A-fiber sensory input induces neuronal cell death in the dorsal horn of the adult rat spinal cord.

Excitotoxicity due to excessive synaptic glutamate release is featured in many neurological conditions in which neuronal death occurs. Whether activation of primary sensory pathways can ever produce sufficient over-activity in secondary sensory neurons in the dorsal horn of the spinal cord to induce cell death, however, has not been determined. In this study, we asked whether activity in myelinated afferents (A fibers), which use glutamate as a transmitter, can induce cell death in the dorsal horn. Using stereological estimates of neuron numbers from electron microscopic sections, we found that stimulation of A-fibers in an intact sciatic nerve at 10 Hz, 20 Hz, and 50 Hz in 10-minute intervals at a stimulus strength that activates both Abeta and Adelta fibers resulted in the loss of 25% of neurons in lamina III, the major site of termination of large Abeta fibers, but not in lamina I, where Adelta fibers terminate. Furthermore, sciatic nerve lesions did not result in detectable neuron loss, but activation of A fibers in a previously sectioned sciatic nerve did cause substantial cell death not only in lamina III but also in laminae I and II. The expansion of the territory of A-fiber afferent-evoked cell death is likely to reflect the sprouting of the fibers into these laminae after peripheral nerve injury. The data show, therefore, that primary afferent A-fiber activity can cause neuronal cell death in the dorsal horn with an anatomical distribution that depends on whether intact or injured fibers are activated. Stimulation-induced cell death potentially may contribute to the development of persistent pain.

Loss of dorsal root ganglion cells concomitant with dorsal root axon sprouting following segmental nerve lesions.

Tight ligation of the fifth and sixth lumbar segmental nerves in the rat provides a model of neuropathic pain. We used this model to assess the changes in primary afferent input to the dorsal horn in neuropathic pain syndromes. Dorsal roots and ganglia were examined for up to 32 weeks following segmental nerve ligation. Stereologic and morphometric techniques revealed a notable decrease in the numbers of dorsal root ganglion cells and unmyelinated dorsal root axons by six weeks post-injury. By 32 weeks following segmental nerve ligations, the numbers of dorsal root ganglion cells have dropped to 50% of pre-ligation levels while the numbers of dorsal root axons have increased to normal levels predominantly due to sprouting of myelinated fibres. These findings indicate that although there is a great loss of dorsal root ganglion cells, there is dramatic sprouting of myelinated fibres and possibly some sprouting of unmyelinated fibres in the dorsal roots. Additionally, a difference in the responses of unmyelinated and myelinated fibres to this peripheral nerve injury is revealed. These changes in dorsal root ganglion cells and their central axons may underlie certain aspects of abnormal pain syndromes because of changes in the types and quantity of input the dorsal horn receives.

Central changes in primary afferent fibers following peripheral nerve lesions.

Cutting or crushing rat sciatic nerve does not significantly reduce the number of central myelinated sensory axons in the dorsal roots entering the fourth and fifth lumbar segments even over very extended periods of time. Unmyelinated axons were reduced by approximately 50%, but only long after sciatic nerve lesions (four to eight months), and reinnervation of the peripheral target did not rescue these axons. This indicates that a peripheral nerve lesion sets up a slowly developing but major shift towards large afferent fiber domination of primary afferent input into the spinal cord. In addition, since myelinated axons are never lost, this is good evidence that the cells that give rise to these fibers are also not lost. If this is the case, this would indicate that adult primary sensory neurons with myelinated axons do not depend on peripheral target innervation for survival.

Sprouting of A beta fibers into lamina II of the rat dorsal horn in peripheral neuropathy.

Cholera toxin beta-subunit conjugated to horseradish peroxidase was used to label the large myelinated (A beta) fiber input to the dorsal horn in a model of peripheral neuropathy induced by tight ligation of the L5 and L6 spinal nerves. Following induction of neuropathy, A beta fibers were present in lamina II of the ipsilateral dorsal horn, a region normally devoid of A beta input. This reorganization of large fiber input to the superficial dorsal horn provides some anatomical basis for sensory changes found in this model of neuropathic pain.

Methods for determining numbers of cells and synapses: a case for more uniform standards of review.

Neuron and synapse numbers are important assays in neuroscience. These numbers are estimated by one of four methods: 1) profile counts, 2) assumption-based methods, 3) serial reconstructions, and 4) stereological methods. The criteria for these methods are diverse. This creates a disparity in that some reviewers accept estimates from any of these methods, while others accept only specific methods. An equally important issue is the diversity of sampling strategies, since unbiased estimates of neuronal or synaptic numbers are contingent upon both counting and sampling techniques. The purpose of this commentary is to institute a dialog that will lead to a better understanding of the strengths and weaknesses of the above methods, and to propose guidelines that should lead to more uniform and thus fairer judging of the studies that provide estimates of neuron or synapse numbers. In addition, adoption of more uniform standards for obtaining unbiased numerical estimates should result in the generation of an unbiased database that will be of considerable use in future studies.

Glutamatergic and GABAergic input to rat spinothalamic tract cells in the superficial dorsal horn.

The distribution of synaptic terminals onto spinothalamic tract cells (types I and II) of the superficial dorsal horn was determined with special reference to the amino acid transmitters glutamate and gamma-aminobutyric acid. Fifteen spinothalamic cells retrogradely labeled from the thalamus with the neuroanatomical tracer wheatgerm agglutinin conjugated to horseradish peroxidase were sectioned for electron microscopy. Serial sections from several levels through each cell were immunostained for glutamate and gamma-aminobutyric acid using a postembedding immunogold technique. Perimeter measurements of spinothalamic cell somata and dendrites and the lengths of apposition for all terminal profiles in contact with the spinothalamic cells were obtained from electron micrographs using a digitizing tablet. These data were used to determine the density of terminals on the soma and dendrites. In addition, the terminal population on these cells was categorized by transmitter content (glutamate, gamma-aminobutyric acid, or unlabeled). The results demonstrate that terminal density increased on dendrites relative to their distance from the soma. Glutamatergic and GABAergic input composed 37% and 20% of the terminal population, respectively, and these percentages remained uniform for the soma and dendrites. There were no significant differences among the 15 cells analyzed for this study. The results, therefore, suggest that both type I and type II STT cells of the superficial DH have similar synaptic organizations.

Effects of sinoaortic denervation on glucose utilization in the subfornical organ and pituitary neural lobe during administration of angiotensin II.

Angiotensin II infused intravenously into sinoaortic-denervated rats induced drinking and increased glucose utilization in the subfornical organ and pituitary neural lobe in amounts not different from those observed in sham-operated animals. We suggest that inputs from baroreceptors have a negligible influence on glucose metabolism in the subfornical organ during infusion of angiotensin II.