Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in <em>Aplysia californica</em>

Abstract

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques 1,2,3,4 . Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica 5 . We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath 5 , so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation 4,6,7 . For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation 8 or in vivo 9. This process can also be applied in other motor pools 10,11,12 in Aplysia or in other animal systems 2,3,13,14 . Video LinkThe video component of this article can be found at http://www.jove.com/video/50189/ Protocol Preparation of Recording Dish1. During the force transducer experiments, the buccal ganglia, cerebral ganglion, and buccal mass are placed in a round Pyrex dish that is specialized for force studies. 2. To induce ingestive-like patterns in the experiments, we need to apply the non-hydrolyzable cholinergic agonist carbachol to the cerebral ganglion 15 . To avoi...

Motor neuronal activity varies least among individuals when it matters most for behavior

“…Previous studies of Aplysia feeding suggested that the duration of biting behavior shortens as animals engage in biting (Rosen et al 1989), and a previous study of variability of swallowing used real-time behavioral durations to characterize variations over time (Lum et al 2005). We hypothesized that behavior duration alone might provide a way of distinguishing responses in a sequence, or one animal from another, and found evidence to support this hypothesis.…”

“…Previous studies have demonstrated that all critical motor neuronal elements for controlling the feeding apparatus (the buccal mass) can be monitored by recording directly from 1) the protractor muscle (I2), whose activation is an obligatory first step for effective feeding movements and whose EMG patterns represent the activity of interneurons/motor neurons B31/B32 and motor neurons B61/B62 (Hurwitz et al 1996); 2) the radular nerve (RN), which contains axons of the motor neurons B8a/B8b controlling closure of the grasper Chiel 1993a, 1993b); 3) buccal nerve 2 (BN2), which contains axons of the motor neurons controlling the jaw musculature, B10, B6, B9, B3, B38, and B43 (Church and Lloyd 1994;Lu et al 2013;Morton and Chiel 1993b); and 4) buccal nerve 3 (BN3), which contains axons of motor neurons controlling some of the intrinsic muscles of the grasper (e.g., B15/B16; Church and Lloyd 1994;Cohen et al 1978) and a multiaction neuron, B4/B5 (Church and Lloyd 1994;Warman and Chiel 1995;Ye et al 2006b). Nerves RN, BN2, and BN3 are referred to as n1, n5, and n4 in Scott et al (1991).…”

“…1, and the criteria used to define the starts and ends of bursts are listed in Table 1. To characterize the key motor features, we determined the start and stop times of the I2 EMG during the protraction phase by measuring both the time of the first spike and the time at which the muscle EMG reached 10 Hz and the stop time when it fell below 5 Hz (Hurwitz et al 1996; 3 measures), the start and stop times of the largest units on RN (corresponding to activity in the B8a/b neurons; Chiel 1993a, 1993b; 2 measures), the start and stop times of the B4/B5 units on BN3 [largest units on BN3 (Warman and Chiel 1995) from the first to the last spike; 2 measures], and the start and stop times of identified motor neurons B6/B9 and B43 (Lu et al 2013;4 measures). Finally, we measured the third-largest units on BN2 (Church and Lloyd 1994;Morton and Chiel 1993b;Ye et al 2006a), focusing on when they began in retraction (1 measure), for a total of 12 different measures of the motor program.…”

“…Variability can obscure connections between motor patterns and behavior (Horn et al 2004;Lum et al 2005). Averaging parameters from many animals may not solve this problem.…”

“…Motor patterns can be recorded in intact, behaving animals (Cullins and Chiel 2010), in which motor neurons can be identified (Lu et al 2013). Biting (an attempt to grasp) and swallowing (food ingestion) are readily quantified (Kupfermann 1974).…”