Secretory leukocyte protease inhibitor reverses inhibition by CNS myelin, promotes regeneration in the optic nerve, and suppresses expression of the transforming growth factor-ß signaling protein Smad2.

After CNS injury, axonal regeneration is limited by myelin-associated inhibitors; however, this can be overcome through elevation of intracellular cyclic AMP (cAMP), as occurs with conditioning lesions of the sciatic nerve. This study reports that expression of secretory leukocyte protease inhibitor (SLPI) is strongly upregulated in response to elevation of cAMP. We also show that SLPI can overcome inhibition by CNS myelin and significantly enhance regeneration of transected retinal ganglion cell axons in rats. Furthermore, regeneration of dorsal column axons does not occur after a conditioning lesion in SLPI null mutant mice, indicating that expression of SLPI is required for the conditioning lesion effect. Mechanistically, we demonstrate that SLPI localizes to the nuclei of neurons, binds to the Smad2 promoter, and reduces levels of Smad2 protein. Adenoviral overexpression of Smad2 also blocked SLPI-induced axonal regeneration. SLPI and Smad2 may therefore represent new targets for therapeutic intervention in CNS injury.

A micro-optrode for simultaneous extracellular electrical and intracellular optical recording from neurons in an intact oscillatory neuronal network.

Fluorescent dyes and proteins have become ubiquitous tools for the study of intracellular function. However, standard techniques used to visualize these indicators in the brain usually require cellular dissociation or fine sectioning of the specimen into slices for viewing under a fluorescence microscope. These actions remove cells from their natural physiological environment and in the case of neurons, sever synaptic connections from other regions of the central and peripheral nervous system. Even with the use of multi-photon excitation microscopy, resolution of neurons in the intact brain beyond depths of around 500 microm is technically difficult to achieve. We have developed a relatively inexpensive small fiber optic probe ('micro-optrode') to simultaneously record extracellular electrical activity and intracellular fluorescence changes in real-time from structures deep within the intact brain. The micro-optrode was tested during experiments in the in situ working heart-brainstem preparation (WHBP) of rat, which allows the study of the intact ponto-medullary neuronal network that controls breathing. In conjunction with calcium-sensitive indicators, we successfully validated the utility of the micro-optrode by recording, for the first time, intracellular calcium dynamics in respiratory neurons of the intact respiratory network under normal conditions and during physiological and pharmacological manipulations.

Temporal profile of arginine vasopressin release from the neurohypophysis in response to hypertonic saline and hypotension measured using a fluorescent fusion protein.

Methods currently employed to study the release of hormones such as arginine vasopressin (AVP), while sensitive, suffer from a low temporal resolution such that the monitoring of AVP release on a moment-to-moment basis is not possible. Here, we describe a new approach to indirectly monitor the temporal profile of AVP release from the neurohypophysis of transgenic rats expressing an AVP-eGFP fusion gene. Using fibre-optic probes (termed 'optrodes') we were able to indirectly monitor AVP release via a reporter moiety in real-time. This method is a major advance over current methods used to monitor AVP release. Intravenous administration of hypertonic saline (3M NaCl) induced a rapid (latency of 2-3s) increase in fluorescence detected in the neurohypophysis that lasted on average for 60s - a response that was highly reproducible. Infusion of sodium nitroprusside induced a rapid fall in blood pressure accompanied by a rapid, stimulus-locked increase in fluorescent signal that returned to baseline with the recovery of blood pressure to pre-stimulus levels - again this response was highly reproducible. Withdrawal of blood (to simulate haemorrhage) also resulted in a stimulus-locked increase in fluorescence that return to baseline after the withdrawn blood was returned to the animal. In conclusion, we developed a highly sensitive approach that allows the indirect measurement of AVP release via the monitoring of a reporter gene in real-time. This technology can be adapted to permit the study of a whole array of neurohormones/chemicals in transgenic animals expressing a fluorescent reporter construct.