In this study, we investigated postnatal changes in Rexed's laminae and distribution of nociceptive afferents in the dorsal horn of the rat lumbar spinal cord at postnatal days 0, 5, 10, 15, 20, and 60. Transverse sections of the L4-L5 segments were processed for triple labeling with isolectin B4 (IB4)-binding as a marker of nonpeptidergic C-fibers, calcitonin gene-related peptide (CGRP) immunoreactivity to label peptidergic nociceptive afferents, and a fluorescent Nissl stain to visualize cells and lamination at different stages of postnatal development. The Nissl staining revealed that the thickness of lamina I (LI) and outer lamina II remained mostly unchanged from birth until adulthood. CGRP afferents terminated mostly in LI and the outer two-thirds of lamina II, whereas the termination area of fibers binding IB4 was centered on the middle one-third of lamina II at all ages studied. In absolute values, the overall width of the bands of intense CGRP and IB4 labeling increased with age but decreased as a percentage of the overall thickness of the dorsal horn with maturation. The overlap of CGRP termination area with that of IB4 afferents increased with age. The consequences of these findings are twofold. First, the size of the different laminae does not grow evenly across the dorsal horn. Second, CGRP and IB4 labeling cannot be considered per se to be reliable markers of lamination during development. These findings have implications for comparing data obtained in immature and mature tissues with respect to localization of structures in the dorsal horn.

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Inhibitory interneurons are an important component of dorsal horn circuitry where they serve to modulate spinal nociception. There is now considerable evidence indicating that reduced inhibition in the spinal dorsal horn contributes to neuropathic pain. A loss of these inhibitory neurons after nerve injury is one of the mechanisms being proposed to account for reduced inhibition; however, this remains controversial. This is in part because previous studies have focused on global measurements of inhibitory neurons without assessing the number of inhibitory synapses. To address this, we conducted a quantitative analysis of the spatial and temporal changes in the number of inhibitory terminals, as detected by glutamic acid decarboxylase 65 (GAD65) immunoreactivity, in the superficial dorsal horn of the spinal cord following a chronic constriction injury (CCI) to the sciatic nerve in rats. Isolectin B4 (IB4) labelling was used to define the location within the dorsal horn directly affected by the injury to the peripheral nerve. The density of GAD65 inhibitory terminals was reduced in lamina I (LI) and lamina II (LII) of the spinal cord after injury. The loss of GAD65 terminals was greatest in LII with the highest drop occurring around 3–4 weeks and a partial recovery by 56 days. The time course of changes in the number of GAD65 terminals correlated well with both the loss of IB4 labeling and with the altered thresholds to mechanical and thermal stimuli. Our detailed analysis of GAD65+ inhibitory terminals clearly revealed that nerve injury induced a transient loss of GAD65 immunoreactive terminals and suggests a potential involvement for these alterations in the development and amelioration of pain behaviour.

Chlorisondamine (CHL) blocks behavioural responses to nicotine for several weeks or months in rats. Persistent blockade has also been demonstrated ex vivo, in assays of nicotine‐evoked striatal dopamine release. Central administration of [3H]‐CHL leads to long‐term retention of radiolabel in nigrostriatal dopaminergic neurons and in few other cell groups. We investigated whether an analogous blockade also occurs in noradrenergic neurons in the brain and in cultured pheochromocytoma (PC12) cells, which have a similar noradrenergic phenotype. Administration of CHL (10 mg kg−1 s.c. or 10 μg i.c.v.), 21 days prior, resulted in a near‐total block of nicotine‐evoked release of hippocampal [3H]‐noradrenaline ([3H]‐NA) from superfused rat synaptosomes; NMDA‐evoked [3H]‐NA release was unaffected. Three weeks after administration of [3H]‐CHL (10 μg i.c.v.), preferential accumulation of radiolabel was observed in the locus coeruleus, which provides the entire noradrenergic innervation to hippocampus, as well as in previously noted structures. In rat pheochromocytoma (PC12) cells, nicotine evoked [3H]‐NA release (EC50 approximately 30 μm). This effect was blocked by co‐incubation with mecamylamine (10 μm) or CHL (1 μm) but was not affected by α‐bungarotoxin. As in the hippocampus, the nicotinic agonist cytisine was at least as efficacious as nicotine. Acute exposure of PC12 cells to CHL 10 or 100 μm (but not 1 μm), followed by 90 min wash‐out, almost completely blocked release evoked by 30 μm nicotine. More prolonged (24 h) exposure to CHL 100 μm (but not 1 or 10 μm), followed by 3 days of wash‐out, partially inhibited release evoked by nicotine, leaving responses to high K+ unchanged. A significant (30%) reduction was also seen 5 days after exposure. We conclude that persistent nicotinic blockade by CHL is neither restricted to mesostriatal dopamine neurons, nor to the CNS, nor to neurons possessing the same nicotinic receptor pharmacology. In addition, the persistent blockade does not appear to result from an acute blocking action, but may be dependent upon intracellular accumulation of the antagonist. British Journal of Pharmacology (1998) 125, 1218–1227; doi:

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