Presynaptic inhibition of primary muscle spindle (group Ia) afferent terminals in motor nuclei of the spinal cord plays an important role in regulating motor output and is produced by a population of GABAergic axon terminals known as P boutons. Despite extensive investigation, the cells that mediate this control have not yet been identified. In this work, we use immunocytochemistry with confocal microscopy and EM to demonstrate that P boutons can be distinguished from other GABAergic terminals in the ventral horn of rat and mouse spinal cord by their high level of the glutamic acid decarboxylase (GAD) 65 isoform of GAD. By carrying out retrograde labeling from lamina IX in mice that express green fluorescent protein under the control of the GAD65 promoter, we provide evidence that the cells of origin of the P boutons are located in the medial part of laminae V and VI. Our results suggest that P boutons represent the major output of these cells within the ventral horn and are consistent with the view that presynaptic inhibition of proprioceptive afferents is mediated by specific populations of interneurons. They also provide a means of identifying P boutons that will be important in studies of the organization of presynaptic control of Ia afferents.GABA ͉ Ia afferent ͉ presynaptic inhibition T he inhibitory transmitter GABA is used by many neurons in the spinal cord and generates both postsynaptic inhibition at axo-dendritic and axo-somatic synapses and presynaptic inhibition at axo-axonic synapses (1). GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD), and Abs against GAD have been used to identify GABAergic axonal boutons in the spinal cord (2, 3). More recently, two forms of the enzyme have been identified, and based on their molecular weights these forms have been named GAD65 and GAD67 (4). Both forms are present in the ventral horn of the rat spinal cord but have a very different distribution (5, 6). The majority of GABAergic boutons throughout the ventral horn show strong GAD67 immunoreactivity and a low level of GAD65. However, there are clusters of boutons in lamina IX that contain very high levels of GAD65. We have suggested (6) that these clusters may correspond to the P boutons that form axo-axonic synapses with terminals of group Ia afferents (7-10). In this work, we have used a variety of approaches to confirm this hypothesis and to identify the cells of origin of these boutons. MethodsAll experiments were approved by the Ethical Review Process Applications Panel of the University of Glasgow or the Animal Care and Protection Committee at the University of Debrecen and were performed in accordance with the U.K. Animals (Scientific Procedures) Act 1986 and the European Communities Council Directives. Immunocytochemical reactions were performed on free-floating 60-m transverse Vibratome sections that had been treated with 50% ethanol for 30 min to enhance Ab penetration. Abs were diluted in PBS with 0.3% Triton X-100 (except on sections used for EM).Primary Afferent Labeling. Transgang...

FMRFamide-related peptides are widespread neurotransmitters or neurohormones regulating somatic or visceral motor activity. Some recent data indicate that these neuropeptides may be involved in the control of cell proliferation and apoptosis. In this work we investigated the possible effect of FMRFamide on cell viability in an invertebrate-type proliferating tissue. As a model, we used the midintestinal gland of the snail, Helix lucorum Linnaeus. Immunohistochemistry demonstrated the direct innervation of the gland cells by FMRFamide-containing nerve fibers. Midintestinal glands of snails were injected with 50 microM FMRFamide and the control with sterile deionised water or bovine serum albumin (BSA). Injections were administrated 4 times. Transmission electron microscopy, annexin V-labeling, thiazolyl blue (MTT) viability tests and ploidy analyses were carried out to define the viable/dead cell ratio in the tissue samples. FMRFamide increased the MTT-reduction of tissues, reduced the amount of apoptotic nuclei and annexin V-labeled cells. Deionised water or BSA injection induced cell death. Cell cycle analysis revealed that FMRFamide significantly elevated the amount of cells in G0/G1 phase, but did not induce mitosis. We conclude, that the FMRFamide can be a life-signal for cells, protect them from apoptosis without altering mitosis.