Expression of CGRP Binding Sites in the Developing Rat Cerebellum L. PROVINI," S. MORARA,b A. ROSINA,b AND G. FORLONI" "Ist. Fisiologia Generale Chimica Bioiogica Facolt6 di Farmacia Universita di Milano Milan, Italy bIst. Fisiologiu Centvi Nervosi C N R Milan, Italy 'Ist. Mario Negri Milan, Italy The distribution and physiological effects of calcitonin gene-related peptide (CGRP) in the adult nervous system suggest its role as an anterograde trophic factor, a differentiation signal, or a neuromodulator. CGRP-like immunoreactivity is undetectable in the adult rat cerebellum, whereas it is transiently expressed at early postnatal stages, confined to specific sets of olivocerebellar fibers.' CGRP binding sites are found in the adult cerebellum, restricted to the molecular layer; the dendrites of Purkinje (Pk) cells, which are extensively innervated by olivocerebellar axons terminating as climbing fibers, are located here. We conducted a quantitative autoradiographic study of the density and distribution of CGRP receptors in rat cerebellum during ontogenesis, to determine their relationship to: (1) the pattern of development of the different cellular components of the cerebellum, and ( 2 ) the transient CGRP-like imrnunoreactivity expressed in the climbing fiber terminals.'
METHODS AND RESULTS Thirty rats (Sprague-Dawley, Charles River) were used on postnatal days (PD) 3, 6, 9, 12, 15, 21, 27, and 40. Binding was performed as described by Kruger and coworkers.* Densitometric quantitative analysis of the autoradiograms was conducted using the RAS 3000 Image Analysis System (Loats System). CGRP binding sites were already present in the cortex of the posterior lobe vermis at PD 9. At this stage, the dendrites of PK cells were developing, so that a nascent molecular layer was recognizable, but binding sites were confined to the Pk cell layer. At PD 12-15, while the molecular layer continued to grow, binding sites were still confined to the Pk cell layer, which was now homogeneously labeled. The density of binding sites remained the same as at PD 9. From PD 15-21, two major changes occurred in the molecular layer: the binding sites increased in density and their localization diffused the entire extent of the layer. A further slight increase in binding densities was recorded at PD 27, when both density and distribution pattern of binding sites became very similar to those of the adult (PD 40). 423
ANNALS NEW YORK ACADEMY OF SCIENCES
424
High levels of binding sites were already found in the white matter at PD 3, with further increases up to PD 12-15. After this age, the binding densities sharply decreased to adult levels at PD 21-24. During all the developmental stages investigated, the levels of CGRP binding sites in the internal and external granular layers were very low.The plots in FIGURE 1 show the developmental time course of CGRP binding densities in the molecular layer and white matter of both anterior and posterior lobe vermis from PD 6 rats to adults.
4.01
A
1.01
t,
0
6
. I I I I
I
I
I
12
18
24
30
12
18
24
30
I
I
I
36 PD
I
42
PMOI -
1.5
-
1.0
-
.5 0
6
36
PD
42
F1 URE 1. Time course of CGRP binding levels (expressed in prnolig of tissu protein) in (A) the molecular layer and (B) the white matter of the cerebellar vermis from rats at PD 6 , 9, 12, 15, 21, 27, and 40. Different symbols indicate different experiments; each point
represents the average of 15-50 sampled layers.
CONCLUSIONS In conclusion, the expression of CGRP binding sites begins in the Pk cell layer while the peptide is still present in the climbing fiber terminals decorating the Pk cell somata (Morara el a/., this volume). Furthermore, a strong modulation of CGRP receptor densities occurs in synchrony with the process of dendritic growth of the Pk cells3 and with the onset of the climbing stage of olivocerebellar fibers4 Therefore, we suggest that CGRP plays an active role in the process of connectivity
PROVINI et al.: CGRP BINDING SITES IN CEREBELLUM
425
reshaping and synapse stabilization which take place during postnatal development. REFERENCES 1 . MORARA,S . , L. PROVINI & A. ROSINA.1989. Brain Res. 504: 315-319. N. C. BRECHA & C. R. MANTYH.1988. 2. KRUGER,L . , P. W. MANTYH,C. STERNINI, Brain Res. 463: 223-244. 3. ALTMAN, J. 1976. J . Comp. Neurol. 165: 65-76. 4. RAMONY CAIAL,S. 191 1 . Histologie du Systeme Nerveux de 1'Hornrne et des VertebrCs. Vol. 2. Maloine. Paris.
METHODS AND RESULTS Thirty rats (Sprague-Dawley, Charles River) were used on postnatal days (PD) 3, 6, 9, 12, 15, 21, 27, and 40. Binding was performed as described by Kruger and coworkers.* Densitometric quantitative analysis of the autoradiograms was conducted using the RAS 3000 Image Analysis System (Loats System). CGRP binding sites were already present in the cortex of the posterior lobe vermis at PD 9. At this stage, the dendrites of PK cells were developing, so that a nascent molecular layer was recognizable, but binding sites were confined to the Pk cell layer. At PD 12-15, while the molecular layer continued to grow, binding sites were still confined to the Pk cell layer, which was now homogeneously labeled. The density of binding sites remained the same as at PD 9. From PD 15-21, two major changes occurred in the molecular layer: the binding sites increased in density and their localization diffused the entire extent of the layer. A further slight increase in binding densities was recorded at PD 27, when both density and distribution pattern of binding sites became very similar to those of the adult (PD 40). 423
ANNALS NEW YORK ACADEMY OF SCIENCES
424
High levels of binding sites were already found in the white matter at PD 3, with further increases up to PD 12-15. After this age, the binding densities sharply decreased to adult levels at PD 21-24. During all the developmental stages investigated, the levels of CGRP binding sites in the internal and external granular layers were very low.The plots in FIGURE 1 show the developmental time course of CGRP binding densities in the molecular layer and white matter of both anterior and posterior lobe vermis from PD 6 rats to adults.
4.01
A
1.01
t,
0
6
. I I I I
I
I
I
12
18
24
30
12
18
24
30
I
I
I
36 PD
I
42
PMOI -
1.5
-
1.0
-
.5 0
6
36
PD
42
F1 URE 1. Time course of CGRP binding levels (expressed in prnolig of tissu protein) in (A) the molecular layer and (B) the white matter of the cerebellar vermis from rats at PD 6 , 9, 12, 15, 21, 27, and 40. Different symbols indicate different experiments; each point
represents the average of 15-50 sampled layers.
CONCLUSIONS In conclusion, the expression of CGRP binding sites begins in the Pk cell layer while the peptide is still present in the climbing fiber terminals decorating the Pk cell somata (Morara el a/., this volume). Furthermore, a strong modulation of CGRP receptor densities occurs in synchrony with the process of dendritic growth of the Pk cells3 and with the onset of the climbing stage of olivocerebellar fibers4 Therefore, we suggest that CGRP plays an active role in the process of connectivity
PROVINI et al.: CGRP BINDING SITES IN CEREBELLUM
425
reshaping and synapse stabilization which take place during postnatal development. REFERENCES 1 . MORARA,S . , L. PROVINI & A. ROSINA.1989. Brain Res. 504: 315-319. N. C. BRECHA & C. R. MANTYH.1988. 2. KRUGER,L . , P. W. MANTYH,C. STERNINI, Brain Res. 463: 223-244. 3. ALTMAN, J. 1976. J . Comp. Neurol. 165: 65-76. 4. RAMONY CAIAL,S. 191 1 . Histologie du Systeme Nerveux de 1'Hornrne et des VertebrCs. Vol. 2. Maloine. Paris.
