Synaptic reliability and temporal precision are achieved via high quantal content and effective replenishment: auditory brainstem versus hippocampus.

KEY POINTS: Auditory brainstem neurons involved in sound source localization are equipped with several morphological and molecular features that enable them to compute interaural level and time differences. As sound source localization works continually, synaptic transmission between these neurons should be reliable and temporally precise, even during sustained periods of high-frequency activity. Using patch-clamp recordings in acute brain slices, we compared synaptic reliability and temporal precision in the seconds-minute range between auditory and two types of hippocampal synapses; the latter are less confronted with temporally precise high-frequency transmission than the auditory ones. We found striking differences in synaptic properties (e.g. continually high quantal content) that allow auditory synapses to reliably release vesicles at much higher rate than their hippocampal counterparts. Thus, they are indefatigable and also in a position to transfer information with exquisite temporal precision and their performance appears to be supported by very efficient replenishment mechanisms. ABSTRACT: At early stations of the auditory pathway, information is encoded by precise signal timing and rate. Auditory synapses must maintain the relative timing of events with submillisecond precision even during sustained and high-frequency stimulation. In non-auditory brain regions, e.g. telencephalic ones, synapses are activated at considerably lower frequencies. Central to understanding the heterogeneity of synaptic systems is the elucidation of the physical, chemical and biological factors that determine synapse performance. In this study, we used slice recordings from three synapse types in the mouse auditory brainstem and hippocampus. Whereas the auditory brainstem nuclei experience high-frequency activity in vivo, the hippocampal circuits are activated at much lower frequencies. We challenged the synapses with sustained high-frequency stimulation (up to 200 Hz for 60 s) and found significant performance differences. Our results show that auditory brainstem synapses differ considerably from their hippocampal counterparts in several aspects, namely resistance to synaptic fatigue, low failure rate and exquisite temporal precision. Their high-fidelity performance supports the functional demands and appears to be due to the large size of the readily releasable pool and a high release probability, which together result in a high quantal content. In conjunction with very efficient vesicle replenishment mechanisms, these properties provide extremely rapid and temporally precise signalling required for neuronal communication at early stations of the auditory system, even during sustained activation in the minute range.

Inhibitory glycinergic neurotransmission in the mammalian auditory brainstem upon prolonged stimulation: short-term plasticity and synaptic reliability.

Short-term plasticity plays a key role in synaptic transmission and has been extensively investigated for excitatory synapses. Much less is known about inhibitory synapses. Here we analyze the performance of glycinergic connections between the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO) in the auditory brainstem, where high spike rates as well as fast and precise neurotransmission are hallmarks. Analysis was performed in acute mouse slices shortly after hearing onset (postnatal day (P)11) and 8 days later (P19). Stimulation was done at 37°C with 1-400 Hz for 40 s. Moreover, in a novel approach named marathon experiments, a very prolonged stimulation protocol was employed, comprising 10 trials of 1-min challenge and 1-min recovery periods at 50 and 1 Hz, respectively, thus lasting up to 20 min and amounting to >30,000 stimulus pulses. IPSC peak amplitudes displayed short-term depression (STD) and synaptic attenuation in a frequency-dependent manner. No facilitation was observed. STD in the MNTB-LSO connections was less pronounced than reported in the upstream calyx of Held-MNTB connections. At P11, the STD level and the failure rate were slightly lower within the ms-to-s range than at P19. During prolonged stimulation periods lasting 40 s, P19 connections sustained virtually failure-free transmission up to frequencies of 100 Hz, whereas P11 connections did so only up to 50 Hz. In marathon experiments, P11 synapses recuperated reproducibly from synaptic attenuation during all recovery periods, demonstrating a robust synaptic machinery at hearing onset. At 26°C, transmission was severely impaired and comprised abnormally high amplitudes after minutes of silence, indicative of imprecisely regulated vesicle pools. Our study takes a fresh look at synaptic plasticity and stability by extending conventional stimulus periods in the ms-to-s range to minutes. It also provides a framework for future analyses of synaptic plasticity.

Biodistribution and efficacy of [131I]A33scFv::CDy, a recombinant antibody-enzyme protein for colon cancer.

In antibody-directed enzyme-prodrug therapy (ADEPT), an antibody-bound enzyme localizes to tumor tissue, where it selectively converts a subsequently administered non-toxic prodrug into a cytotoxic drug. A33scFv::CDy is a bifunctional fusion construct comprising a single chain antibody against the gpA33 antigen and the prodrug-converting enzyme cytosine deaminase. gpA33 is highly and homogeneously expressed in >95% of all colorectal cancers. Here we describe the biodistribution and tumor-targeting capacity of 131I labeled A33scFv::CDy. 131I labeling of A33scFv::CDy was performed by the chloramine-T method, and the properties of the resulting [131I]A33scFv::CDy conjugate were determined in vivo and in vitro, including biodistribution studies in nude mice bearing human LIM1215 colon carcinoma xenografts. The [131I]A33scFv::CDy conjugate bound specifically to colorectal cancer cells in vitro with KD = 15.8 nM as determined by a saturation assay. in vivo, the tumor uptake of [131I]A33scFv::CDy peaked at 87% injected dose/g 47 h post injection. Normal tissue uptake was low, and activity in blood was lower than in tumor at all time-points studied (6-92 h). The tumor-to-blood ratio increased over time with a maximum of 8.1 at 67 h post injection. [131I]A33scFv::CDy thus shows a biodistribution that makes it attractive for both radioimmunotherapy (RIT) and ADEPT. Preliminary therapeutic experiments showed a significant reduction of tumor size in mice treated with the A33scFv::CDy-5-fluorocytosine/5-fluorouracil ADEPT system. This work demonstrates the feasibility of ADEPT and RIT based on the A33scFv::CDy recombinant construct.

Ipsilateral submandibular lymphadenectomy does not prolong orthotopic corneal graft survival in mice.

PURPOSE: To explore the impact of selective lymphadenectomy on the outflow from the outer eye and corneal graft survival in mice. METHODS: BALB/c mice underwent submandibular lymphadenectomy either on the left side 7 or 21 days before the experiments or bilaterally 7 days in advance. First, (99m)Tc colloidal albumin (Nanocoll) was given as a subcutaneous lower-lid, upper-lid or subconjunctival injection, and count rates were determined 24 h later in the eyes, submandibular lymph nodes, spleen, liver and blood. Second, corneal graft survival was assessed in lymphadenectomised and control mice, and IFN-gamma secretion was determined in draining lymph nodes at the time of transplant rejection. RESULTS: Following subconjunctival Nanocoll injection, count rates/min/mg tissue were significantly higher in the ipsilateral submandibular lymph node than in the other tissues or blood (<0.01). Ipsilateral lymphadenectomy prior to injection resulted in considerably increased count rates in contralateral nodes ( P<0.01), which were higher after injections into upper than into lower lids ( P=0.004). Bilateral submandibular lymphadenectomy led to enhanced count rates in the eyes, blood, spleen and liver (all P<0.01). Removal of the ipsilateral lymph node prior to corneal transplantation did not prolong allograft survival ( P>0.05) and considerably increased IFN-gamma secretion in contralateral nodes after prior removal of the ipsilateral ones paralleled transplant rejection. CONCLUSIONS: In mice, removed submandibular lymph nodes are functionally completely replaced by the contralateral nodes. These studies demonstrate for the first time lymphatic drainage crossing the midline of the body. Consequently, unilateral lymphadenectomy does not improve corneal graft survival.

Ballistic CTLA4 and IL-4 gene transfer into the lower lid prolongs orthotopic corneal graft survival in mice.

PURPOSE: To explore outflow from the eye and to determine and modulate the influence of lymphatic drainage on corneal graft survival in mice. METHODS: Tracer experiments were conducted in BALB/c mice using the (99m)Tc colloidal albumin Nanocoll. Count rates were determined in the eyes, submandibular lymph nodes, spleen, liver and blood 24 h after subconjunctival, intracorneal, intracameral (anterior chamber), intravenous and subcutaneous lower-lid or upper-lid injections ( n=6 each). Four groups of BALB/c mice ( n=8) received corneal transplants from C3H mice; two of them were treated ballistically with vector CTLA4+IL-4 onto the leg or the lower lid, one group was untreated and the other control group was treated with an empty minimalistic, immunologically defined, gene expression (MIDGE) vector. RESULTS: Radioactivity was detected in the liver, spleen and ipsilateral submandibular lymph node after intracameral injection as follows: 91.9%, 6.6% and 1.2% respectively. Radioactivity uptake of the ipsilateral submandibular lymph node was also low after intravenous injection (0.1%) but high after intracorneal (33.8%), lower-lid (62.0%) and subconjunctival (71.2%) injection. Vector CTLA4+IL-4 treatment of the lower lid but not of the leg prolonged graft survival ( P=0.004). CONCLUSION: These tracer studies confirmed for the first time identical lymphatic drainage from the cornea and the lower lid. Logically, lymphatic drainage could be manipulated and graft survival improved by gene transfer to the lower lid.