Putative Neurotransmitters Research Articles

In vivo monitoring of cerebral agmatine by microdialysis and capillary electrophoresis

Agmatine is a putative neurotransmitter in the brain. Current analytical techniques do not allow the detection of agmatine in extracellular fluid, making it difficult to study its physiological role. However, a new method for in vivo monitoring agmatine in the brain was developed. Capillary zone electrophoresis and laser induced fluorescence detection (CZE–LIFD) was used to measure nanomolar concentrations of agmatine in submicroliter sample volumes. This analytical technique proved to detect 0.49 attomole of agmatine improving the sensitivity of previous analytical techniques. On the other hand, the hippocampus is a brain region well known for having a population of agmatine containing neurons. Therefore, intracerebral microdialysis was performed in the hippocampus and agmatine was extracted from the extracellular environment. Detectable amounts of agmatine were found in dialysates from probes located in the hippocampus but not from the probes located in the lateral ventricle. Furthermore, extracellular agmatine was calcium and impulse dependent and depolarization of hippocampal neurons increased extracellular agmatine concentration. The methods reported here are sensitive enough to study the physiological role of brain agmatine in freely moving animals.

Spatial learning-induced accumulation of agmatine and glutamate at hippocampal CA1 synaptic terminals

Agmatine, the decarboxylated metabolite of l-arginine, is considered to be a novel putative neurotransmitter. Recent studies have demonstrated that endogenous agmatine may directly participate in the processes of spatial learning and memory. Agmatine-immunoreactivity has been observed within synaptic terminals of asymmetric excitatory synapses in the hippocampal CA1 stratum radiatum (SR), suggesting that agmatine may be colocalized with glutamate. In the present study we demonstrate, using immunofluorescence confocal microscopy, that agmatine is colocalized with glutamate within CA1-CA3 hippocampal pyramidal cell bodies, in young Sprague-Dawley rats. Subcellular investigation, using postembedding electron microscopy-immunogold cytochemistry, has also revealed that agmatine is colocalized with glutamate in most synaptic terminals in the SR region of CA1. Ninety-seven percent of all agmatinergic profiles were found to contain glutamate, and 92% of all glutamatergic profiles contained agmatine (n=6; 300 terminals). Alterations in colocalized agmatine and glutamate levels in the SR synaptic terminals, following 4 days Morris water maze training, were also investigated. Compared with swim only control rats, water maze-trained rats had statistically significant increases in both agmatine (78%; P<0.01) and glutamate (41%; P<0.05) levels within SR terminals synapsing onto CA1 dendrites. These findings provide the first evidence that agmatine and glutamate are colocalized in synaptic terminals in the hippocampal CA1 region, and may co-participate in spatial learning and memory processing.

Gaba Transport Inhibitors and Seizure Protection: The Past and Future

Since it was first reported approximately 40 years ago that putative amino acid neurotransmitters, including GABA, would likely be inactivated by synaptic high-affinity transporters, there has been an exponential increase in interest in delineating the pharmacological characteristics of these transporters. During the 1980s and 1990s a large series of publications was devoted to a detailed characterization of neuronal and astroglial GABA transporters demonstrating important differences between these, a notion that turned out to be of relevance for the development of anticonvulsants targeting GABA transporters. The cloning era, leading to the identification of four proteins capable of transporting GABA across plasma membranes, has further boosted this research. Ultimately the clinically active antiepileptic drug, tiagabine, was developed and it was established that its mechanism of action involved inhibition of the GABA transporter-1 (GAT1). Current and future research is directed towards a better understanding of how extrasynaptic GABA receptors may be regulated via manipulation of extrasynaptic GABA levels, possibly involving extrasynaptic GABA transporters, most likely non-GAT1 transporters.

The cardiac neuronal hierarchy and susceptibility to arrhythmias

The cardiac neuronal hierarchy and susceptibility to arrhythmias

Spatial learning‐induced increase in agmatine levels at hippocampal CA1 synapses

Agmatine, a metabolite of L-arginine, is considered as a novel putative neurotransmitter. It has been detected in axon terminals that synapse with pyramidal cells in the hippocampus, a brain region that is critically involved in spatial learning and memory. However, the role of agmatine in learning and memory is poorly understood. Recently, we demonstrated water maze training-induced increases in tissue levels of agmatine in the CA1 subregion of the hippocampus. This finding has raised an issue whether an endogenous agmatine could directly participate in learning and memory processes as a neurotransmitter. In the present study, quantitative immunogold-labeling and electron-microscopical techniques were used to analyze the levels of agmatine in CA1 stratum radiatum (SR) terminals (n = 600) of male Sprague-Dawley rats that had been trained to find a hidden escape platform in the water maze (WM) task or forced to swim (SW) in the pool with no platform presented. Agmatine levels were significantly increased by ∼85% in the synaptic terminals of SR of trained WM group compared with the SW control group (all P < 0.001). These results, for the first time, demonstrate spatial learning-induced elevation in agmatine levels at synapses in the hippocampus and provide evidence of its participation in learning and memory processing as a novel neurotransmitter.

Spontaneous Electrical Activity in the Nervous System and its Modification by Biogenic Amines in the Silkworm &lt;i&gt;Bombyx Mori &lt;/i&gt;(L)

The present study was carried out to examine the effects of biogenic amines on the spontaneous electrical activity of the nervous system in the silkworm Bombyx mori. The activity recorded from different segments of the ventral nerve cord differed in the frequency and number of spike categories firing. The activity was highest between the subesophageal ganglion and 1st thoracic ganglion. Lower activity was recorded in the thoracic part of the cord and the activity increased in the abdominal cord. More or less the same level of activity was recorded from all the abdominal segments of the cord. The above trend was the same from the 1st day to the 7th day of the 5th instar of the silkworm. However, the overall level of activity increased from the 1st day to the 7th day in tune with the increasing complexity of the peripheral nervous system in the silkworm as the 5th instar progressed. Treatment of the cord with solutions of putative neurotransmitter substances, viz. epinephrine (EP), norepinephrine (NEP), dopamine (DA) and 5-hydroxytryptamine (5-HT) at different concentrations from 1x10-8 M to 1x10-4 M showed elevation at lower concentrations anddecrease at 1x10-4 M. The concentration at which maximum elevation could be elicited varied between the above transmitters. Biochemical analyses revealed the presence of these biogenic amines in the silkworm nervous system. Thus the possibility remains that one or more of these substances could act as neurotransmitters in the silkworm nervous system.Keywords: Silkworm; 5th Instar; Spontaneous electrical activity;Biogenic amines; Neurotransmitters.

The agmatine-degrading enzyme agmatinase: a key to agmatine signaling in rat and human brain?

Agmatinase, an ureohydrolase belonging to the arginase family, is widely expressed in mammalian tissues including the brain. Here, it may serve two different functions, the inactivation of the arginine derivative agmatine, a putative neurotransmitter, and the formation of the diamine putrescine. In order to identify the cellular sources of agmatinase expression in the brain, we generated a polyclonal monospecific antibody against recombinant rat agmatinase. With immunocytochemistry, selected areas of rat and human brain were screened. Clearly, in both species agmatinase-like immunoreactivity was predominantly detected in distinct populations of neurons, especially cortical interneurons. Also, principal neurons in limbic regions like the habenula and in the cerebellum robustly expressed agmatinase protein. When comparing the overall agmatinase expression with immunocytochemical data available for agmatine and polyamine biosynthetic enzymes, the observed pattern may argue in favor of an agmatine inactivating function rather than fueling the alternative pathway of polyamine synthesis. The putative neurotransmitter agmatine is seemingly involved with mental disorders. Therefore, agmatinase may be similarly important for pathogenesis. The normal expression profile of the protein as described here may therefore be altered under pathological conditions.

Feline Immunodeficiency Virus as a Gene Transfer Vector in the Rat Nucleus Tractus Solitarii

Gene transfer has been used to examine the role of putative neurotransmitters in the nucleus tractus solitarii (NTS). Most such studies used adenovirus vector-mediated gene transfer although adenovirus vector transfects both neuronal and non-neuronal cells. Successful transfection in the NTS has also been reported with lentivirus as the vector. Feline immunodeficiency virus (FIV), a lentivirus, may preferentially transfect neurons and could be a powerful tool to delineate physiological effects produced by altered synthesis of transmitters in neurons. However, it has not been studied in NTS. Therefore, we sought to determine whether FIV transfects rat NTS cells and to define the type of cell transfected. We found that injection of FIV encoding LacZ gene (FIVLacZ) into the NTS led to transfection of numerous NTS cells. Injection of FIVLacZ did not alter immunoreactivity (IR) for neuronal nitric oxide synthase, which we have shown resides in NTS neurons. A majority (91.7 +/- 3.9%) of transfected cells contained IR for neuronal nuclear antigen, a neuronal marker; 2.1 +/- 3.8% of transfected cells contained IR for glial fibrillary acidic protein, a glial marker. No transfected neurons or fibers were observed in the nodose ganglion, which sends afferents to the NTS. We conclude that FIV almost exclusively transfects neurons in the rat NTS from which it is not retrogradely transported. The cell-type specificity of FIV in the NTS may provide a molecular method to study local physiological functions mediated by potential neurotransmitters in the NTS.

Storage and secretion of β‐NAD, ATP and dopamine in NGF‐differentiated rat pheochromocytoma PC12 cells

In nerve-smooth muscle preparations beta-nicotinamide adenine dinucleotide (beta-NAD) has emerged as a novel extracellular substance with putative neurotransmitter and neuromodulator functions. beta-NAD is released, along with noradrenaline and adenosine 5'-triphosphate (ATP), upon firing of action potentials in blood vessels, urinary bladder and large intestine. At present it is unclear whether noradrenaline, ATP and beta-NAD are stored in and released from common populations of synaptic vesicles. The answer is unattainable in complex systems such as nerve-smooth muscle preparations. Adrenal chromaffin cells are thus used here as a single-cell model to examine mechanisms of concomitant neurosecretion. Using high-performance liquid chromatography techniques with electrochemical and fluorescence detection we simultaneously evaluated secretion of dopamine (DA), ATP, adenosine 5'-diphosphate, adenosine 5'-monophosphate, adenosine, beta-NAD and its immediate metabolites ADP-ribose and cyclic ADP-ribose in superfused nerve growth factor-differentiated rat pheochromocytoma PC12 cells. beta-NAD, DA and ATP were released constitutively and upon stimulation with high-K(+) solution or nicotine. Botulinum neurotoxin A tended to increase the spontaneous secretion of all substances and abolished the high-K(+)-evoked release of beta-NAD and DA but not of ATP. Subcellular fractionation by continuous glycerol and sucrose gradients along with immunoblot analysis of the vesicular marker proteins synaptophysin and secretogranin II revealed that beta-NAD, ATP and DA are stored in both small synaptic-like vesicles and large dense-core-like vesicles. However, the three substances appear to have different preferential sites of release upon membrane depolarization including sites associated with SNAP-25 and sites not associated with SNAP-25.

Development of central neurotransmitter-specified neuronal systems: implications for pediatric neuro-psychiatric disorders.

An increasing focus on the mechanism of synaptic neurochemistry in pediatric neurology, may lead to a better understanding of the pathophysiology of many disorders and result in a more rational approach to their pharmacotherapy. With the burgeoning list of putative neurotransmitters in brain, and the growing evidence of co-localization of many of these neurotransmitters, chemical neurotransmission likely involves a higher degree of complexity than appreciated heretofore. The potential role of neurotransmitter dysfunction in the pathophysiology of neurologic and behavior disorders of children, should not be considered as restricted to those disorders that involve selective neuronal loss, but may encompass a much wider spectrum of syndromes due to metabolic abnormalities, as well as disturbances of the finer features of chemical neurotransmission.

Differential expression of canonical (classical) transient receptor potential channels in guinea pig enteric nervous system

The canonical transient receptor potential (TRPC) family of ion channels is implicated in many neuronal processes including calcium homeostasis, membrane excitability, synaptic transmission, and axon guidance. TRPC channels are postulated to be important in the functional neurobiology of the enteric nervous system (ENS); nevertheless, details for expression in the ENS are lacking. Reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry were used to study the expression and localization of TRPC channels. We found mRNA transcripts, protein on Western blots, and immunoreactivity (IR) for TRPC1/3/4/6 expressed in the small intestinal ENS of adult guinea pigs. TRPC1/3/4/6-IR was localized to distinct subpopulations of enteric neurons and was differentially distributed between the myenteric and submucosal divisions of the ENS. TRPC1-IR was widely distributed and localized to neurons with cholinergic, calretinin, and nitrergic neuronal immunochemical codes in the myenteric plexus. It was localized to both cholinergic and noncholinergic secretomotor neurons in the submucosal plexus. TRPC3-IR was found only in the submucosal plexus and was expressed exclusively by neuropeptide Y-IR neurons. TRPC4/6-IR was expressed in only a small population of myenteric neurons, but was abundantly expressed in the submucosal plexus. TRPC4/6-IR was coexpressed with both cholinergic and nitrergic neurochemical codes in the myenteric plexus. In the submucosal plexus, TRPC4/6-IR was expressed exclusively in noncholinergic secretomotor neurons. No TRPC1/3/4/6-IR was found in calbindin-IR neurons. TRPC3/4/6-IR was widely expressed along varicose nerve fibers and colocalized with synaptophysin-IR at putative neurotransmitter release sites. Our results suggest important roles for TRPC channels in ENS physiology and neuronal regulation of gut function.

DOPA cyclohexyl ester, a DOPA antagonist, blocks the depressor responses elicited by microinjections of nicotine into the nucleus tractus solitarii of rats

Nicotinic cholinergic receptors play a role in cardiovascular regulation in the lower brain stem. Herein, we present evidence that l-3,4-dihydroxyphenylalanine (DOPA), a putative neurotransmitter in the central nervous system, is involved in the depressor response to microinjection of nicotine into the nucleus tractus solitarii (NTS). Microinjection of nicotine into the medial area of the NTS led to decreases in arterial blood pressure and heart rate in anesthetized rats. Mecamylamine, a nicotinic receptor antagonist, microinjected into NTS, blocked the depressor and bradycardic responses to nicotine. Nicotine-induced depressor and bradycardic responses were blocked by DOPA cyclohexyl ester (DOPA CHE), an antagonist for DOPA. DOPA CHE did not modify the action of carbachol on excitatory postsynaptic potential in rat cortical slices. These results suggest that endogenous DOPA is involved in nicotine-induced depressor responses in the NTS of anesthetized rats.

Non‐N‐methyl‐D‐aspartate glutamate receptors in the paraventricular nucleus of hypothalamus mediate the pressor response evoked by noradrenaline microinjected into the lateral septal area in rats

The lateral septal area (LSA) is a part of the limbic system and is involved in cardiovascular modulation. We previously reported that microinjection of noradrenaline (NA) into the LSA of unanesthetized rats caused pressor responses that are mediated by acute vasopressin release. Magnocellular neurons of the paraventricular (PVN) and supraoptic (SON) of the hypothalamus synthesize vasopressin. In the present work, we studied which of these nuclei is involved in the pressor pathway activated by unilateral NA injection into the LSA as well as the local neurotransmitter involved. Chemical ablation of the SON by unilateral injection of the nonspecific synapses blocker cobalt chloride (1 mM/100 nl) did not affect the pressor response evoked by NA (21 nmol/200 nl) microinjection into the LSA. However, the response to NA was blocked when cobalt chloride (1 mM/100 nl) was microinjected into the PVN, indicating that this hypothalamic nucleus is responsible for the mediation of the pressor response. There is evidence in the literature pointing to glutamate as a putative neurotransmitter activating magnocellular neurons. Pretreatment of the PVN with the selective non-N-methyl-D-asparate (NMDA) antagonist NBQX (2 nmol/100 nl) blocked the pressor response to NA microinjected into the LSA, whereas pretreatment with the selective NMDA antagonist LY235959 (2 nmol/100 nl) did not affect the response to NA. Our results implicate the PVN as the final structure in the pressor pathway activated by the microinjection of NA into the LSA. They also indicate that local glutamatergic synapses and non-NMDA glutamatergic receptors mediate the response in the PVN.

Modalities of GABA and Glutamate Neurotransmission in the Vertebrate Inner Ear Vestibule

GABA and glutamate have been postulated as afferent neurotransmitters at the sensory periphery inner ear vestibule in vertebrates. GABA has fulfilled the main criteria to act as afferent neurotransmitter but may also be a putative efferent neurotransmitter, mainly due to cellular localization of its synthesizing enzyme glutamate decarboxylase derived from biochemical, immunocytochemical, in situ hybridization and molecular biological techniques, whereas glutamate afferent neurotransmission role is supported mainly by pharmacological evidences. GABA and Glu could also act as afferent co-neurotransmitters based upon immunocytochemical techniques. This multiplicity was not considered earlier and postulates a peripheral modulation of afferent information being sent to higher vestibular centers. In order to make a definitive cellular assignation to these putative neurotransmitters it is necessary to have evidences derived from immunocytochemical and pharmacological experiments in which both substances are tested simultaneously.

Glutamate, GABA and Acetylcholine Signaling Components in the Lamina of the Drosophila Visual System

Synaptic connections of neurons in the Drosophila lamina, the most peripheral synaptic region of the visual system, have been comprehensively described. Although the lamina has been used extensively as a model for the development and plasticity of synaptic connections, the neurotransmitters in these circuits are still poorly known. Thus, to unravel possible neurotransmitter circuits in the lamina of Drosophila we combined Gal4 driven green fluorescent protein in specific lamina neurons with antisera to γ-aminobutyric acid (GABA), glutamic acid decarboxylase, a GABAB type of receptor, L-glutamate, a vesicular glutamate transporter (vGluT), ionotropic and metabotropic glutamate receptors, choline acetyltransferase and a vesicular acetylcholine transporter. We suggest that acetylcholine may be used as a neurotransmitter in both L4 monopolar neurons and a previously unreported type of wide-field tangential neuron (Cha-Tan). GABA is the likely transmitter of centrifugal neurons C2 and C3 and GABAB receptor immunoreactivity is seen on these neurons as well as the Cha-Tan neurons. Based on an rdl-Gal4 line, the ionotropic GABAA receptor subunit RDL may be expressed by L4 neurons and a type of tangential neuron (rdl-Tan). Strong vGluT immunoreactivity was detected in α-processes of amacrine neurons and possibly in the large monopolar neurons L1 and L2. These neurons also express glutamate-like immunoreactivity. However, antisera to ionotropic and metabotropic glutamate receptors did not produce distinct immunosignals in the lamina. In summary, this paper describes novel features of two distinct types of tangential neurons in the Drosophila lamina and assigns putative neurotransmitters and some receptors to a few identified neuron types.

Development of enteric and vagal innervation of the zebrafish (Danio rerio) gut

The autonomic nervous system develops following migration and differentiation of precursor cells originating in the neural crest. Using immunohistochemistry on intact zebrafish embryos and larvae we followed the development of the intrinsic enteric and extrinsic vagal innervation of the gut. At 3 days postfertilization (dpf), enteric nerve cell bodies and fibers were seen mainly in the middle and distal intestine, while the innervation of the proximal intestine was scarcer. The number of fibers and cell bodies gradually increased, although a large intraindividual variation was seen in the timing (but not the order) of development. At 11-13 dpf most of the proximal intestine received a similar degree of innervation as the rest of the gut. The main intestinal branches of the vagus were similarly often already well developed at 3 dpf, entering the gut at the transition between the proximal and middle intestine and projecting posteriorly along the length of the gut. Subsequently, fibers branching off the vagus innervated all regions of the gut. The presence of several putative enteric neurotransmitters was suggested by using markers for neurokinin A (NKA), pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP), nitric oxide, serotonin (5-hydroxytryptamine, 5-HT), and calcitonin gene-related peptide (CGRP). The present results corroborate the belief that the enteric innervation is well developed before the onset of feeding (normally occurring around 5-6 dpf). Further, the more detailed picture of how development proceeds at stages previously not examined suggests a correlation between increasing innervation and more regular and elaborated motility patterns.

Regulation of basal tone, relaxation and contraction of the lower oesophageal sphincter. Relevance to drug discovery for oesophageal disorders

The lower oesophageal sphincter (LOS) is a specialized region of the oesophageal circular smooth muscle that allows the passage of a swallowed bolus to the stomach and prevents the reflux of gastric contents into the oesophagus. The anatomical arrangement of the LOS includes semicircular clasp fibres adjacent to the lesser gastric curvature and sling fibres following the greater gastric curvature. Such anatomical arrangement together with an asymmetric intrinsic innervation and distinct proportion of neurotransmitters in both regions produces an asymmetric pressure profile. The LOS tone is myogenic in origin and depends on smooth muscle properties that lead to opening of L-type Ca(2+) channels; however it can be modulated by enteric motor neurons, the parasympathetic and sympathetic extrinsic nervous system and several neurohumoral substances. Nitric oxide synthesized by neuronal NOS is the main inhibitory neurotransmitter involved in LOS relaxation. Different putative neurotransmitters have been proposed to play a role together with NO. So far, only ATP or related purines have shown to be co-transmitters with NO. Acetylcholine and tachykinins are involved in the LOS contraction acting through acetylcholine M(3) and tachykinin NK(2) receptors. Nitric oxide can also be involved in the regulation of LOS contraction. The understanding of the mechanisms that originate and modulate LOS tone, relaxation and contraction and the characterization of neurotransmitters and receptors involved in LOS function are important to develop new pharmacological tools to treat primary oesophageal motor disorders and gastro-oesophageal reflux disease.

The Role of Carcinine in Signaling at the Drosophila Photoreceptor Synapse

The Drosophila melanogaster photoreceptor cell has long served as a model system for researchers focusing on how animal sensory neurons receive information from their surroundings and translate this information into chemical and electrical messages. Electroretinograph (ERG) analysis of Drosophila mutants has helped to elucidate some of the genes involved in the visual transduction pathway downstream of the photoreceptor cell, and it is now clear that photoreceptor cell signaling is dependent upon the proper release and recycling of the neurotransmitter histamine. While the neurotransmitter transporters responsible for clearing histamine, and its metabolite carcinine, from the synaptic cleft have remained unknown, a strong candidate for a transporter of either substrate is the uncharacterized inebriated protein. The inebriated gene (ine) encodes a putative neurotransmitter transporter that has been localized to photoreceptor cells in Drosophila and mutations in ine result in an abnormal ERG phenotype in Drosophila. Loss-of-function mutations in ebony, a gene required for the synthesis of carcinine in Drosophila, suppress components of the mutant ine ERG phenotype, while loss-of-function mutations in tan, a gene necessary for the hydrolysis of carcinine in Drosophila, have no effect on the ERG phenotype in ine mutants. We also show that by feeding wild-type flies carcinine, we can duplicate components of mutant ine ERGs. Finally, we demonstrate that treatment with H3 receptor agonists or inverse agonists rescue several components of the mutant ine ERG phenotype. Here, we provide pharmacological and genetic epistatic evidence that ine encodes a carcinine neurotransmitter transporter. We also speculate that the oscillations observed in mutant ine ERG traces are the result of the aberrant activity of a putative H3 receptor.

Comparative analysis of the effect of endogenous antibiotic defensin NP-1 and aminoglycoside antibiotic gentamicin on synaptic transmission in receptors of the frog vestibular apparatus

The effect of human and rabbit neutrophilic defensins NP-1 and amonoglycoside antibiotic gentamicin on the synaptic transmission in the afferent synapse of isolated vestibular apparatus of the frog has been comparatively studied. Both defensins proved active in the concentration range of 0.0001 to 1 nM and efficiently decreased the impulse frequency in the afferent nerve fibers in a concentration-dependent manner. No significant differences in the efficiency of rabbit and human defensin NP-1 have been revealed in these experiments. Gentamicin also had an inhibitory effect on the afferent discharge in the concentration range of 10-500 microM (0.5-25 mg/kg). The inhibitory effect of gentamicin on the impulse activity of the vestibular nerve was observed at therapeutic doses. The excitatory effect of the putative neurotransmitter L-glutamate was considerably inhibited by defensin NP-1. These findings suggest that the mechanism of defensin action involves a modification of the synaptic transmission the hair receptor and is mediated by L-glutamate.

Neurotransmitter systems of commissural interneurons in the lumbar spinal cord of neonatal rats

The circuits that generate rhythmic locomotor activities are located in the ventromedial area of the lumbar spinal cord and comprise commissural interneurons necessary for left–right alternation during walking movements. In this study we injected biotinylated dextran amine (BDA) into the ventromedial gray matter of the lumbar spinal cord of neonatal rats to label commissural interneurons. Anterogradely labeled axons arose from the site of injection, crossed the midline in the anterior commissure and arborized extensively in the contralateral ventral horn of the spinal cord. The presence of neurotransmitter systems in labeled axon terminals of commissural interneurons was investigated by using antibodies raised against specific transmitter-related proteins. Boutons potentially containing inhibitory amino acids were identified by applying glutamic acid decarboxylase (GAD65/67) and glycine transporter 2 antibodies. Out of 1146 BDA-labeled axon terminals, 663 boutons were assumed on this basis to be inhibitory; 76% of these terminals were immunoreactive for glycine transporter, 53% were immunoreactive for GAD and about 30% of inhibitory boutons might contain both inhibitory amino acids. Boutons potentially containing putative excitatory neurotransmitter were revealed with antibodies raised against vesicular glutamate transporters 1 and 2. Out of 590 BDA-labeled boutons about one fourth (158) were immunoreactive for glutamate transporters. These mammalian commissural interneurons are compared to the glycinergic commissural interneurons in the swimming CPGs of lamprey and the Xenopus tadpole. Our results show that commissural interneurons in the mammalian spinal cord form a heterogeneous group including glutamatergic excitatory and GABAergic and glycinergic inhibitory neurons.