Immunohistochemical characterization of TH13‐L2 spinal ganglia neurons in sheep (Ovis aries)

Abstract

Spinal ganglia (SG) neurons are commonly classified according to various specific features. The most widespread classification based on morphological and ultrastructural features subdivides SG neurons into light and small dark neurons. Using immunohistochemical, histochemical and lectin methods, it is possible to further subdivide the small dark neurons into two subpopulations: peptidergic and nonpeptidergic neurons. The majority of studies on SG neurons were carried out on mice and rats; there are few or no studies on large mammals. In this study, some of the widely used neuronal markers, neurofilament 200 kDa (NF200), substance P (SP), calcitonin gene-related peptide (CGRP) and isolectin B4 (IB4), were employed to characterize neuronal nitric oxide synthase (nNOS)-immunoreactivity (-IR) in sheep (Ovis aries) SG (Th13-L2) neurons. The majority of the SG neurons were IB4-labeled (79 +/- 10%), followed by NF200- (45 +/- 4%), NOS- (44 +/- 10%), SP- (42 +/- 5%) and CGRP-IR (35 +/- 7%) neurons. The triple staining experiments showed that a higher percentage (75 +/- 16%) of NOS-IR neurons bound both IB4 and CGRP, or both IB4 and SP (49 +/- 6%). The IB4 marker showed an unexpected staining pattern; in fact, IB4-labeled neurons largely colocalized with NF200, usually considered a marker of light SG neurons, and with CGRP and SP. For this reason, IB4 cannot be employed in sheep to differentiate between light and dark neurons, or between peptidergic and nonpeptidergic neurons. These results suggest the importance of being cautious when comparing data among different species.

Quantitative analysis of afferents expressing substance P, calcitonin gene‐related peptide, isolectin B4, neurofilament 200, and Peripherin in the sensory root of the rat trigeminal ganglion

“…Most IB41 fibers were unmyelinated, and about 5% were small myelinated, corroborating previous electrophysiological (Gerke and Plenderleith, 2001;Fang et al, 2006) and LM immunohistochemical (Wang et al, 1994;Russo et al, 2010) evidence in the DRG. Previous EM studies in the peripheral nerve and the spinal cord reported that IB4 is expressed exclusively in unmyelinated fibers (Streit et al, 1986;Kitchener et al, 1993;Murinson et al, 2005), possibly because of loss of myelin sheath in the IB41 myelinated fibers in the periphery.…”

Sympathetic innervation of the ileocecal junction in horses

“…The anti-nNOS antibody (Santa Cruz Biotechnology, Santa Cruz, CA; (A-11) sc-530) employed in this study was tested in a double-staining protocol with a polyclonal anti-nNOS antibody raised against amino acids 1414-1434 of human nNOS (Biomol, Plymouth Meeting, PA; SA227). The two antibodies showed a full correspondence, with the same fibers and neurons stained, as recently shown on sheep DRG (Russo et al, 2010).…”

Distribution of Nitrergic Neurons in the Dorsal Root Ganglia of the Bottlenose Dolphin (Tursiops truncatus)

“…In this study, immunoperoxidase and double immunofluorescence techniques were used to determine the distribution and the number of nitrergic neurons in the DRGs. As nitrergic neurons of the DRGs contain tachykinins (Aimi et al, 1991;Zhang et al, 1993;Ruscheweyh et al, 2006;Tan et al, 2008;Russo et al, 2010) in others mammals, double immunofluorescence was used to determine if nNOS is colocalized with SP also in Tursiops truncatus.…”

“…NO regulates many functions in the DRGs, including transmission of visceral sensation, primary afferent neurons development and differentiation, communication between neurons and satellite glial cells, and neuroprotection (Aimi et al, 1991;Thippeswamy and Morris, 2002;Tan et al, 2008;Russo et al, 2010). In addition, immunohistochemical, electrophysiological, and pharmacological studies have suggested that NO acts as a neu-rotransmitter or neuromodulator in the processing of nociceptive stimuli.…”