Synaptic structural modification following changes in activity induced by tetanus neurotoxin in cat abducens neurons

Abstract

A low or a high dose of tetanus neurotoxin (TeNT) injected in the lateral rectus muscle of the cat causes respectively, functional block of inhibitory synapses only or of both inhibitory and excitatory synapses simultaneously in abducens neurons (González-Forero et al. [2003] J. Neurophysiol. 89:1878-1890). As a consequence, neuronal firing activity increases (at low dose) or decreases (at high dose). We investigated possible structural modifications of inhibitory synapses in response to these activity alterations induced by TeNT. We used immunofluorescence against postsynaptic (gephyrin) and presynaptic (vesicular gamma-aminobutyric acid [GABA] transporter [VGAT]) markers of inhibitory synapses in combination with cell type markers for abducens motoneurons (calcitonin gene-related peptide or choline acetyltransferase) or internuclear neurons (calretinin). Seven days after high-dose treatment, the number of gephyrin-immunoreactive (IR) clusters per 100 microm of membrane perimeter was reduced on the soma of abducens motoneurons by 55.3% and by 60.1% on internuclear neurons. Proximal dendritic gephyrin-IR clusters were also significantly altered but to a lesser degree. Partial synaptic re-establishment was observed 15 days post injection, and complete recovery occurred after 42 days. Coverage by VGAT-IR terminals was reduced in parallel. In contrast, a low dose of TeNT caused no structural alterations. With electron microscopy we estimated that overall synaptic coverage was reduced by 40% in both types of neurons after a high dose of TeNT. However, F-type terminals with postsynaptic gephyrin were preferentially lost. Thus, the ratio between F and S terminals diminished from 1.28 to 0.39 on motoneurons and from 1.26 to 0.47 on internuclear neurons. These results suggest that the maintenance of proximal inhibitory synaptic organization on central neurons is best related to neuronal activity and not to the level of inhibitory synaptic function, which was equally blocked by the high or low dose of TeNT.

Complementary Actions of BDNF and Neurotrophin-3 on the Firing Patterns and Synaptic Composition of Motoneurons

“…Dual or triple color immunofluorescence for choline acetyltransferase (ChAT) (for motoneurons), synaptophysin (SYN) (for synaptic boutons) and glial fibrillary acidic protein (GFAP) (for astrocytes) was performed by mixing the following primary antibodies and dilutions: ChAT [goat polyclonal antibody (pAb); 1:300; Millipore], SYN (rabbit pAb; 1:500; Zymed Laboratories) and GFAP [mouse monoclonal antibody (mAb) 7a; 1:800; Sigma]. To determine which fraction of the boutons was inhibitory, a series of experiments used the marker vesicular GABA/glycine transporter (VGAT) (Dumoulin et al, 1999;González-Forero et al, 2004), which is the vesicular ␥-aminobutyric acid transporter also known as vesicular inhibitory amino acid transporter (VIAAT) that transports GABA and glycine into synaptic vesicles (rabbit pAb; 1:500, Millipore). Labeling of terminals with vesicular glutamate transporters 1 and 2 (VGLUT1 and VGLUT2) (guinea pig pAb; 1:300 -1:1000; Millipore) yielded very low number of boutons on abducens motoneurons and no further attempts to characterize them were done.…”

Nerve Growth Factor Regulates the Firing Patterns and Synaptic Composition of Motoneurons

“…Primary antisera cocktails were performed by mixing the following primary antibodies and dilutions: ChAT (goat pAb, 1:300; Millipore), SYN (rabbit pAb, 1:500; Zymed Laboratories), GFAP (mouse mAb G-A-5, 1:800; Sigma), TrkA (directed against a specific peptide mapping adjacent to the carboxy terminus of the TrkA receptor; rabbit pAb, 1:400; Santa Cruz Biotechnology) and p75 NTR (rabbit, 1:200; a gift from Dr. Louis Reichardt). VGAT immunoreactivity was used to calculate which fraction of the total number of boutons was inhibitory (González-Forero et al, 2004). VGAT is the vesicular transporter of GABA and glycine (rabbit pAb, 1:500; Millipore).…”

Tetanus toxin reduces local and descending regulation of the H-reflex

“…25,26 Nerve conduction velocities were not measured directly by the recording procedure, but the latency between the stimulation artifact and the initiation of the M-or H-waves showed no differences after injection of the toxin, similar to studies using analogous human peripheral nerves. 27,28 The total duration of the M-and Hwaves waves, which might be altered if tetanus toxin interfered directly with the intrinsic excitability of a-motor neurons 29 or neuromuscular transmission, 30,31 was similarly unaffected by the toxin. The slopes of the recruitment curves for the M- Table 1.…”