T U T O R I A L
Cesar D. Fermin, Ph.D. Tulane Pathology (504) 584-2521, Fax 504 587-7389

Picker-uppers

The typical hair cell is a complicated unit. In addition to transforming mechanical movements into neural stimuli, hair cells respond to insults of chemicals including neurotransmitters, antibiotics and diuretics. When the hair cells are damaged, the sensations of sound, movement, equilibrium and orientation are affected. Unfortunatelly, demonstrating that one molecule affect a single hair cells does not tell us how that hair cell work in harmony with other hair cells and the rest of the epithelia of the inner ear.

Polarization of receptors

Hair cells are organized in a such manner as to permit detection of movement in any direction. Hair cells in some areas are turned on whereas adjacent cells are turned off by the same movement

Bipolar Neurons

The specialized bipolar neurons were studied in detail back in the 80's (see Fermin and Igarashi Publications). Bipolar neurons form the auditory and vestibular ganglia, but only the bipolar vestibular neurons survive deafferentation longer than 3 years in large enough numbers to suggest possible functional attributes after repair. Normal detection of acceleration depends thus in hair cells, bipolar neurons and central nuclei integrity, among other variables. Any change to these and other indirect contributing pathways affects equilibrium.

Central Processing Units

Despite the complexity of their environment, the ultimate control and modulation of stimuli occurs in the brain.In this simplified diagram, the complexity of interconnections and cross talk among neurons and groups of neurons in the brain can be appreciated. We analyze hair cells (microphones), neurons of the nerve (cables) and processor (brain) to gain insights into properties that affect hearing a equilibrium. Experiments to understand the processes that permit detection of these sensations generally include provisions to extrapolate the results to the in vivo system (black box).

Processing unit parts

The vestibular system is organized symmetrically. Both sides of the brain stem contain redudant switches to ensure that malfunction in one side is compensated quickly and efficiently. To understand the basis of neural function, individual connections must be understood. Unforntunately even the most sophisticated tools today DO NOT permit complete isolation while retaining the in vivo integrity of the system as a whole (block box). Aside from directionality (which inpulse goes where?), there are redudant contributors to a single path (a single neuron could received let's say 5 inputs). Recording from the neuron despited here green with electrodes could tell about a few but not all inputs. Visualizing the inputs (histochemical reaction of proteins at the point of contact) can not tell about the functional attributes or the cell structure which is destroyed during processing. We push forward with multidisciplinary approaches to deduce some but not all the essentials.

S100 in Utricle

S100 beta is a calcium binding protein and neurotrophic factor (see Fermin and Martin,CMB 1995). Its presence in nerve terminals of the afferent neurons (cables) of the inner ear suggest some role at that location. The cables at these locations pick up information from the hair cells. We assume that the immunoreaction is NOT contributed by myelin because these nerve fibers loose the myelin before penetrating the basement membrane. This is true for birds and mammals but not for lower vertebrates.

S100b & GABA in Crista

S100 beta is a calcium binding protein and neurotrophic factor (see Fermin and Martin,CMB 1995). GABA is a transmitter of the inner ear. Localization of molecule with neurotropic potential requires its colocalization with a known neurotransmitter, thus the experiment yielding this image.

GABA in Utricle

The utricle is one of the organs in the inner ear that detects acceleration and helps to maintain equilibrium. The two types of hair cells described (Color Diagrams) do not react with anti-GABA equally. Hair cells with large chalice endings and those with small terminals are differentially stained.

S100-GABA-Striola

In the striola (central area of the utricle) hair colocalization of GABA & S100 is differential among hair cells type I and II. S100 stain is light brown GABA is red.

S100-NF

It was noted earlier that S100beta reactivity inside the epithelia was not contributed by the myelin (S100 was originally isolated from glial proteins and thus stains myelin). Interesting, colocalization of S100beta with neurofilament protein (cytoskeletal component of all nerves) showed a higher expression of NF than S100 at the afferent contacts with hair cells. Myelin outside the basement membrane stain with S100 still supporting our interpretation of S100 specificity at the contact points with hair cells.

Neurofilament protein

We use antibodies to NF to outline the path that afferent fibers take to the hair cells in the inner ear organs. This area of the saccule (a gravity detecting organ) shows intricate path of the fibers, suggesting that during development fibers follow random directions in search for target to innervate.

S100

Neurons of the vestilar ganglion are bipolar because they have two axons; one goes to the brain and the other to the hair cells. The group of neurons is called a ganglion and in the ganglion there are different types of neurons that stain different with the histological stains like this pentachrome stain. Previous work by the author showed that the vestibular neurons are more plastic (they can recuperate from insults) than the adjacent auditory neurons. Such plasticity may be related to the multiple factors (neutrophic-transmitters-hormonal, etc).

S100 in Bipolar neurons

S100 beta is expressed in only some neurons of the vestibular ganglion, suggesting that at the time of death different neurons expressed different quantities of this molecule. We are investigating the implications of this finding, which was described in mammals as well. For more details see Recent publications

Built-in Control

Nonspecific reactivity of S100 or GABA is controlled for by incorporation of control tissues known to express these molecules. Thus, the inner ear is reacted in the same slide with cerebelar neurons with terminal contacts that express GABA. These are Purkinje neurons stained with GABA (red) and S100beta (brown). In addition, we always react samples for comparison together, at the same time and under identical conditions. When embryological comparisons are made individuals of different ages are processed together and pre-absorption of the antibody is used for a negative control.

S100 in brain neurons

Different neurons in the brain centers express S100beta differently. Here a Neuron from the Tangential nucleus of the chick expressed S100 & GABA in identical manner. Note brown (S100) stain over myelin where is supposed to be according to the properties of the molecule.