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Simple mechanisms of learning in vitro
THE PROJECT
Virtually all the spinal circuits in humans and vertebrates are subjected to modulation not only during embryonic and neonatal development, in which circuits are shaped, but also during the whole lifespan-time. Plasticity of spinal circuits involves both functional and anatomical changes in different cellular compartments. Action potential threshold, conduction velocity and efficacy in synaptic transmission can be varied. Such a plastic modifications of spinal circuits are caused by signals that come from both
sensory afferents and descending pathways. They are the physiological basis of habituation and sensitization induced by different experimental protocols.
Our research is aimed to uncover the mechanisms of plasticity at the first relay station in spinal cord. Various biological preparations have been considered in our laboratory.
a) in vitro co-cultures consisting of dorsal root ganglion (DRG) neurons grown with dorsal horn
(DH) spinal neurons of rat (Formenti & Arrigoni, unpublished results). The advantages of these co-cultures are mainly represented by their structural essentiality, the accessibility to means of investigation (electrodes), the easy and fast change of the extracellular solution and the possibility to identify the molecular targets with immunocytochemical techniques.
Two variants of this preparation have been implemented. In the first preparation, a fragment of DRG has been cultivated on DH neurons grown in a monolayer (see figure 'Formenti Lab' in the upper left corner of this page) The DRG cells send axons to make synapses on the DH cells. In the second DRG-DH coculture the DRG cells are dissociated. In the culture they can be distinguished from the DH cells becose they are bigger. (see figure below, left)
b) The upper figure on the right shows another in-vitro preparation: A transvers rat spinal cord slice. In the upper part of the figure note the dorsal root stumps with the associated ganglia.
c) An isolated in-vitro whole spinal cord preparation (see figure below)
The isolated rat spinal cord in vitro was used
to investigate the mechanisms of habituation and sensitization of the monosynaptic reflex.
METHODS: Sprague Dawley rats, 7 to 14 days old, were decapitated and the spinal cord was carefully dissected out of the body cavity and placed in a Sylgard-lined Petri dish in cooled (4°C) artificial CSF (ACSF) containing (in mM): 125 CholineCl, 1.3 KCl, 1 CaCl2, 1 MgCl2, 25 glucose, Hepes 10 at pH 7.4, oxygenated with 100% O2. After the dura mater was opened and removed to facilitate access of ACSF to the cord, the dorsal and ventral roots were identified and pinned out with small insect pins. Preparations were left to recover at room temperature for 30–60 min before the onset of experimentation in a different ACSF, containing (in mM): 130 NaCl, KCl 3, 2 CaCl2, 1.3 MgSO4, 0.58 NaH2PO4, 25 NaHCO3, 10 glucose. Glass suction electrodes were used for stimulation and recording L6 dorsal root. The stimulus was delivered at low intensity to elicit in L6 ventral root only the early response (4-5 ms latency) that is widely accepted (see Pinco and Lev-Tov, J. Neurophysiol 1993, 70 (1), 406-417 for reference) to be a monosynaptic reflex (MR), without any later component. Size of the MR, typically triphasic (right panel in fig. 1), was evaluated measuring the underlying area.
Our results show that the monosynaptic reflex in in-vitro rat spinal cord shows several features of the behavioral habituation, including, the size decrement during repetitive stimulation, the dependence of habituation from the stimulus frequency and intensity, the spontaneous recovery after the stimulation is discontinued, the recovery induced by another converging input (dishabituation), indicating that monosynaptic reflex in the isolated in vitro rat spinal cord is a good model for studying the neural mechanisms of simple learnings.
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Alessandro Formenti, Luciano Zocchi (2014) Error signals as powerful stimuli for the operant conditioning-like process of the fictive respiratory output in a brainstem spinal cord preparation from rats. Behavioural Brain Research 272 8-15
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