Neuroscience and Behavioral Physiology, Vol. 43, No. 2, February, 2013
Substance P-Immunopositive Neurons in Rat Spinal Nerve Sensory Ganglia during Postnatal Ontogeny V. V. Porseva,1 V. V. Shilkin,1 M. B. Korzina,2 A. A. Korobkin,2 and P. M. Maslyukov2
UDC 611.892.018.8:612.65:599.323.4
Translated from Morfologiya, Vol. 141, No. 1, pp. 75–77, January–February, 2012. Original article submitted May 27, 2011. We report here immunohistochemical studies addressing substance P (SP)-containing neurons in the spinal nerve sensory ganglia of rats (n = 30) aged 10–90 days. The results showed that SP-immunopositive neurons are present in these ganglia from the moment of birth. The number of SP-containing neurons decreased with age to day 10 of life. SP-immunopositive neurons were very small and small cells. Keywords: neurons, spinal ganglia, substance P, immunohistochemistry, ontogeny.
under urethane anesthesia (3 g/kg, i.p.). neurons in the sensory ganglion of the second thoracic spinal nerve were studied. SP-containing neurons were identified using labeled antibodies (Abcam, USA, diluted 1:1000) as described previously [1, 5]. Secondary antibodies were conjugated with the fluorochrome fluorescein isothiocyanate (FITC) (Jackson, USA), which gives a green fluorescence. The proportion of immunopositive neurons was determined using not only the label for SP, but also a label for the whole neuron population, i.e., the fluorochrome NeuroTrace (Molecular Probes, USA), which fluoresces red. Specimens were analyzed using a Mikromed 2 version 12 fluorescence microscope (LOMO, Russia) with the appropriate set of light filters and an MDS320 CCD camera (Scope-Tec, China). The ImageJ program (NIH, USA, http://rsb.info.nih.gov.ij/) was used to analyze digital images of histological sections of ganglia at a magnification of ×200, with identification of the cross-sectional areas of nerve cell bodies; cells were counted in rectangular areas of 100 μm2 using a grid attachment. Proportions of immunopositive neurons were identified in terms of the ratio of these cells to the total number of neurons, which was taken as 100%. Ganglion neurons were classified into five size classes in terms of cross-sectional area: up to 300 μm2 (very small), 301–600 μm2 (small), 601–900 μm2 (intermediate), 901–1200 μm2 (large), and 1201–1500 μm2 (very large). Neurons whose sections passed through the nucleus with
The neurophysiological mechanisms of pain involve peripheral, segmental, and suprasegmental structures. A number of peptides and their receptors have roles in the perception of pain, these being located in different structures of the nervous system [2, 4, 6]. Data on the neurochemical mechanisms of nociceptor activation are widely known; however, there are as yet no reports on the characteristics of the cellular composition and morphological features of the population of substance P (SP)-containing sensory neurons during postnatal ontogeny. The aim of the present work was to study the morphometric characteristics of SP-immunopositive neurons in the sensory ganglia of spinal nerves during postnatal ontogeny in rats. Materials and Methods Experiments were performed on 30 white female Wistar rats aged 3, 10, 20, 30, 60, and 90 days in compliance with the “Regulations for Studies Using Experimental Animals” (USSR Ministry of Health Decree No. 755 of August 12, 1977). Animals were subjected to euthanasia 1 Department
of Human Anatomy (Director: Professor V. V. Shilkin); e-mail:
[email protected]. 2 Department of Normal Physiology with Biophysics (Director: Professor V. N. Volovenko), Yaroslavl State Medical Academy, Russian Ministry of Health; e-mail:
[email protected].
150 0097-0549/13/4302-0150 ©2013 Springer Science+Business Media New York
Substance P-Immunopositive Neurons in Rat Spinal Nerve Sensory Ganglia
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Fig. 1. Substance P-immunopositive neurons in the sensory ganglion of the second thoracic spinal nerve of rats aged 3 days (a) and 10 days (b). Immunocytochemical reaction. Objective ×20; ocular ×7.
TABLE 1. Morphometric Characteristics of Substance P-Immunopositive Neurons (x ± sx) Proportions of neurons of different size classes (%)
Age (days)
Proportion of neurons in ganglion (%)
Cross-sectional area (μm2)
3
9.5 ± 0.8
10
6.4 ±
0.4*
20
up to 300 μm2
301–600 μm2
217 ± 7
88.4 ± 0.6
12.6 ± 0.6
218 ± 9
82 ± 4
18 ± 4
6.1 ± 0.4*
225 ± 9
77.5 ± 0.4
22.5 ± 0.4
30
5.11 ± 0.24*
256 ± 9*
73.3 ± 0.6
26.7 ± 0.6
60
6.7 ± 0.4*
304 ± 9*
52.4 ± 0.7
47.6 ± 0.7
90
6.5 ± 0.4*
281 ± 8*
64.2 ± 0.5
35.8 ± 0.5
* Significant differences compared with age 3 days, p < 0.05.
visible nucleoli were included in the analysis. Statistical analysis included calculation of the arithmetic mean and standard error. Significant differences were identified using Student’s t test with a criterion of p < 0.05. Results SP-immunopositive neurons were found in the sensory ganglia in rats of all the study groups (Fig. 1). Increases in age were accompanied by decreases in the numbers of SPimmunopositive neurons: by 10 days, the number decreased 1.5-fold, after which there were no further changes to the end of the experiment (Table 1). The cross-sectional areas of SP-containing neurons increased 1.3-fold over the whole of the observation period, reaching a maximum by 60 days of age, after which there was a minor decrease by age 90 days (see Table 1). Analysis of the cellular composition of immunopositive neurons showed that in rats of all age groups studied,
these neurons were of only two size classes: very small and small (see Table 1). At ages 3–30 days, populations of immunopositive neurons were dominated by very small cells – more than 80% at ages 3 and 10 days and more than 70% at ages 20 and 30 days. At age 60 days, the proportion of very small cells decreased, increasing again at age 90 days to a level of greater than 60% (see Table 1). Discussion The results obtained here show that only a small proportion (less than 10%) of neurons in the thoracic spinal nerve sensory ganglion in rats of different ages contain SP, which is consistent with data reported by other investigators using adult animals [3]. SP-immunopositive neurons in sensory ganglia were characterized in terms of their morphological features and were very small and small neurons. The cross-sectional area of SP-immunopositive neurons increased as rats aged, though the proportion of small neu-
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rons was high (greater than 50%) throughout the observation period. Thus, the population of neurons in the sensory ganglia of spinal nerves were already heterogeneous in terms of size and immunohistochemical characteristics by birth. That part of the nociceptive system including SP-immunopositive structures thus appears to be functional in rats at birth. Final formation of this system is complete by age 10 days. This study was supported by grants from the Young Scientists Support Program of the President of the Russian Federation and the Federal Targeted Program on Scientific and Teaching Staff for an Innovative Russia in 2009–2013.
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