Molecular Brain Research 80 (2000) 233–236 www.elsevier.com / locate / bres Short communication Aggregates of cAMP-dependent kinase RIa characterize a type of cholinergic neurons in the rat brain * Carla Mucignat-Caretta Dipartimento di Anatomia e Fisiologia Umana, Universita’ di Padova, Padova, Italy Accepted 30 May 2000 Abstract Acetylcholine is synthesized by different types of neurons, showing a distinct biochemical phenotype. Aggregates of RIa regulatory subunit of cAMP-dependent protein kinases are visualized by immunohistochemistry only in some cholinergic neurons, since they tightly colocalize with two different markers, choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT). These neurons are present mainly in brain areas related to the limbic system. None of the other regulatory subunits of cAMP dependent kinases colocalize with cholinergic markers.  2000 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Acetylcholine Keywords: Protein kinase A; Acetylcholine; Brain; Fluorescent cyclic nucleotides Cholinergic neurons are characterized by the presence of The distribution of peculiar dot-like insoluble aggregates choline acetyl transferase (ChAT), a key enzyme in the of RIa has been previously described in neurons [6,12], synthesis of acetylcholine [1], and the vesicular acetyl- only in the basal forebrain (substantia innominata) and in choline transporter (VAChT), which concentrates acetyl- the brainstem reticular formation, RIa immunoreactivity is choline inside secretory vesicles [2]. diffuse also to the whole neuronal cytoplasm, with some The second messenger cAMP commonly acts through insoluble clusters of aggregated RIa [12]. cAMP-dependent protein kinases, composed of four In this paper it is shown that RIa aggregates can subunits, two catalytic and two regulatory, that can phos- characterize a class of cholinergic neurons, since they are phorylate a variety of target proteins [9,17]. Four types of present inVAChT and ChAT immunolabelled neurons, and regulatory subunits have been described: RIa, RIb, RIIa in some clusters exactly colocalize with ChAT. and RIIb; they have different biochemical properties, The present experiments were carried out according to although no specific function can be assigned to each of European laws on animal experiments (86/609/EEC). them [4,7]. Brain and spinal cord from 25 male rats (30–50 day old) In the brain all the four regulatory subunit isoforms are were processed as previously described [12]. Alternate present [3], partly in soluble form and partly bound to the coronal sections were incubated at room temperature for cytoskeleton [10]. 12 h in one of the following antibodies against: RIa, RIb, RIIa, RIIb, ChAT (Chemicon and Santa Cruz Biotechnol- ogy, made in rabbit), or VAChT (Santa Cruz Biotechnol- Abbreviations: ChAT, Choline acetyl transferase; RI, cAMP-dependent protein kinase regulatory subunit type I; RII, cAMP-dependent protein ogy, made in goat) and then incubated at 378C for 30 min kinase regulatory subunit type II; SAF-cAMP, 8-(5-thioacetamidofluores- with fluorescent secondary antibody. Since most antibodies cein)-adenosine 39-59cyclic monophosphate; SAR-cAMP, 8-(5- were obtained from rabbit, co-localization experiments in thioacetamidotetramethylrhodamine)-adenosine 39-59cyclic monophos- the same section were possible withVAChT antibodies, but phate; VAChT, vesicular acetylcholine transporter were also performed by covering sections with 8-(5- *Tel.: 139-049-827-5304; fax: 139-049-827-5301. E-mail address: mucignat@ux1.unipd.it (C. Mucignat-Caretta). thioacetamidofluorescein)-adenosine 39:59cyclic mono- 0169-328X / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0169-328X(00)00127-3