Evidence for GABAergic interneurons in the red nucleus of the painted turtle

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Abstract
Immunocytochemical and electrophysiological evidence supporting the presence of GABAergic interneurons in the turtle red nucleus is presented. Injections of HRP into the spinal cord produced labeling of large neurons in the contralateral red nucleus. The peroxidase-antiperoxidase (PAP) method revealed smaller cells immunoreactive to an antibody against glutamate decarboxylase (GAD), the synthetic enzyme for the inhibitory neurotransmitter GABA, that were interspersed among larger immunonegative neurons. Similar small neurons were densely immunostained by antibodies to GABA-glutaraldehyde conjugates obtained from different sources and applied according to pre-embedding and postembedding protocols. Rubrospinal neurons retrogradely labeled with HRP measured 16 and 27 μm in mean minor and major cell body diameters, while GABA-like immunopositive neurons situated within the red nucleus measured 7 and 13 μm. There was very little overlap in soma size between the two cell populations. Therefore, we suggest that the GAD- and GABA-positive neurons may be local inhibitory interneurons. This nition is further supported by observations of pre-embedding immunostaining for GAD and postembedding immunostaining for GABA showing that the turtle red nucleus is amply innervated by immunoreactive axon terminals. These puncta are closely apposed to cell bodies and dendrites of both immunonegative large neurons and immunopositive small neurons. Moreover, immunogold staining at the electron microscopic level demonstrated that GABA-like immunoreactive axon terminals with pleomorphic synaptic vesicles formed symmetric synapses with cell bodies and dendrites of the two types of red nucleus cells. These ultrastructural features are commonly assumed to indicate inhibitory synapses. A moderately labeled bouton with round vesicles and asymmetric synapses was also observed. In addition, the two types of red nucleus neurons received asymmetric axosomatic and axodendritic synapses with GABA-negative boutons provided with round vesicles, features usually associated with excitatory functions. To obtain electrophysiological evidence for inhibition, intracellular recordings from red nucleus neurons were conducted using an in vitro brainstem-cerebellum preparation from the turtle. Small, spontaneous IPSPs were recorded from 7 out of 14 red nucleus cells studied. These morphological and physiological results provide strong support for concluding that the turtle red nucleus, like its mammalian counterpart, contains GABAergic inhibitory interneurons. While we have not indentified the main source of input to these interneurons, in view of the scarce development of the reptilian cerebral cortex, this input is unlikely to come from the motor cortex as it does in mammals. Current concepts of the role of these interneurons in motor control must take into account the presence of this interneuronal system that phylogenetically precedes the emergence of the sensorimotor cortex. © Wiley-Liss, Inc.