Neurochemical classification and projection targets of CART peptide immunoreactive neurons in sensory and parasympathetic ganglia of the head

Abstract

The aims of the present study were to determine if there is neuronal Cocaine and amphetamine regulated transcripts (CART) peptide expression (CART+) in parasympathetic (sphenopalatine (SPG); otic (OG)) and sensory (trigeminal (TG)) ganglia of the head and to examine the neurochemical phenotype (calcitonin gene-related peptide (CGRP), neurofilament 200 (NF200), isolectin B4 (IB4) binding, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY) and enkephalin (ENK) immunoreactivity) and projection targets (lacrimal gland (LG), parotid gland (PG), nasal mucosa (NM), temporomandibular joint (TMJ), middle cerebral artery (MCA) and middle meningeal artery (MMA)) of CART expressing neurons in these ganglia. We found CART+ neurons in both the SPG (5.25 ± 0.07%) and OG (4.32 ± 0.66). A significant proportion of these CART+ neurons contained VIP, NPY or ENK (34%, 26% and 11%, respectively). SPG neurons retrogradely labelled from the lacrimal gland (29%) were CART+, but we were unable to demonstrate CART+ labelling in any of the SPG or OG neurons labelled from other targets. This supports a role for CART peptides in lacrimation or regulation of vascular tone in the lacrimal gland, but not in salivation or nasal congestion. CART+ neurons were also present in the trigeminal ganglion (1.26 ± 0.38%), where their size distribution was confined almost completely to neurons smaller than 800 μm² (mean = 410 μm²; 98% < 800 μm²), and were almost always CGRP+, but did not bind IB4. This is consistent with a role for CART peptides in trigeminal pain. However, there were few CART+ neurons amongst any of the trigeminal neurons retrogradely labelled from the targets we investigated and thus we cannot comment on the tissue type where such pain may have originated. Our study shows that some specialization of CART peptide expression (based on neurochemical phenotype and target projection) is evident in sensory and parasympathetic ganglia of the head.

Introduction

Cocaine and amphetamine regulated transcripts (CART) are mRNA that encode for the CART propeptide (proCART). ProCART has a long form and a short form, and each can undergo post-translational processing that results in the production of a number of biologically active CART peptides see (Rogge et al., 2008). Two of these CART peptides (CART 55–102 and CART 62–102) have been isolated in rat tissues and derive from the long form of proCART (Thim et al., 1999). In humans, only the short form of proCART is produced and the active peptides derived from this short form are called CART 42–89 and CART 49–89 (Rogge et al., 2008). The amino acid sequences of CART 55–102 and CART 62–102 are in fact identical to CART 42–89 and CART 49–89, respectively (the different amino acid numbering system is based on whether peptides are derived from the long or short form of proCART).

CART peptide immunoreactivity has been reported in dense core vesicles within nerve terminals (Dun et al., 2002, Smith et al., 1997), suggesting CART peptides are secreted from nerve terminals and might act as neurotransmitters or neuromodulators. CART mRNA and peptides have a wide distribution throughout the mammalian central nervous system. They have been reported in the nucleus accumbens, amygdala, olfactory bulbs, hypothalamus, somatosensory cortical barrels, thalamus, brain stem and cerebellum, and supraspinal and spinal cord territories associated with pain processing (Douglass et al., 1995, Koylu et al., 1998, Koylu et al., 1997). Given this wide distribution, it is not surprising that CART peptides have been implicated in a variety of physiological processes, including reward and reinforcement, feeding/apetite, stress, anxiety, autonomic regulation, sensory–motor processing and pain (Dominguez et al., 2004, Hunter et al., 2004, Koylu et al., 2006, Koylu et al., 1998, Koylu et al., 1997, Stanek, 2006, Vicentic and Jones, 2007).

In the peripheral nervous system, CART peptides have been reported in small diameter sensory neurons in the dorsal root ganglia (DRG) (Kozsurek et al., 2007), vagal afferent neurons in the nodose ganglia (de Lartigue et al., 2007, De Lartigue G et al., 2010), autonomic efferent neuronal somata in the cardiac ganglia (parasympathetic) and fibres in the stellate ganglia (sympathetic) (Gonsalvez et al., 2010, Richardson et al., 2006). These findings are consistent with a role for CART peptides in pain processing and autonomic regulation of the vasculature. However, there is only a single report of CART peptide expression in peripheral ganglia of the head. Superior cervical ganglion contains CART immunoreactive fibres that originate in preganglionic sympathetic neurons of the upper thoracic spinal cord (Gonsalvez et al., 2010). The aim of the present study was to determine if there is neuronal CART peptide expression in parasympathetic (sphenopalatine (SPG); otic (OG)) and sensory (trigeminal (TG)) ganglia of the head and examine the characteristics (neurochemical phenotype and projection targets) of CART expressing neurons in these ganglia.