Acetylcholinesterase-positive and paraformaldehyde-induced-fluorescence-positive innervation in the upper eyelid of the sheep (Ovis aries)

This is the first study which describes the innervation of some eyelid structures, such as the glands of Moll and the glands of Zeiss. It is also the first to investigate the innervation pattern of the eyelid as a whole. We have studied the acetylcholinesterase-positive and paraformaldehyde-induced-fluorescence-positive (FIF+) innervation pattern of the different structures that constitute the upper eyelid of the sheep. There is widespread acetylcholinesterase-positive innervation in the epithelium, but not such an abundant FIF+ innervation. Both types of innervation are represented in the connective tissue by trunks or fibers that are distributed towards the different structures immersed within them. In the glands of Zeiss, cholinesterasepositive innervation is much more widespread than FIF innervation. On the contrary, the glands of Moll present denser FIF+ innervation than acetylcholinesterasepositive innervation. The Meibomian glands and the lachrymal glands show a rich acetylcholinesterasepositive and FIF+ innervation. Eyelid muscle innervation is mainly acetylcholinesterase-positive. In the conjunctive membrane there is no acetylcholinesterase-positive innervation, and only scarce FIF+ fibers can be demonstrated

Characterization by immunocytochemistry of ionic channels in Helix aspersa suboesophageal brain ganglia neurons

The aim of this work was to characterize several ionic channels in nervous cells of the suboesophageal visceral, left and right parietal, and left and right pleural brain ganglia complex of the snail Helix aspersa by immunocytochemistry. We have studied the immunostaining reaction for a wide panel of eleven polyclonal antibodies raised against mammal antigens as follows: voltage-gated-Na+ channel; voltagegated- delayed-rectifier-K+ channel; SK2-smallconductance- Ca2+-dependent-K+ channel apamin sensitive; SK3 potassium channel; charybdotoxinsensitive voltage-dependent potassium channel; BKCamaxi- conductance-Ca2+-dependent-K+ channel; hyperpolarization-activated cyclic nucleotide-gated potassium channel 4; G-protein-activated inwardly rectifying potassium channel GIRK2 and voltage-gatedcalcium of L, N and P/Q type channels. Our results show positive reaction in neurons, but neither in glia cells nor in processes in the Helix suboesophageal ganglia. Our results suggest the occurrence of molecules in Helix neurons sharing antigenic determinants with mammal ionic channels. The reaction density and distribution of immunoreactive staining within neurons is specific for each one of the antisera tested. The studies of colocalization of immunoreaction, on alternate serial sections of the anterior right parietal ganglion, have shown for several recognized mapped neurons that they can simultaneously be expressed among two and seven different ionic protein channels. These results are considered a key structural support for the interpretation of Helix aspersa neuron electrophysiological activity

Localization of connexins in neurons and glia cells of the Helix aspersa suboesophageal brain ganglia by immunocytochemistry

The aim of the present study was to examine the distribution of cells expressing connexin 26 (Cx26) in the suboesophageal visceral, left and right parietal and left and right pleural ganglia of the snail Helix aspersa by immunocytochemistry. Altogether we have found approximately 452 immunoreactive neurons which represent the 4.7% of the total neurons counted. The stained large neurons (measured diameter 55-140 μm) occurred mostly on the peripheral surface of the ganglia while the small immunostained cells (5-25 μm diameter) were observed in groups near the neuropil. The number of large neurons giving positive Cx26-like immunostaining was small in comparison with that for medium (30-50 μm diameter) and small sized cells. The expression of Cx26 was also observed in the processes of glia cells localized among neurons somata and in the neuropil showing that the antiserum recognized epitopes in both protoplasmic and fibrous glia cells of Helix aspersa. The neuropils of all ganglia showed fibers densely immunostained. While we have observed a good specificity for Cx26-antiserum in neurons, a lack of reaction for Cx43 antiserum was observed in neurons and glia cells. The reaction for enolase antiserum in neurons was light and non-specific and a lack of reaction in glia cells and processes for GFAP antiserum was observed. Although the percentage of positive neurons for Cx26 antiserum was low is suggested that in normal physiological conditions or under stimulation the results can be considered of interest in the interpretation of Helix aspersa elemental two neuron networks synchronizing activity, observed under applied extremely low frequency magnetic fields