AB139 Sigma-AldrichAnti-Glycine Antibody

Rabbit eyeblink conditioning is a well characterized model of associative learning. To identify specific neurons that are part of the eyeblink premotor pathway, a retrograde transsynaptic tracer (pseudorabies virus) was injected into the orbicularis oculi muscle. Four time points (3, 4, 4.5, and 5 d) were selected to identify sequential segments of the pathway and a map of labeled structures was generated. At 3 d, labeled first-order motor neurons were found in dorsolateral facial nucleus ipsilaterally. At 4 d, second-order premotor neurons were found in reticular nuclei, and sensory trigeminal, auditory, vestibular, and motor structures, including contralateral red nucleus. At 4.5 d, labeled third-order premotor neurons were found in the pons, midbrain, and cerebellum, including dorsolateral anterior interpositus nucleus and rostral fastigial nucleus. At 5 d, labeling revealed higher-order premotor structures. Labeled fourth-order Purkinje cells were found in ipsilateral cerebellar cortex in cerebellar lobule HVI and in lobule I. The former has been implicated in eyeblink conditioning and the latter in vestibular control. Labeled neurons in anterior interpositus were studied, using neurotransmitter immunoreactivity to classify individual cell types and delineate their interconnectivity. Labeled third-order premotor neurons were immunoreactive for glutamate and corresponded to large excitatory projection neurons. Labeled fourth-order premotor interneurons were immunoreactive for GABA (30%), glycine (18%), or both GABA and glycine (52%) and form a functional network within anterior interpositus involved in modulation of motor commands. These results identify a complete eyeblink premotor pathway, deep cerebellar interconnectivity, and specific neurons responsible for the generation of eyeblink responses.

The synaptic connections between neurokinin 1 (NK1) receptor-like immunoreactive (LI) neurons and γ-aminobutyric acid (GABA)-, glycine (Gly)-, serotonin (5-HT)- or dopamine-β-hydroxylase (DBH, a specific marker for norepinephrinergic neuronal structures)-LI axon terminals in the rat medullary dorsal horn (MDH) were examined under electron microscope by using a pre-embedding immunohistochemical double-staining technique. NK1 receptor-LI neurons were observed principally in laminae I and III, only a few of them were found in lamina II of the MDH. GABA-, Gly-, 5-HT-, or DBH-LI axon terminals were densely encountered in laminae I and II, and sparsely in lamina III of the MDH. Some of these GABA-, Gly-, 5-HT-, or DBH-LI axon terminals were observed to make principally symmetric synapses with NK1 receptor-LI neuronal cell bodies and dendritic processes in laminae I, II and III of the MDH. The present results suggest that neurons expressing NK1 receptor within the MDH might be modulated by GABAergic and glycinergic inhibitory intrinsic neurons located in the MDH and 5-HT- or norepinephrine (NE)-containing descending fibers originated from structures in the brainstem.

BACKGROUND: Rhythmic motor patterns for locomotion in vertebrates are generated in spinal cord neural networks known as spinal Central Pattern Generators (CPGs). A key element in pattern generation is the role of glycinergic synaptic transmission by interneurons that cross the cord midline and inhibit contralaterally-located excitatory neurons. The glycinergic inhibitory drive permits alternating and precisely timed motor output during locomotion such as walking or swimming. To understand better the evolution of this system we examined the physiology of the neural network controlling swimming in an invertebrate chordate relative of vertebrates, the ascidian larva Ciona intestinalis.

Identification of shared features between avian and mammalian auditory brainstem circuits has provided much insight into the mechanisms underlying early auditory processing. However, previous studies have highlighted an apparent difference in inhibitory systems; synaptic inhibition is thought to be slow and GABAergic in birds but to have fast kinetics and be predominantly glycinergic in mammals. Using patch-clamp recordings in chick brainstem slices, we found that this distinction is not exclusively true. Consistent with previous work, IPSCs in nucleus magnocellularis (NM) were slow and mediated by GABA(A) receptors. However, IPSCs in nucleus laminaris (NL) and a subset of neurons in nucleus angularis (NA) had rapid time courses twofold to threefold faster than those in NM. Furthermore, we found that IPSCs in NA were mediated by both glycine and GABA(A) receptors, demonstrating for the first time a role for fast glycinergic transmission in the avian auditory brainstem. Although NM, NL, and NA have unique roles in auditory processing, the majority of inhibitory input to each nucleus arises from the same source, ipsilateral superior olivary nucleus (SON). Our results demonstrate remarkable diversity of inhibitory transmission among the avian brainstem nuclei and suggest that differential glycine and GABA(A) receptor activity tailors inhibition to the specific functional roles of NM, NL, and NA despite common SON input. We additionally observed that glycinergic/GABAergic activity in NA was usually depolarizing and could elicit spiking activity in NA neurons. Because NA projects to SON, these excitatory effects may influence the recruitment of inhibitory activity in the brainstem nuclei.,

The nuclei of the auditory brainstem harbor a diversity of neuronal cell types and are interconnected by excitatory as well as inhibitory ascending, descending, and commissural pathways. Classically, neurons have been characterized by size and shape of their cell body and by the geometry of their dendrites. Our study is based on the use of axonal tracers in combination with immunocytochemistry to identify and distinguish neuronal subtypes by their molecular signature in dorsal and ventral cochlear nucleus, lateral superior olive, medial superior olive, medial nucleus of the trapezoid body, and inferior colliculus of the adult rat. The presumed neurotransmitters glutamate, glycine, and GABA were used alongside the calcium-binding proteins parvalbumin, calretinin, and calbindin-D28k as molecular markers. Our data provide distinct extensions to previous characterizations of neuronal subtypes and reveal regularities and differences across auditory brainstem nuclei that are discussed for their functional implications.

We have characterized the vascular, neuronal, and glial changes in oxygen-induced retinopathy, a model of retinopathy of prematurity (ROP). Newborn Sprague-Dawley rats were exposed to either 80% +/- 2% oxygen to postnatal day P11 and then room air until P18 (ROP) or room air for the entire duration (controls). Retinal structure was examined under the light microscope and following postembedding immunocytochemistry in central, midperipheral, and peripheral regions. Müller cells were evaluated immunocytochemically with glial fibrillary acidic protein. The extent of vascularization was established histologically. ROP caused significant thinning of the inner cellular and plexiform layers, which became more pronounced in the peripheral inner nuclear layer of ROP animals (11.3% loss vs. 25.4% loss). Amacrine cell amino acid levels were particularly vulnerable in the peripheral retina; bipolar cells showed similar but less prominent changes. Müller cells had elevated glutamine levels and were most gliotic in the periphery. The vasculature extended to peripheral retinal regions at P18 in controls but not in ROP rats. The most striking pattern of change was evident in the midperipheral transition zone of ROP animals. Areas close to blood vessels showed neurochemical properties that were similar to those of the central retina, indicating a local protective effect of the inner retinal blood supply. We find that ROP produces complex vascular, neural, and glial changes that relate to the proximity of inner retinal blood vessels.

The wave-type African weakly electric fish Gymnarchus niloticus produces electric organ discharges (EODs) from an electric organ in the tail that is driven by a pacemaker complex in the medulla, which consists of a pacemaker nucleus, two lateral relay nuclei and a medial relay nucleus. The prepacemaker nucleus (PPn) in the area of the dorsal posterior nucleus of the thalamus projects exclusively to the pacemaker nucleus and is responsible for EOD interruption behavior. The goal of the present study is to test the existence of inhibition of the pacemaker nucleus by the PPn. Immunohistochemical results showed clear anti-GABA immunoreactive labeling of fibers and terminals in the pacemaker nucleus, but no apparent anti-glycine immunoreactivity anywhere in the pacemaker complex. GABA injection into the pacemaker nucleus could induce EOD interruptions that are comparable to the interruptions induced by glutamate injection into the PPn. Application of the GABA(A) receptor blocker bicuculline methiodide reversibly eliminated the effects of stimulation of the PPn. Thus the EOD interruption behavior in Gymnarchus is mediated through GABAergic inhibition of the pacemaker nucleus by the PPn.

Three series of experiments were carried out to characterize interneurons located within the trigeminal motor nucleus of young rats aged 5-24 days. Cholera toxin injections were made bilaterally into the masseter and, sometimes, digastric muscles to label motoneurons. In the first set of experiments, thick slices were taken from the pontine brainstem and cholera toxin-positive and cholera toxin-negative neurons located inside the trigeminal motor nucleus were filled with biocytin through whole-cell recording patch electrodes. Positively identified motoneurons (cholera toxin+) of various shapes and sizes always had a thick, unbranched axon that entered the motor root following a tight zigzag course. Many cholera toxin-negative neurons were also classified as motoneurons after biocytin filling based on this particularity of their axon. These are probably either fusimotor motoneurons or motoneurons supplying other jaw muscles. The cholera toxin-negative neurons classified as interneurons differed markedly from motoneurons in that they had thin, usually branched axons that supplied the ipsilateral reticular region surrounding the trigeminal motor nucleus (peritrigeminal area), the main trigeminal sensory nucleus, the trigeminal mesencephalic nucleus, the medial reticular formation of both sides, and the contralateral medial peritrigeminal area. Most often, their dendrites were arranged in bipolar arbors that extended beyond the borders of the trigeminal motor nucleus into the peritrigeminal area. Immunohistochemistry against glutamate, GABA and glycine was used to further document the nature and distribution of putative interneurons. Immunoreactive neurons were uniformly distributed throughout the rostro-caudal extent of the trigeminal motor nucleus. Their concentration seemed greater toward the edges of the nucleus and they were scarce in the digastric motoneuron pool. Glutamate- outnumbered GABA- and glycine-immunoreactive neurons. There was no clear segregation between the three populations. In the final experiment, 1,1'-dioctadecyl-3,3,3',3'-tetra-methylindocarbocyanine perchlorate crystals were inserted into one trigeminal motor nucleus in thick slices and allowed to diffuse for several weeks. This procedure marked commissural fibers and interneurons in the contralateral trigeminal motor nucleus. Together these results conclusively support the existence of interneurons in the trigeminal motor nucleus.

The volume, total neuron number, and number of GABA- and glycine-expressing neurons in the sensory trigeminal nuclei of the adult rat were estimated by stereological methods. The mean volume is 1.38+/-0.13 mm3 (mean+/-SD) for the principal nucleus (Vp), 1.59+/-0.06 for the n. oralis (Vo), 2.63+/-0.34 for the n. interpolaris (Vip), and 3.73+/-0.11 for the n. caudalis (Vc). The total neuron numbers are 31,900+/-2,200 (Vp), 21,100+/-3,300 (Vo), 61,600+/-8,300 (Vip), and 159,100+/-25,300 (Vc). Immunoreactive (-ir) neurons were classified as strongly stained or weakly stained, depending on qualitative criteria, cross-checked by a densitometric analysis. GABA-ir cells are most abundant in Vc, in an increasing rostrocaudal gradient within the nucleus. Lower densities are found in Vip and Vp. The mean total number of strongly labeled GABA-ir neurons ranges between 1,800 in Vp to 7,800 in Vip and 22,900 in Vc, and varies notably between subjects. Glycine-ir neurons are more numerous and display more homogeneous densities in all nuclei. Strongly labeled Gly-ir cells predominate in all nuclei, their total number ranging between 9,400 in Vp to 24,300 in Vip and 34,200 in Vc. A substantial fraction of immunolabeled neurons in all nuclei coexpress GABA and glycine. In general, all neurons strongly immunoreactive for GABA are small, while weakly GABA-ir cells which coexpress Gly are larger. In Vc, one-third of all neurons are immunoreactive: 16.6% of them are single-labeled for GABA and 31.6% are single-labeled for glycine. The remaining 51.8% express GABA and glycine in different combinations, with those showing strong double labeling accounting for 22.6%.

Data from humans and animal models provide evidence for an age-dependent impairment in the ability to localize sound. The lateral superior olive (LSO) in the ascending auditory pathway is one important center involved in processing of binaural auditory stimuli. To identify potential age-dependent changes we characterized the LSO in young ( 15 months) and old (> or =3 years) gerbils with a special emphasis on the expression of GABA- and glycine-like immuno-reactivity. The dimensions of the LSO, as well as the number and density of glycine- and GABA-immuno-reactive neurons, were not significantly different between young and old gerbils. The size of glycine- and GABA-immuno-reactive neurons was significantly reduced in the high-frequency (medial) limb of the LSO. Over all, age-dependent changes in the LSO of the gerbil were small.