Co-localization of Gamma-Aminobutyric Acid and Glutamate in Neurons of the Spider Central Nervous System

Abstract

Spider sensory neurons with cell bodies close to various sensory organs are innervated by putative efferent axons from the central nervous system (CNS). Light and electronmicroscopic imaging of immunolabeled neurons has demonstrated that neurotransmitters present at peripheral synapses include γ-aminobutyric acid (GABA), glutamate and octopamine. Moreover, electrophysiological studies show that these neurotransmitters modulate the sensitivity of peripheral sensory neurons. Here, we undertook immunocytochemical investigations to characterize GABA and glutamate-immunoreactive neurons in three-dimensional reconstructions of the spider CNS. We document that both neurotransmitters are abundant in morphologically distinct neurons throughout the CNS. Labeling for the vesicular transporters, VGAT for GABA and VGLUT for glutamate, showed corresponding patterns, supporting the specificity of antibody binding. Whereas some neurons displayed strong immunolabeling, others were only weakly labeled. Double labeling showed that a subpopulation of weakly labeled neurons present in all ganglia expresses both GABA and glutamate. Double labeled, strongly and weakly labeled GABA and glutamate immunoreactive axons were also observed in the periphery along muscle fibers and peripheral sensory neurons. Electron microscopic investigations showed presynaptic profiles of various diameters with mixed vesicle populations innervating muscle tissue as well as sensory neurons. Our findings provide evidence that: (1) sensory neurons and muscle fibers are innervated by morphologically distinct, centrally located GABA- and glutamate immunoreactive neurons; (2) a subpopulation of these neurons may co-release both neurotransmitters; and (3) sensory neurons and muscles are innervated by all of these neurochemically and morphologically distinct types of neurons. The biochemical diversity of presynaptic innervation may contribute to how spiders filter natural stimuli and coordinate appropriate response patterns.

The distribution of cholinergic neurons and their co-localization with FMRFamide, in central and peripheral neurons of the spider Cupiennius salei

“…Many ChAT labeled cell bodies were found in similar locations to GABA and/or glutamate immunoreactive neurons in previous studies (Barth 2002; Fabian-Fine et al 2015). One example is given by the anterolateral neuron clusters (ANC) of the leg ganglia that were shown here to have 56 % of somata expressing ChAT.…”

“…Therefore, it is likely that, as in other arthropods, the major fast acting excitatory motor neurons in spiders are not cholinergic, but more likely use glutamate as their primary transmitter (Takeuchi and Takeuchi 1964; Usherwood et al 1968, Jan and Jan 1976). Further evidence for this is that many large neurons in the subesophageal ganglia were previously shown to be immunoreactive to glutamate and/or GABA, and both transmitters were also present in axon terminals innervating the leg muscle (Fabian-Fine et al 2015). However, some of the smaller Type D neurons that were also labeled by ChAT may be cholinergic motoneurons that act on muscarinic ACh receptors and modulate muscle excitability either directly or presynaptically via other motor neurons.…”

“…Previous studies have described immunoreactivity to other neurotransmitters in some of the same visual areas. For example, all C. salei optic neuropils were immunoreactive to GABA (Becherer and Schmid 1999) and many globuli cells were labeled by antisera for both GABA and glutamate (Fabian-Fine et al 2015). Extensive labeling with anti-FMRFamide was also found in all optic neuropils while proctolin and octopamine limmunoractive cells were less common (Seyfarth et al 1993; Becherer and Schmid 1999: Loesel et al 2011).…”

“…After removal of muscle tissue, the underlying hypodermis containing sensory neurons attached to the cuticle was gently pulled away from the cuticle, with part of the hypodermis left attached to the cuticle to prevent damage to the delicate hypodermis during processing. After the immunolabeling procedure was completed, the hypodermis was detached from the cuticle and mounted on glass slides in Mowiol medium, prepared as described previously (Fabian-Fine et al 2015). The preparations were left to set overnight at 6°C.…”

“…It is also likely that some efferent fibers contain more than one neurotransmitter, as in many neurons across all animal phyla (Trudeau 2004; Salio et al 2006). Recently, the spider CNS was shown to contain neurons immunoreactive to both GABA and glutamate as well as their transporters (Fabian-Fine et al 2015). Some of these neurons are probably efferents innervating the sensory neurons.…”