999). This holds true for Purkinje cells as well (Soha and Herrup,

1995). However, activity dependent mechanisms, particularly the signalling from

afferents accounts for the most active period of dendritic growth (Zhang and Poo,

2001; Wong and Ghosh, 2002). The importance of activity-dependent mechanisms

resonates most soundly for Purkinje cells, since the greatest amount of dendritic

growth occurs during the most active period of synaptogenesis, which in the rat,

occurs from the second postnatal week onwards. In relation to electrical signalling

also trophic factors play a dynamic role in either inducing or repressing dendritic

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growth (McAllister, 2001). A yet unexplored possible trophic agent of Purkinje cell

development is corticotropin releasing factor (CRF) (Vale et al., 1981).

In the cerebellum, CRF is selectively localised in climbing and mossy fibres (Palkovitz

et al., 1987). In adulthood, CRF released from climbing fibres is crucial for the

induction of long term depression at the parallel fibre-Purkinje cell synapse, a type

of synaptic plasticity considered to be the cellular correlate of motor learning.

Interestingly, cerebellar CRF (Bishop and King, 1999) and the receptors to which it

binds (Chan et al., 1993) are expressed already at early stages of cerebellar

development, prior to any functional synapses being formed. CRF augments the effects

of glutamate (Bishop, 1990), a neurotransmitter directly implicated in activity

dependent dendritic development (Metzger et al., 1998), suggesting that climbing

fibre released CRF might first function as a trophic agent. We have recently shown

that a newer member of the CRF family of peptides, urocortin (Vaughan et al., 1995)

is also expressed in the rat cerebellum (Swinny et al., 2002). The major sites of

expression were the Purkinje cells, and climbing and parallel fibres. Our earlier

observations show that urocortin is also expressed in the developing rat cerebellum

suggesting that both CRF and urocortin could play a trophic role in Purkinje cell

development. Both CRF and urocortin act via two G-protein coupled receptors, namely

CRF receptor 1 (CRF-R1) and CRF receptor 2 (CRF-R2) with urocortin having a

greater binding affinity for CRF-R2. In the Purkinje cell, CRF-R1 is localised on the

dendrites whilst CRF-R2 is restricted to the somata (Bishop et al., 2000; Bishop and

King, 2002; Swinny et al.,2003). Since CRF and urocortin couple to the two CRF

receptors with different binding affinities (Vaughan et al., 1995), these two peptides

might subserve different trophic roles. To explore this postulate, we have used

organotypic slice cultures of rat cerebellum to study the effects of CRF and urocortin

on Purkinje cell dendritic differentiation in vitro.

METHODS

Materials

Human and rat CRF, rat urocortin and alpha-helical CRF (a non-selective CRF receptor

antagonist) were obtained from Sigma (Zwijndrecht, The Netherlands). KT 5720 (a

selective PKA antagonist) and PD98059 (a selective MAPK antagonist) were obtained

from Calbiochem (Breda, The Netherlands). Antalarmin was a generous gift of KC

Rice and H Habib (NIH). Chemicals were dissolved in a 50:50 mixture of DMSO and

ethanol. Minimal essential medium (MEM), Hank’s balanced salt solution (HBSS),

glutamax I and horse serum were purchased from Invitrogen (Breda, The Netherlands).

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Organotypic slice cultures of rat cerebellum

Approval to conduct the study was obtained from the Ethics Committee on Animal

Experimentation, University of Groningen. All efforts were made to minimise the

number of animals used and their suffering. The preparation of cerebellar slices was

performed according to a protocol modified from Metzger et al. (2000). In brief, 8-

day-old Black-hooded Lister rat pups were decapitated and the brain aseptically

removed. The cerebellum was rapidly dissected in ice-cold preparation medium (MEM

containing 2 mM glutamax I, pH 7.3) and the meninges were carefully removed.

Sagittal slices of 400 µm were cut using a McIllwain tissue cutter, separated with

fine forceps and transferred onto humidified transparent membranes (Millicell-CM,

Millipore). They were cultured on a liquid layer of MEM containing HBSS (25%),

horse serum (25%), glutamax I (2 mM) and NaHCO3 (5 mM), HEPES (10 mM),

pH 7.3, in a humidified atmosphere with 5% CO2 at 37 °C.

CRF receptor desensitization is time-dependent, with ~ 50% down-regulation

occurring within 3 hours in Y79 cells (Dautzenberg et al., 2002). We therefore explored

the effects of intermittent exposure or constant exposure of CRF and urocortin on

Purkinje cell dendritic development. Thus, slices were either exposed to

pharmacological agents for 12 hours per day for 10 days in vitro (DIV) or continuously

for 10 DIV.

Immunocytochemistry

Slice cultures were fixed after 10 DIV in phosphate buffer (100 mM) containing 4%

paraformaldehyde. All further steps were performed in 100 mM phosphate buffer

(PB), pH 7.3. The slices were permeabilised and non-specific binding sites blocked

with Triton X-100 (0.3%) and normal goat serum (2%). Monoclonal antibody against

calbindin D-28K (Sigma) was applied (1: 1000) and the slices incubated at 4 °Covernight. After washing, the slices were incubated with goat-antimouse Alexa Fluor

633 (Molecular Probes), (1: 500) for 2 hours. The slices were washed again and

mounted with Mowiol (Calbiochem). Slices were investigated by fluorescence

microscopy.

Quantitative morphological analysis of Purkinje cell dendritic trees

Purkinje cells with little overlap with neighbouring cells were selected in order to

fully quantify the entire dendritic trees as described (Metzger and Kapfhammer, 2000;

Schrenk et al., 2002). Purkinje cells were photographed with a high-resolution CCD

camera at a 200x magnification. The images were analysed using a morphometric

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program supplied by analySIS Soft Imaging System (Munster, Germany). The

following parameters were determined from micrographs according to the definitions

of Metzger and Kapfhammer (2000) and Schrenk et al. (2002): (i) the number of

primary dendrites; (ii) the number of dendritic branching points; (iii) the length of the

longest dendrite (i.e. distance between the cell body and the most distal dendritic

ending); (iv) the total dendritic tree area by connecting the ends of all terminal dendritic

tips of a single Purkinje cell including the soma with straight lines. Additionally, the

branching density, the area and the number of branching points per dendrite were

calculated for each Purkinje cell from the obtained raw data.

Calculations and statistics

Individual experiments were performed with 5-6 cerebellar slices per sample and

repeated 2-3 times using matched controls, and the obtained data were pooled. From

each sample, 15-40 Purkinje cells were evaluated. Results are expressed in the text

and graphically as mean ± standard error of the mean (SEM). The statistical

significance of differences in parameters was assessed by parametric one-way analysis

of variance (ANOVA) followed by Dunnett’s multiple comparison test using SPSS

statistical software (Chicago).

RESULTS

Intermittent exposure of slices to CRF and urocortin stimulate Purkinje cell

dendritic outgrowth and elongation

Since CRF receptors are rapidly down-regulated (Hauger et al., 2000) by G-protein

receptor kinase 3 (Dautzenberg et al., 2001; 2002), we investigated the effects of

intermittent and continuous exposure of CRF and urocortin, and hence, different

degrees of CRF receptor desensitisation, on Purkinje cell dendritic development. We

therefore treated cerebellar slice cultures for each 12 hours per day with CRF or

urocortin. Generally, treatment with CRF, urocortin or any antagonist used in this

study had no discernable effect on Purkinje cell survival (data not shown). Intermittent

exposure of cerebellar slices to CRF and urocortin had overall stimulatory effects on

Purkinje cell dendritic growth and development. At 4 DIV, cells from control slices

exhibited numerous immature or unbranched dendritic processes. In comparison, CRF

and urocortin treated cells had longer primary dendrites that branched more extensively.

At 10 DIV, both control and treated cells showed the characteristic morphology of

Purkinje cells. However, cells treated with CRF and urocortin had, on average more

primary dendrites, which were longer and branched more (figure 1). The effects of

picomolar (pM), nanomolar (nM) and micromolar (µM) concentrations of CRF and

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Figure 1. Comparative morphologies of calbindin-immunostained Purkinje cells from slicestreated with the CRF and urocortin over 4 and 10 DIV daily for 12 hours. (A) a cell fromcontrol experiments at 4 DIV exhibiting relatively short unbranched primary dendrites. (Band C) in comparison, CRF and urocortin (1 nM) treated cells had longer primary dendriteswith apparently more branches (D) shows the characteristic morphology of a Purkinje cellafter 10 DIV. (E and F) show cells treated with CRF and urocortin respectively at a concentra-tion of 1 nM. In both treatment groups, more primary dendrites are evident with these beingmore elongated compared to controls. Scale bar = 50 µm.

Figure 2. Quantitative morphological analysis of Purkinje cell dendritic trees showing themeans and SEM following treatment of cerebellar slices with different concentrations of CRF

and urocortin for 12 hours per day for 10 DIV.*** P < 0.01 versus respective controls.

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urocortin on Purkinje cell dendritic growth and differentiation were quantified. Both

CRF and urocortin caused a concentration dependent increase in the number of primary

dendrites with 1 nM concentration resulting in the most significant amount of primary

dendritic outgrowth (fig. 2 A). At 10 DIV, urocortin, at 1 nM concentration was the

most potent in inducing dendritic outgrowth (mean of 5.3 vs 3.1 primary dendrites

for control). One nM concentrations of CRF and urocortin also resulted in the most

significant increases of branching points per Purkinje cell (fig. 2 B). However, there

were no significant differences between branching points per primary dendrite

suggesting that both factors had no effect on the induction of new branching (fig. 2

C). Compared to controls, CRF and urocortin treated cells had significantly longer

dendrites indicating that CRF at 1 nM was most potent in inducing dendritic elongation

(mean length 174 vs 139 µm in controls) (fig 2 D). Furthermore, CRF (1pM and

1nM) caused a statistical significant increase in total dendritic area (fig 2 E) whereas

cells treated with urocortin (1 nM and 1 µM) showed significantly lower areas per

dendrite (fig. 2 F).

An apparent discrepancy occurred between the effects of CRF and urocortin on the

dendritic branching and area (revealing the branching density of the dendritic tree)

when calculated per Purkinje cell or single dendrite (see fig. 2). We therefore performed

a linear correlation analysis to correlate the branching density either per cell or per

single dendrite. The analysis per Purkinje cell revealed very poor correlations under

control conditions as well as after CRF or urocortin treatment (fig. 3A, table 1) whereas

the calculation per single dendrite greatly improved this correlation (fig. 3B, table 1).

This supports an idea that dendritic branching is a stoichiometric process driven by

factors influencing dendritic lengthening, in this case, CRF. The correlation analyses

further supported the calculation per dendrite as relevant because the apparent number

of primary dendrites (y-intersects in fig. 3) only in this analysis revealed values

qualitatively fitting to our observations since we observed Purkinje cells always bearing

one to seven primary dendrites in our cultures (table 1). Using this analysis we observed

a highly significant increase in the branching density of 55 % after CRF treatment as

compared to controls whereas urocortin did not reveal any change in branching density

but showed a tendency to induce more primary dendrites although this difference did

not reach significance (table 1). These data indicate that CRF and urocortin might

exert different effects on Purkinje cell dendritic development with CRF being more

active in already-formed dendrites, influencing their further differentiation while

urocortin initiates dendritic outgrowth. For clarity, in the rest of the manuscript we

therefore only show data calculated per single dendrite.

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corticotropin releasing factor and Urocortin

Table 1: Linear regression analysis of morphological parameters. Correlations betweendendritic area and branching points (see fig. 3) were calculated per cell or per primarydendrite. Correlations were fitted to a straight line equation revealing a slope (branchingdensity) and the y-intercept (primary dendrites emerging from the cell body where dendritearea is zero). ** p<0.01 as assessed by unpaired t-test. R indicates quality of fit.

Treatment Slope Y-intercept R

Evaluation per cellControl 0.85 ± 0.18 16.0 ± 4.3 0.366CRF 1 nM 1.07 ± 0.26 30.1 ± 8.0 0.494Urocortin 1 nM 1.01 ± 0.39 30.1 ± 9.6 0.319

Evaluation per dendriteControl 1.35 ± 0.13 1.6 ± 1.1 0.663CRF 1 nM 2.09 ± 0.21** 0.3 ± 1.6 0.802Urocortin 1 nM 1.45 ± 0.23 4.5 ± 1.4 0.636

Figure 3 (for colorinfor-mation: see page 149)Linear regression analysis ofthe correlation betweendendritic area and branch-ing. Data for the dendritictree area and the numberof branching points wereeither calculated either perPurkinje cell (A) or per sin-gle dendrite (B) and theobtained data fitted to astraight line equation(dashed lines) revealingslope (branching density)and y-intercept (primarydendrites/cell). For numericdata, see table 1.

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In order to gauge how soon the stimulatory effects of CRF and urocortin are manifested,

we quantified the morphological parameters at 4 DIV. Cells treated with CRF and

urocortin had on average more primary dendrites although these differences were not

significantly different to control values (fig. 4 A). Application of both CRF and

urocortin resulted in cells having significantly longer primary dendrites (129 and 114

respectively vs 79 µm in controls) (fig. 4 B). At 4 DIV, CRF-treated cells had

significantly more branching points per dendrite (fig. 4 C) and a greater area per

dendrite (fig. 4 D). Compared to the 10 DIV cultures, these data suggest that acute

exposure of CRF and urocortin in vitro has a more pronounced effect on dendritic

elongation whilst long-term exposure is required to maintain dendrites.

Constant exposure of CRF and urocortin to cerebellar slices inhibits Purkinje

cell dendritic development

Instead of exposing slices to CRF or urocortin for 12 hours per day, slices were

continuously exposed to the peptides at 1 µM concentration for 10 DIV in order to

mimic a state of continuous and complete CRF receptor down-regulation. The constant

exposure of CRF and urocortin resulted in Purkinje cells showing significantly less

growth than cells from control slices. Cells had shorter primary dendrites and less

Figure 4. Comparison of quantitative data for short-term vs. long-term treatments of slicecultures with CRF or urocortin. Cerebellar slices were treated with CRF or urocortin (each 1nM) for 12 hours per day for 4 or 10 DIV and Purkinje cell dendrites morphologically ana-lysed. * P < 0.05; ** P < 0.01; *** P < 0.001 versus respective control.

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corticotropin releasing factor and Urocortin

Figure 5. Fluorescence micrographs of Purkinje cells from a control slice and slices treatedwith 1 µM of CRF and urocortin continuously for 10 DIV. Purkinje cells from treated sliceshave relatively shorter primary dendrites and less dendritic branching particularly in urocortintreated slices. Scale bar = 50 µm.

Figure 6. Quantitative morphological analysis of Purkinje cell dendritic trees showing themeans and SEM following treatment of cerebellar slices with CRF and urocortin 1 µM continu-ously for 10 DIV. Continuous exposure of cerebellar slices to CRF and urocortin, had, ingeneral, inhibitory effects on Purkinje cell growth. * P < 0.05; *** P < 0.001 versus control.

dendritic branching, with cells from urocortin effecting the most significant amount

of inhibition (fig. 5).

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The continuous exposure of slices to 1µM urocortin had a potent inhibitory effect on

all morphological parameters evaluated. The number of primary dendrites growing

out was reduced by urocortin (2.0 vs 3.4 for control; fig. 6 A). Dendrites were

significantly shorter when treated with CRF and urocortin (127 and 107 µm

respectively vs 146 µm for control (fig. 6 B). Compared to control values, 1 µM

concentrations of CRF and urocortin also significantly decreased the total number of

dendritic branches per primary dendrite (fig. 6 C) with urocortin significantly

decreasing the area per dendrite (fig. 6 D). These data indicate that CRF receptors

were down regulated by continuous peptide application and that CRF receptor

signalling is necessary for proper dendrite outgrowth in cerebellar slice cultures.

Blockade of CRF receptors and secondary messenger cascades

CRF receptor antagonists

The specificity of the CRF and urocortin effects on Purkinje cell dendrite outgrowth

was tested using receptor-specific blockers. a-helical-CRF (a-h-CRF), which inhibits

both known CRF receptors (CRF-R1 and CRF-R2) ( Menzaghi et al., 1994) was used

as a non-specific CRF receptor antagonist. Antalarmin (Webster et al., 1994) was

used as a selective CRF-R1 antagonist.

A-h-CRF alone did not have any significant effects on Purkinje cell dendritic growth

and differentiation when compared with cells from control slices (fig. 7 A). Cells

from slices treated with CRF and urocortin (1 nM) in the presence of a-h-CRF had

less primary dendrites, which were shorter and branched less, when compared with

cells from slices treated with CRF and urocortin alone (figs. 7 B and C. compare with

fig. 1). In the presence of a-h-CRF, the CRF and urocortin induced outgrowth of

primary dendrites was attenuated (3.4 and 3.1 for CRF and urocortin respectively).

Although a-h-CRF significantly reduced the total number of branch points per cell

(data not shown), there was no significant reduction in branching points per dendrite

(fig. 8 B). A-h-CRF significantly reduced the dendritic length of CRF-treated cells

(174 µm reduced to 150 µm). However, the area per dendrite was unaffected by a-h-

CRF (fig. 8 C).

The CRF-R1-specific antagonist antalarmin alone did not alter normal Purkinje cell

dendritic development in vitro when compared with cells from untreated slices (fig. 7

D. see fig 1A). In contrast, the CRF and urocortin-induced dendritic lengthening and

branching effects were markedly inhibited by antalarmin (fig 7 E and F). The addition

of antalarmin to CRF and urocortin treated slice cultures resulted in cells having

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corticotropin releasing factor and Urocortin

Figure 7. Comparative morphologies of Purkinje cells from slices treated with the CRF-R1and CRF-R2 antagonist a-h-CRF alone, antalarmin, the specific CRF-R1 antagonist alone orwith CRF and urocortin in the presence of a-h-CRF and antalarmin for 12 hours per day for12 DIV. (A) cells from slices treated with a-h-CRF resembled cells from untreated slices (seefig. 1 A). (B and C) however, the presence of a-h-CRF in CRF and urocortin treated slicesattenuated the stimulatory effects of CRF and urocortin (see also fig. 1 B and C). (D-F)antalarmin significantly reduced the dendritic length of CRF and urocortin treated cells. Scalebar = 50 µm.

significantly less primary dendrites (2.7 and 3.9 respectively) compared with those

treated CRF and urocortin alone (fig. 8 A). Antalarmin significantly reduced the total

number of dendritic branches of CRF and urocortin-treated cells (65.7 and 68.5

respectively reduced to 38 and 40 respectively). However, the branching points per

dendrite of CRF and urocortin treated cells were unaffected by antalarmin treatment

(fig. 8 B). The mean dendritic length of CRF-treated cells (174 µm to 151 µm) as

well as the mean total dendritic area (32607 µm2 to 18530 µm2) was significantly

reduced by antalarmin. However, the area per primary dendrite was unaffected by

antalarmin alone (fig. 8 C) although it abolished the urocortin effect.

CRF receptor secondary messenger cascades

Both CRF receptors couple to G proteins resulting in cAMP stimulation and protein

kinase A (PKA) activation (Chang et al., 1993; De Souza, 1995). However, CRF and

urocortin have been shown to activate diverse signalling cascades, such as the mitogen

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activating protein kinase (MAPK) pathways (Rossant et al., 1999; Blank et al., 2003).

We used the specific PKA inhibitor, KT 5720 (Kase et al., 1987) and the specific

MAPK inhibitor, PD 98059 (Alessi et al., 1995) to assess the roles of the PKA and

MAPK pathways in the CRF and urocortin-induced dendritic differentiation. The

culturing of slices in the presence of KT 5720 and PD 98059 alone did not significantly

alter the normal growth of Purkinje cells when compared with cells from untreated

slices. In the presence of KT 5720, the stimulatory effects of CRF 1 nM and urocortin

1 nM on Purkinje cell dendritic outgrowth was significantly reduced (4.5 and 5.3

respectively reduced to 3.4 and 4.0 mean number of primary dendrites per cell

respectively; fig.8A). PD 98059 was more potent in inhibiting the CRF and urocortin

Figure 8. Quantitative morphological analysis of Purkinje cell dendritic trees showing themeans and SEM following treatment of cerebellar slices with a-h-CRF (CRF-R1 and 2 antago-nist), antalarmin (CRF-R1 antagonist), KT 5720 (PKA antagonist) and PD 98059 (MAPK an-tagonist) (1 µM) alone or with CRF and urocortin (1 nM) for 12 hours per day for 10 DIV. Thedata represents the means and SEM of parameters assessed. (A) a-h-CRF significantly at-tenuated the urocortin-induced outgrowth of primary dendrites, to a greater degree thanantalarmin. PD 98059 was most potent in reversing the CRF and urocortin-induced dendriticoutgrowth. (B and C) following treatment with the antagonists, the parameters of branchingpoints per dendrite and area per dendrite were comparable to control values. ** P < 0.01;*** P< 0.001.

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corticotropin releasing factor and Urocortin

induced dendritic outgrowth (reduced to 2.6 and 2.9 mean number of primary dendrites

per cell respectively). Whilst the total number of dendritic branches per cell, dendritic

length and total dendritic areas of CRF and urocortin treated cells were all significantly

reduced to levels of untreated controls, the branching points per primary dendrite and

area per dendrite were not significantly altered by the treatment of slices with CRF

and urocortin concomitantly with KT 5720 and PD 98059 (figs. 8 B and C). In fact,

the inhibitory effect of urocortin on the dendritic area per primary dendrite was

abolished in the presence of either K/ 5720 or PD 98059 (fig.8C).

DISCUSSION

In the current study, we have used a well-established model of Purkinje cell dendritic

development (Metzger and Kapfhammer, 2000; Schrenk et al., 2002; Gundlfinger et

al., 2003) to show that CRF and urocortin dynamically effect dendritic outgrowth and

elongation. The constant exposure of cerebellar slices to CRF and urocortin, which,

one could reasonably expect to lead to sustained receptor desensitisation (Hauger et

al., 2000), resulted in grossly under-developed dendritic trees. Conversely, intermittent

exposure of these peptides stimulated Purkinje cell dendritic outgrowth and elongation,

indicating that the expression of CRF-like peptides and the functional status of the

CRF receptor system are crucial for normal dendritic development. CRF and urocortin

most likely modulate different phases of the dendritic developmental process.

Induction of dendritic outgrowth

Based on the quantitative morphological assessment, urocortin is a potent inducer of

dendritic outgrowth, more so than CRF. Indeed, urocortin caused a 70% increase in

number of primary dendrites compared with control values, and 17% increase when

compared with cells from slices treated with CRF alone (see figure 2). The effects of

urocortin appear to predominate in the initial phases of dendritic development, namely

the initial outgrowth, since its effects on parameters concerned with later dendritic

differentiation, such as elongation and branching were always less than that of CRF.

Both CRF and urocortin are expressed in climbing fibres (Swinny et al., 2002) which

form synapses on Purkinje cell somata as early as embryonic day 19 (Morara et al.,

2001) and then translocate to the proximal dendritic regions (Scelfo et al., 2003).

CRF-like peptides act via two G-protein coupled receptors with urocortin showing

selectivity for CRF-R2. We have recently shown that, postnatally, CRF-R2 is restricted

to the Purkinje cell somata, while CRF-R1 enjoys a more dynamic ontogenic

distributional profile (Swinny et al., 2003). Indeed, at early postnatal stages, CRF-R1

protein was first localised in the cell body, but infused into the developing dendritic

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shafts. Based on the localisation data of CRF receptors (Bishop et al., 2000; King and

Bishop, 2002; Swinny et al., 2003) and the current study, a delicate interplay probably

ensues between climbing fibre co-localised CRF and urocortin. At the initial stage of

climbing fibre synaptogenesis on Purkinje cell somata, the effects of urocortin, via

CRF-R2 probably predominate, initiating dendritic outgrowth.

Dendritic elongation

The current study shows unequivocally that CRF and urocortin elongate primary

dendrites. CRF had the most influence on dendritic length, with cells from CRF-

treated slices having dendrites 25% longer than untreated cells and 9% longer than

urocortin treated cells. Furthermore, the dendritic lengthening effects of CRF appear

to be mediated by CRF-R1. The similar degree of inhibition of dendrite elongation by

the CRF-R1 and CRF-R2 receptor antagonist, a-h-CRF and the specific CRF-R1

antagonist, antalarmin, implies that CRF-R1 primarily mediates this process. This is

also consonant with localisation data that show CRF-R1 primarily distributed in the

growing dendritic shafts, closely trailing the upward migration of climbing fibres

(King and Bishop, 2002; Swinny et al., 2003). Furthermore, both CRF and urocortin

significantly increased total dendritic branching per cell. However, only CRF

significantly increased the number of branch points per primary dendrite showing

that the enhanced total dendritic branching effect of urocortin is a consequence of the

additional primary dendrites and not directly attributable to specific branching effects,

as is the case with CRF. Hence, after the phase of dendritic outgrowth, which is

probably heavily influenced by urocortin acting via CRF-R2, the effects of CRF

predominate, in concert with the upwardly-migrating CRF-R1, resulting in the

elongation and branching of the newly established dendrites.

Secondary messenger cascades

Metzger and Kapfhammer, (2000) have shown that protein kinase C (PKC) is critical

in dendritic development, with the activation of this pathway resulting in the inhibition

of normal dendritic development. In an attempt to understand the signal transduction

pathways involved in the CRF or urocortin-induced dendritic outgrowth, we used

specific antagonists against the two main secondary messenger pathways implicated,

namely the cAMP-PKA and MAPK pathways. Blockade of the PKA pathway caused

a 5 and 26% decrease in the CRF and urocortin induced dendritic outgrowth

respectively, whilst inhibition of the MAPK pathway resulted in a 42 and 45% decrease

respectively, suggesting that signals mediating the stimulatory effects of CRF and

urocortin on dendritic outgrowth are predominantly transduced via the MAPK pathway.

This is in accordance with an earlier study by Cibelli et al. (2001) who also showed

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corticotropin releasing factor and Urocortin

the importance of PKA and MAPK in mediating the stimulator effects of CRF on

dendritic outgrowth in locus coeruleus-derived neurons.

The precise signal transduction pathway involved in CRF and urocortin-induced

dendritic elongation is somewhat ambiguous, based on the data emanating from this

study. PKA and MAPK antagonism resulted in similar inhibition of the CRF-induced

dendritic elongation. However, blockade of the MAPK showed stronger inhibition in

the urocortin-treated cells compared with PKA blockade. Notwithstanding, the PKA

pathway is reputed to be crucial in dendritic organisation. Microtubule–associated

protein (MAP) 2 is involved in microtubule assembly and the stabilisation of dendrites

(Kaech et al., 1996). MAP 2 is also one of the major PKA-anchoring proteins in

dendrites (Vallee et al., 1981). Not surprisingly, Harada et al. (2002) have shown that

MAP 2 and PKA signal transduction are essential for proper dendritic elongation.

Since G-protein coupled receptor signalling does not consist of solitary, linear

pathways (Belcheva and Coscia, 2002), and the fact that urocortin is not selective for

only CRF-R2 (Vaughan et al., 1995), the results of the current study are probably due

to “noise” from cross-talk between the two CRF receptors and the different

intracellular signalling cascades.

In conclusion, the study shows that intermittent exposure of Purkinje cells to CRF

and urocortin has a strong trophic action, stimulating dendritic outgrowth and

elongation. In converse, prolonged exposure results in underdeveloped dendritic trees.

This overt inhibition of dendritic structure, and probably synaptic remodelling, might

render an explanation, and potential therapeutic modalities, for pathologies where

early life stressful events or sustained stressful stimuli are know to manifest in

conditions like anxiety or major depression (De Souza, 1995), in which perturbations

of the CRF peptide/receptor system has been implicated.

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