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We used confocal laser scanning microscopy and fluorescent immunohistochemistry to study the developmental pattern and distribution of specific neuronal phenotypes within the intrinsic cardiac nervous system in whole-mount atrial preparations from newborn to 5 week old rats. Individual ganglia and neuronal cell bodies were localized by means of two general neuronal markers: protein gene product 9.5 (PGP) and microtubule-associated protein two (MAP). In rats < or =2 weeks old there were two main subpopulations of intrinsic neurons located in the intraatrial septum and around the origin of the superior vena cava. The more abundant was a population of strongly tyrosine hydroxylase (TH) immunoreactive (IR) neurons (10-40 microm in diameter) most of which were also PGP-IR. The second, less numerous (approximately 60-70% than the TH-IR group) type of neurons exhibited ChAT-IR which colocalized with MAP-IR. Towards the end of the second postnatal week and during the third, the ganglia containing these neurons became more numerous and their localization also included tissues around the origins of the inferior vena cava and the pulmonary veins, as well as both atrial walls close to the AV junction. During the second and third postnatal weeks, when the extrinsic innervation of the adrenergic and cholinergic phenotypes largely increases, the intrinsic innervation also changed greatly, and around the 21st postnatal day it appeared to acquire mature characteristics. The TH-IR neurons changed their characteristics and formed two types of ganglia. The larger ganglia containing large cells (20-40 microm in diameter) expressed TH-IR mostly close to their inner body surface (approximately 80-90% of identified neurons). Most of these neurons also expressed neuropeptide Y (NPY)-IR, specifically around their nuclei. The second type of small strongly TH-IR neurons (approximately 10% of all identified neurons) were contained in smaller groups (20-50 cells) which were usually embedded into much larger ganglia (100-400 cells), containing large (20-50 microm) neurons. Unlike all other intrinsic neurons, these small TH-IR cells did not exhibit any PGP-IR or MAP-IR. The number of ChAT-IR neurons increased at this stage, reaching approximately 90% of the neurons identified by the general neuronal markers. These neurons were surrounded by a rich network of cholinergic varicose nerve fibers, some of which were likely of an extrinsic origin. We have also identified relatively small ganglia expressing immunoreactivity to vasoactive intestinal polypeptide (VIP), and to substance P (SP). The presented data indicate that the phenotypes of intrinsic neurons in the rat heart change greatly during the first month of postnatal development. This may be at least partially related to the development and maturation of functional extrinsic nervous control of the heart.

Functional data indicate that neurons in distinct regions of the heart exert preferential regional cardiac control. To date the regional distribution of specific types of neurons within the intrinsic cardiac nervous system remains unknown, as does their associations with distinct neurotransmitter and/or neuromodulatory profiles. This study was designed to ascertain: (1) the distribution of different classes of neurons within the intrinsic cardiac nervous system as determined by microscopic analysis; (2) the neurochemical profiles of neurons in differing atrial loci; (3) which neurochemicals are co-localized within specific populations of intrinsic cardiac neurons; and (4) the distribution of specific sub-populations of neurons expressing specific immunoreactivities. Taking advantage of confocal laser scanning microscopy and distinct immunoreactive fluorescent markers in various double-label combinations, several sub-populations of intrinsic cardiac neurons were identified. Of all identified neurons, 85-90% were located in ganglia (ganglionic neurons), the rest being isolated (individual neurons). The two general neuronal markers protein gene product 9.5 (PGP 9.5) and microtubule-associated protein (MAP-2) were associated with neurons clustered primarily in the interatrial septum and around the origins of the two vena cavae. Ganglia (group 1) contained three sub-populations of neurons: approx. 80% of ganglionic neurons were large (15-40 microm diameters; group 1a) and approx. 20% had smaller diameters (less than 15 microm; group 1b). All of these neurons were PGP-immunoreactive, exhibiting choline acetyltransferase (ChAT) immunoreactivity (IR), tyrosine hydroxylase (TH) IR, neuropeptide Y (NPY) IR, vasoactive peptide (VIP) IR and substance P (SP) IR. The remaining 5% of ganglionic neurons were small (group 1c; less than 20 microm). These displayed TH immunoreactivity but not MAP, PGP, CHAT, NPY or SP immunoreactivity. Ten to fifteen percent of all neurons loosely distributed outside of ganglia were small (10-25 microm) and located primarily around the origin of the superior vena cava. They displayed immunoreactivity to TH, ChAT, VIP, NPY and SP, but not to MAP-2 or PGP 9.5. These data provide anatomical and immunohistochemical evidence for specific localization of differing populations of intrinsic cardiac neurons with respect to their size, ganglionic distributions and capacity to express multiple neurotransmitters. Although the functional importance of such a regional distribution of differing populations of intrinsic cardiac neurons remains unknown, these anatomical data support the thesis that unique clustering of specific populations of neurons within this nervous system represents the anatomical substrate for complex local cardiac regulatory phenomena occurring at the level of the target organ.