Graciela Meza

Neurochemical evidence for afferent gabaergic and efferent cholinergic neurotransmission in the frog vestibule

Glutamate decarboxylase and choline acetyltransferase activities with magnitudes similar to those of their homologous enzymes in frog nervous tissue were found in homogenates of the frog labyrinth. Transection of the vestibular nerve resulted in a gradual diminution of choline acetyltransferase activity until it reached an 88% decrease 6 weeks after surgery. In contrast, glutamate decarboxylase activity did not suffer any alteration at any time after nerve excision. The presence of their enzymes of synthesis is evidence of the neurotransmitter participation of GABA and acetylcholine in the frog vestibule; the observed decrease of choline acetyltransferase following vestibule nerve excision supports the efferent synaptic bouton localization of choline acetyltransferase. The suggestion that glutamate decarboxylase is located in a cell type (or compartment) that may well be the hair cell is supported by the fact that this enzyme does not suffer any modification after surgery. These results are in accordance with an efferent cholinergic neurotransmission and a putative afferent role of GABA in the frog vestibule.

GABA synthesis in isolated vestibulary tissue of chick inner ear.

This study was undertaken to assess the possible participation of GABA in neurotransmission in the inner ear of higher vertebrates utilizing isolated chick vestibulary cristae as a model. GABA synthesis was investigated as a suitable criterion for postulating this amino acid as a putative neurotransmitter. Enzyme-mediated GABA synthesis was demonstrated implying a possible neurotransmitter role of this amino acid in the chick vestibule.

Distribution of GABA-like immunoreactivity in guinea pig vestibular cristae ampullaris

Post-embedding immunocytochemical techniques were used to assess distribution of gamma-aminobutyric acid (GABA) in the guinea pig cristae ampullaris. GABA-like immunoreactivity (GABA-LIR) was found in the cytoplasm of both type I (HCI) and type II hair cells (HCII), in the afferent calyx (AC) contacting HCI and some myelinated fibers in the subjacent stroma. HCI and its calyceal contacts showed variation in GABA-LIR, suggesting different populations in HCI and AC. These results support a putative afferent neurotransmitter role of GABA in HC and a possible degradation site of GABA in AC.

Ontogenetic approach to cellular localization of neurotransmitters in the chick vestibule

Gamma-aminobutyric acid (GABA) and acetylcholine (Ach) have been implicated in afferent and efferent neurotransmission, respectively, in the vestibular sensory periphery. Assuming that glutamate decarboxylase (GAD), the GABA-synthesizing enzyme, and choline acetyltransferase (ChAT), the enzyme for synthesis of acetylcholine, are located in distinct cell types of the inner ear whose maturation occurs at different times during ontogenesis, we measured these enzymes in the ampullary cristae of embryonic chicks at different stages of development. By making these measurements in parallel with electron-microscopic studies of the different cell elements of the chick vestibular sensory periphery, we found that the values of GAD activity were nearly the same from the earliest stage studied, i.e., the 13th day of ontogeny to day 18 of embryonic development, paralleling the morphologically mature appearance of the hair cells and their afferent synapses. A slight increase in enzymatic activity from day 19 of ontogeny to one day after hatching corresponded to a rise in the number of afferent synapses. In contrast, ChAT activity was practically undetectable up to day 17 of embryonic development, but rose suddenly on the 19th day, reaching 1-day-old levels by day 20 of ontogenesis in coincidence with an elevation in the number of well-developed efferent boutons. These results are in accord with the localization of GAD in the sensory cells and a localization of ChAT in the efferent nerve endings. These findings suggest that GABA and Ach are the respective neuromediators for the afferent and efferent systems.

Immunocytochemical evidence for an afferent GABAergic neurotransmission in the guinea pig vestibular system

To implicate gamma-aminobutyric acid (GABA) as an afferent neurotransmitter (AN), the localization of GABA synthesizing and degradation enzymes; L-glutamate decarboxylase (GAD) and GABA transaminase (GABA-T) was investigated by light and electron microscopy immunocytochemistry in guinea pig vestibular cristae and ganglion cells (GC). GAD-like immunoreactivity was exclusively confined to the sensory hair cell (HC) cytoplasm, suggesting that GAD synthesizes GABA in the HC. GABA-T like immunoreactivity was found within HC, nerve calyces, nerve fibers, and GC, suggesting its participation in terminating transmitter action. These results demonstrate the existence of a GABAergic system in the guinea pig vestibule and strongly support GABA as a vestibular AN.

Development of vestibular and auditory function: Effects of hypothyroidism and thyroxine replacement therapy on nystagmus and auditory evoked potentials in the pigmented rat

The functional development of semicircular canals and some brainstem structures of the auditory system was followed in parallel with time in control and propylthiouracyl‐induced hypothyroid pigmented rats by respective recording of postrotatory nystagmus response and auditory evoked brainstem potentials, with the aim of discovering the timing of permanent alterations of these responses in congenital hypothyroidism. A group of hypothyroid rats which underwent thyroxine‐replacement therapy from postnatal day 12 onward was also included in our studies to corroborate the involvement of thyroid hormones in these effects. Postrotatory nystagmus and auditory evoked responses were absent in congenital hypothyroid rats. In the thyroxine‐replaced group postrotatory nystagmus values showed no differences from the control group from postnatal day 28 onward. Auditory evoked potentials in thyroxine‐replaced animals could not be elicited at 30 dB, but by increasing the intensity of stimulus to 70 dB, values of latencies of the four waves composing the response were indistinguishable from controls from postnatal day 39 and thereafter. These results show that hypothyroidism affects both semicircular canal and auditory function, the latter more severely than the former, but that these effects can be prevented when thyroxine replacement treatment is started in early stages of postnatal development.

Comparative studies on glutamate decarboxylase and choline acetyltransferase activities in the vertebrate vestibule.

1. Vestibular putative neurotransmitters GABA and acetylcholine synthesizing enzymes were quantified in four vertebrate species to find a correlation between all-vertebrate vestibular hair cell II (HCII) and synaptic contacts and appearance of hair cell I (HCI) and related synapses in terrestrial species. 2. Glutamate decarboxylase (GAD) and choline acetyltransferase (ChAT) values were: 3.76; 15.38; 21.68; 27.78 and 9.44; 450; 720; 970 n(pico)mol/mg protein/hr (min) in, respectively, frogs, guinea pigs, rats and chicks. 3. GAD and ChAT omnipresence may indicate constant GABAergic HCII and its cholinergic efferent synapses, their raised content, appearance of GABA-containing HCI and related cholinergic boutons in higher vertebrates.

Specific [3H]γ-aminobutyric acid binding to vestibular membranes of the chick inner ear

Abstract To support a postulated neurotransmitter character of γ-aminobutyric acid (GABA) in the vertebrate vestibule, [ 3 H]GABA binding was measured in a crude membrane preparation of chick inner ear ampullary cristae. In the absence of divalent cations bound [ 3 H]GABA was displaced by unlabeled GABA, muscimol or bicuculline, but it was not displaced by (±)-baclofen. A single population of [ 3 H]GABA binding sites with an equilibrium constant of 19.4 nM and a maximum binding capacity of 0.58 pmol/mg protein was found. These results suggest the possible existence of a synaptic GABA A receptor in the chick inner ear membranes and sustain the neurotransmitter role of GABA in the chick vestibule.

Recovery of vestibular function in young guinea pigs after streptomycin treatment: Glutamate decarboxylase activity and nystagmus response assessment

Fifty‐day streptomycin (STP) treatment in guinea pigs causes specific vestibular hair cell (VHC) types I and II (HCI; HCII) degeneration, depletion of glutamate decarboxylase (GAD) and a gradual disappearance of postrotatory nystagmus response (PRNR), which is a sign of vestibular function alteration. In order to look for a possible spontaneous reversibility and its time course guinea pigs receiving 300 mg/kg STP daily were monitored for PRNR and vestibular GAD loss. Once PRNR was lost, STP was interrupted and the animal was allowed to recover; at the time that PRNR was completely reestablished, vestibular GAD was measured.

Possible cholinergic neurotransmission in the cristae ampullares of the chick inner ear

The presence of choline acetyltransferase (ChAT) was investigated in the chick inner ear in order to assess the possible role of acetylcholine (ACh) in neurotransmission within the vestibular labyrinth. ChAT activity found in homogenates of isolated chick vestibular cristae ampullares is of the same order of magnitude and has similar properties as its homologous enzyme in nervous tissue from various regions. These findings are in accord with a probable neurotransmitter role of ACh in the chick vestibular labyrinth.