Differential distribution of parvalbumin- and calbindin-D28K-immunoreactive neurons in the rat periaqueductal gray matter and their colocalization with enzymes producing nitric oxide

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• 作者：Paolo Barbaresi ; Emanuela Mens脿 ; Vincenzo Lariccia ; Arm ; a Pugnaloni ; Salvatore Amoroso ; Mara Fabri
• 关键词：Calcium binding proteins ; Electron microscopy ; Periaqueductal gray matter ; NADPH-d ; Double-labeling
• 刊名：Brain Research Bulletin
• 出版年：October, 2013
• 年：2013
• 卷：99
• 期：Complete
• 页码：48-62
• 全文大小：5101 K

文摘

The distribution, colocalization with enzymes producing nitric oxide (NO), and the synaptic organization of neurons containing two calcium-binding proteins (CaBPs) - parvalbumin (Parv) and calbindin-D28K (Calb) - were investigated in the rat periaqueductal gray matter (PAG). Parv-immunopositive (Parv_IP) neurons were detected in the mesencephalic nucleus and rarely in the PAG. Calb_IP neurons were found both in the dorsolateral (PAG-dl) and ventrolateral PAG (PAG-vl); their size ranged from 112.96 渭m² (PAG-dl) to 125.13 渭m² (PAG-vl). Ultrastructurally Parv and Calb immunoreactivity was mostly found in dendritic profiles. Axon terminals containing each of the two CaBPs formed symmetric synapses. Moreover both Parv and Calb were used to label a subpopulation of NO-producing neurons. Colocalization was investigated using two protocols: (i) a combination of Calb and Parv immunocytochemistry (Icc) with nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry (Hi) and (ii) neuronal NO synthase-Icc (nNOS) (immunofluorescence).

Both techniques demonstrated a complete lack of colocalization of Parv and NADPH-d/nNOS in PAG neurons.

Double-labeled (DL) neurons (Calb-NADPH-d; Calb-nNOS) were detected in PAG-dl. NADPH-d-Hi/Calb-Icc indicated that 41-47% of NADPH-d-positive neurons contained Calb, whereas 17-23% of Calb_IP cells contained NADPH-d. Two-color immunofluorescence revealed that 53-66% of nNOS_IP cells colocalized with Calb and 24-34% of Calb_IP neurons contained nNOS. DL neuron size was 104.44 渭m²; neurons labeled only with NADPH-d or Calb measured 89.793 渭m² and 113.48 渭m², respectively.

Together with previous findings () these data suggest that:

(i) PAG-dl and PAG-vl contain fast CaBPs, (ii) a high degree of heterogeneity exists in PAG-dl, (iii) two subpopulations of NO-producing neurons containing distinct CaBPs are found in PAG-dl.

Therefore the important aspect of the PAG intrinsic organization emerging from this and previous double-labeling studies is the chemical diversity of NO-synthesizing neurons, which is likely related to the different functions in which these neurons are involved.