Articles in PresS. J Neurophysiol (March 18, 2015). doi:10.1152/jn.00135.2015
1 2 3 Compensatory Plasticity Restores Locomotion Following Chronic Removal of Descending Projections 4 5 6 7 8 9 10 11 12 13
by
Cynthia M. Harley1, Melissa G. Reilly1, Christopher Stewart3, Chantel Schlegel1, Emma Morley1, Joshua G. Puhl2, Christian Nagel1, Keven M. Crisp3 and Karen A. Mesce1,2
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Affiliations: 1Department of Entomology and 2Graduate Program in Neuroscience University of Minnesota, Saint Paul, MN, 55108; 3Department of Biology and Neuroscience Program, Saint Olaf College, Northfield, MN 55057
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Abbreviated title: Compensatory Plasticity and the Recovery of Locomotion
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Corresponding author: Dr. Karen A. Mesce, Departments of Entomology and Neuroscience, Graduate Program in Neuroscience, University of Minnesota, 219 Hodson Hall, 1980 Folwell Avenue, St. Paul, MN 55108 Email: [email protected] Phone: (612) 624-3765; (612) 624-3734; Fax: (612) 625-5299
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Number of figures: 9 figures
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Foundation (IOS-0924155 to K.A.M. and K.M.C.). We are especially grateful to William
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B. Kristan, Jr. and members of his laboratory for sharing their valuable insights
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throughout the course of this work. We also thank Irene (Rani) Thomas and Kathleen
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Klukas for conducting preliminary experiments, which facilitated the studies reported
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here. We thank Syed Jabbar Haider for his help with behavioral video collection.
Number of pages: Total = 37 pages (32 text pp and 5 ref pp) Number of words: Abstract (233), Introduction (493), Discussion (1,575) Acknowledgements: This work was supported by grants from the National Science
1 Copyright © 2015 by the American Physiological Society.
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Abstract
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Homeostatic plasticity is an important attribute of neurons and their networks, enabling
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functional recovery after perturbation. Furthermore, the directed nature of this plasticity
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may hold a key to the restoration of locomotion after spinal cord injury. Here, we
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studied the recovery of crawling in the leech, Hirudo verbana, after descending cephalic
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fibers were surgically separated from crawl central pattern generators shown previously
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to be regulated by dopamine. We observed that immediately following nerve cord
46
transection leeches were unable to crawl, but remarkably, after a day to weeks, animals
47
began to show elements of crawling and intersegmental coordination. Over a similar
48
time course, excessive swimming due to the loss of descending inhibition returned to
49
control levels. Additionally, removal of the brain did not prevent crawl recovery,
50
indicating that connectivity of severed descending neurons was not essential. Following
51
crawl recovery, a subset of animals received a second transection immediately below
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the anterior-most ganglion remaining. Similar to their initial transection, a loss of
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crawling with subsequent recovery was observed. These data, in recovered individuals,
54
support the idea that compensatory plasticity directly below the site of injury is essential
55
for the initiation and coordination of crawling. We maintain that the leech provides a
56
valuable model to understand the neural mechanisms underlying locomotor recovery
57
after injury because of its experimental accessibility, segmental organization and
58
dependence on higher-order control involved in the initiation, modulation and
59
coordination of locomotor behavior.
60
2
61
Introduction
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Neural circuits are remarkably resilient. Over a permissible range of perturbations, they
63
can become reconfigured to maintain seemingly normal activity (Marder 2011;
64
Turrigiano 2012; 2011). This ability is largely attributed to cellular- and network-level
65
homeostatic processes that can retune a perturbed system and restore its functional
66
state (Marder and Goaillard 2006; Pozo and Goda 2010; Turrigiano 2012). The idea of
67
manipulating state-preserving homeostatic plasticity for restoring locomotor function
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after spinal cord injury appears promising, especially when repair or regrowth of
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neuronal connections across sites of injury is not possible (Edgerton et al. 2004;
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Rossignol and Frigon 2011). While such strategies may hold keys to significant
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biomedical advances for locomotor recovery, the cellular mechanisms underlying these
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and most other homeostatic or compensatory processes are just beginning to be
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understood (Husch et al. 2012; Pozo and Goda 2010; Sakurai and Katz 2009).
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We propose that the leech serves as a valuable and highly tractable system in
75
which to investigate homeostatic mechanisms contributing to functional locomotor
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recovery. Invertebrate preparations have long been pivotal in illuminating the cellular
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bases of rhythmic-pattern generation, its modulation, and stability (Marder et al. 2005;
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Marder and Calabrese 1996; Selverston 2010). For almost half a century, the medicinal
79
leech, in particular, has served as a model organism to illustrate how neural circuits are
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organized and modulated to produce locomotor behaviors (Briggman and Kristan 2008;
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Brodfuehrer et al. 1995; Kristan et al. 2005; Mullins et al. 2011; Puhl and Mesce 2008;
82
2010). Similar to the functions of vertebrate spinal projections (Grillner 2006a; b;
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Grillner and Wallen 2002; Kiehn 2011), higher-order fibers descending from the leech’s
2
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cephalic ganglion (i.e., brain) have been known to be vital for the initiation, modulation
85
and coordination of crawling across iterated locomotor pattern generators (Puhl et al.
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2012). Thus, in this report, we examined whether medicinal leeches (Hirudo verbana)
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could recover their ability to crawl after their nerve cords were fully transected, while
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noting whether or not regrowth of descending axons may have contributed to locomotor
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recovery. By observing leeches for days to months following such transections, we
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determined whether they could recover their ability to crawl in a coordinated manner,
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despite the loss of previously essential control elements for that coordination.
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In mammals, compensatory plasticity is not always beneficial if it becomes
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undirected during severe supra-spinal injury (Beauparlant et al. 2013); thus discovering
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therapeutic strategies and how they work to steer beneficial features of plasticity hold
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promise for promoting adaptive locomotor recovery, especially those related to
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neuromodulatory intervention (Guertin 2014; Musienko et al. 2011; Sharples et al. 2014).
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In the leech, we found that the crawl pattern-generating networks distal to the site of
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injury were able to achieve adaptive solutions for their reconfiguration and
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intersegmental coordination, independent of descending fibers from the brain becoming
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reconnected. Understanding how the leech nervous system correctly reconfigures itself
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post injury, while making occasional misdirected mistakes, will likely provide valuable
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insights about the cellular substrates of locomotor plasticity across animal species,
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including humans.
3
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Materials and Methods
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Animals and Surgical Procedures
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Adult medicinal leeches (hermaphrodites), Hirudo verbana (Carena 1820; Siddall et al.
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2007) were obtained from Niagara Medical Leeches (Westbury NY, USA) and stored at
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room temperature in a glass terrarium filled with water from a natural well at the
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University of Minnesota. After surgery, leeches were housed in individual containers (17
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cm wide x 8 cm tall) with a thin layer of aquarium gravel covering the bottom and well
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water at 4-5 cm in depth.
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Leeches chosen for behavioral assays weighed 2.45 +/- 0.48 g. Briefly, the leech
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CNS consists of: 1) a compound cephalic ganglion comprising the supra-and sub-
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esophageal (SEG) ganglion (i.e., the brain); 2) the 21 segmentally distributed ganglia
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(M1-M21) and the connectives that link them together (i.e., the ventral nerve cord); 3)
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the compound terminal ganglion (‘tail brain’). Prior to surgery, leeches were
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anesthetized on ice until they were no longer responsive to tactile stimuli. Once
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anesthetized, they were pinned onto a dissecting tray lined with bees’ wax ventral side
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up. One thin pin was placed through the leech’s mouth, avoiding damage to the brain,
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and the other one placed through the caudal sucker. A small vertical incision spanning
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2-3 annuli was made to access the nerve cord for complete transection. The incision
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was closed using one or two stitches made with 7-0 or smaller suture thread. A post-
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mortem examination was conducted in all animals to confirm that the nerve cord
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(connectives) had remained fully transected (Fig. 1A far right). Herein, we present data
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from individuals with several different types of manipulations, defined as follows: 1)
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Transections- A small incision was made 5 annuli below the mouth, which permitted a 4
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complete transection of the nerve cord between the brain and first ganglion (M1) (see
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Fig. 1A) or between M1 and the second ganglion (M2). No differences in behavior were
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observed between these individuals. Three individuals were tested at 40 days and
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another four tested at 48-50 days following transection; these individuals were
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combined into a single grouping in the figures labeled as being tested at 40-50 days
132
following transection; 2) M1 removal- M1 was surgically extirpated from the leech (see
133
Fig. 6A); 3) SEG removal- The subesophageal ganglion was removed by surgically
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detaching it from the supraesophageal ganglion and M1 (see Fig. 7A); 4) Second
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Transection- Leeches that showed a complete recovery of crawling behavior were
136
anesthetized and transected again at 40-50 days following the first transection (between
137
the brain and M1). The second transection was made between M1 and M2 (see Fig.
138
8A); 5) Midbody- This type of transection was made between M9 and M10 or M10 and
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M11, resulting in the removal of all descending inputs to the posterior half of the leech
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while preserving those to the anterior half of the animal (see Fig. 9A inset); 6) Sham-
141
Operated Controls- These surgeries were performed as a control for all of the above
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conditions, whereby the animal received identical surgical manipulations (e.g., skin and
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muscles cut and nerve cord tugged), except the nerve cord was not severed.
144 145
Behavioral Arena and Video Acquisition
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Individuals were tested before and at multiple points following transection. The order in
147
which the tests were presented was randomly chosen. The behavioral arena was a
148
cylindrical acrylic dish, 20.3 cm in diameter by 8 cm in height. It was illuminated with
149
LED lighting to approximately 750 lux. Water within the arena ranged from 19-21˚C. 5
150
Videos were filmed at 38 fps using a DCC1455M high resolution CMOS camera
151
(Thorlabs, Newton NJ, US).
152
Behavior under the following conditions was examined: Deeper water- the arena
153
was filled with well water to a depth of 4 cm. Under this condition a leech was able to
154
swim or crawl. Shallow water- the arena was filled with 15 ml of well water, which was
155
just enough to coat the bottom of the arena with a thin layer of water 1-2 mm high.
156
Under this condition the leech was only able to crawl. Under either condition, the
157
leech’s behavior in the arena was recorded for the 6 minutes immediately following
158
placement of the animal in the arena. Evoked crawling- To initiate crawling, a round no.
159
4 artist’s paintbrush was used for two minutes. The surface of the leech was touched
160
with the brush in an anterior-to-posterior direction to stimulate crawling behavior. In
161
response to this stimulus, a crawl or crawl-like behavior was counted if the leech
162
performed one or more crawl-like cycles (note: an elongation and contraction phase
163
equals one cycle) (Fig. 1D).
164
Behavioral Descriptions and Analysis:
165
Swim and crawl locomotor states were easily separable by visual inspection, defined as
166
follows: Swim- the whole body is flattened and moves with a series of quasi-sinusoidal
167
dorso-ventral movements progressing caudally (Brodfuehrer et al. 1995; Stent et al.
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1978). During swimming, unlike crawling, neither of the leech’s suckers attaches to the
169
substrate. Crawling and Crawl-like behavior- A defining feature of normal crawling is
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that during each of its phases—elongation and contraction— the individual body
171
segments are coordinated (Puhl and Mesce 2010). During elongation, each segment
172
sequentially elongates (lengthens) in a stereotypic anterior-to-posterior directed wave. 6
173
During contraction, individual segments contract (shorten) in the same posteriorly-
174
directed manner. Often, there is a delay of a few seconds before the next cycle is
175
initiated. Here, we use the term ‘crawl-like’ to expand the traditional definition of crawling
176
to include some of the atypical locomotor activity we observed during the recovery
177
process. To be counted as crawl-like behavior, animals had to exhibit a body elongation
178
phase followed by a body contraction phase with no body flattening typically associated
179
with swimming. However, the crawl-specific coordination during each elongation or
180
contraction phase (i.e., the caudally-directed metachronal wave) was not a requirement.
181
For example, a reversal in wave direction, a partial wave or an uncoordinated wave
182
would not limit use of the ‘crawl-like’ designator. Using recorded videos, swim and crawl
183
behaviors were quantified by measuring the duration of bouts of swimming or crawling
184
(in deep water) or by counting the number of crawl cycles (in shallow water) within a 6
185
minute (min) test period.
186
Crawl movement analysis: To understand the recovery process better, videos of
187
representative crawl cycles were used to generate a series of frames depicting the
188
kinematics of the elongation and contraction phases. These analyses were based on
189
the fact that, during the elongation and contraction phases, the body narrows and
190
widens respectively—a consequence of hydroskeletal dynamics. Representative crawl
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phases were divided into 8 images (Fig. 4). The first and last silhouettes indicate the
192
leech’s position at the beginning and end of a crawl cycle. The intervening images were
193
separated by intervals of 15% of the total cycle duration (e.g., about 0.35 s for the pre-
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lesion series). These images (for example, Fig. 4A i) were then analyzed in ImageJ by
195
applying a threshold filter, which outlined the leech in black against the white
7
196
background (Fig. 4A ii). In those videos with some glare, the silhouettes contained a
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peppering of open spaces after thresholding (Fig. 4A ii). Such areas were subsequently
198
filled in by using the line-and-fill tools in ImageJ so as not to cause errors during
199
automated thickness (width) colorization. This process allowed for the local thickness
200
plugin to be run. A reference of a 50 pixel-wide line was placed into the image such that
201
all images could be calibrated to the same scale for coloration (Fig. 4A iii). The local
202
thickness algorithm colorized images according to the largest circle that fit into a
203
particular area, i.e., the leech’s silhouette. The local thickness algorithm was run and the
204
image colorized for individual images such that blue-purplish colors represented less
205
wide (narrower) areas of the leech and orange-yellow-white areas represented wider
206
regions of the leech’s body-- again, elements that correspond to the elongation and
207
contraction phases of crawling (Fig. 4B).
208
Coordination analysis: To assess acquisition of the posteriorly-directed
209
coordination of the crawl elongation phase, a given leech was visually divided into four
210
approximately equal sections. When a body quarter was active during elongation, it
211
would appear thinner due to the consequence of circular muscle contractions. For
212
these analyses, up to 25 crawl elongations were measured for each individual in a 6 min
213
window in shallow water, with the activity and order of body quarters in close succession
214
counted. Only the elongation phases were used for analysis because they typically
215
occupied a greater proportion of a given crawl cycle and active body movements were
216
readily observed, assuring a more accurate measure of active body quarters and their
217
coordination. For such analyses we used individuals that were available for all trials (n
218
= 8 for transections; n = 4 for sham-operated controls). 8
219
Statistics and graphing methods for behavioral analyses
220
Unless otherwise noted, the statistical test used was the Skillings-Mack test (SM) that,
221
when significant, was followed by a post-hoc Dunn’s comparison; results are reported
222
from this Dunn’s comparison where applicable (Skillings and Mack 1981). For swim bout
223
durations, this test required that we take the mean of each individual’s swim bout
224
duration and look at its change over time. The statistical test used for data presented in
225
Fig. 1 was the Kolmogorov-Smirnov (K-S) test (Justel et al., 1997), a two sample
226
analysis to compare non-parametric means with small sample sizes. Box Plots—
227
Because many of the data sets included non-normal data and relatively small sample
228
sizes, we used the Tukey box and whisker method of plotting data. In this method, the
229
box surrounds the 25-75 percentiles. A line is present at 50% (the median). The
230
whiskers extend to 1.5 times the interquartile ratio (Tukey 1977). For illustration
231
purposes only, within these plots, the arithmetic mean is marked with a + sign. Outliers
232
are marked by diamonds. Unless otherwise noted, all figures with measures of crawl or
233
swim cycle number, cycle duration, or duration of uninterrupted locomotor cycles (bouts)
234
incorporated the above mentioned box plots.
235
Results
236
The time course and process of crawl recovery after injury
237
The brain (cephalic ganglion) exerts a hierarchical control over behavior in the form of
238
descending axons whose firing rate biases the animal toward (or against) the
239
expression of swimming or crawling (Brodfuehrer and Friesen 1986; Esch et al. 2002;
240
Mesce et al. 2008; Mullins et al. 2012; Puhl et al. 2012). While disconnection of the
9
241
brain has been shown to promote swimming and result in a loss of crawl coordination
242
(Puhl et al. 2012; Puhl and Mesce 2010), these studies have only examined leeches
243
with acute lesions (i.e., within hours of surgery). Here, we examined the recovery of
244
normal function for weeks to months following transection of the nerve cord above M1
245
or M2 (n = 14), and in sham controls where the nerve cord was not severed (n = 7).
246
Consistent with prior studies, transection of the leech nerve cord between the
247
brain and M1 (Fig. 1A) caused a 10 fold increase in the bout duration of swimming (Fig.
248
1B) (n = 14, p0.9, KS test). In contrast, one day
254
after surgery, transected animals showed a significant decrease in the number of crawl-
255
like cycles, with only one transected individual of 14 exhibiting a single spontaneous
256
crawl cycle (Fig. 1C, p0.08, KS test). Sham-operated
10
263
individuals never showed a significant change in crawling (p>0.5, KS test) or swimming
264
behavior (p>0.9, KS test).
265
Having determined that leeches show an initial deficit in crawling after transection,
266
but exhibit the behavior readily at a later date, we examined the time course of recovery.
267
First we wanted to examine when leeches begin to exhibit crawl cycles. These data are
268
indicated in Figure 2A, where each leech is represented by a single row and each day
269
following surgery is represented by a column. Prior to transection all leeches (n= 14)
270
exhibited crawl behavior; however, following transection only a single individual
271
exhibited one crawl cycle before Day 4. In contrast, all sham-operated individuals (n = 7)
272
exhibited crawling the day following their surgery. In transected animals, by 40-50 days
273
following surgery, all 8 leeches tested were able to exhibit multiple crawl cycles (Fig. 2A).
274
To understand better the time course of crawl recovery, the specific number of crawl
275
cycles exhibited was quantified when leeches were placed in either shallow water (Fig.
276
2B) or deeper water conditions (Fig. 3B). In shallow water, transected leeches exhibited
277
a significant decrease in the number of crawl cycles performed following surgery (Fig.
278
2B, p0.95, SM test).
300
While leeches regained an ability to generate crawl-like cycles with two phases
301
(i.e., elongation-contraction) after transection, we wanted to determine if within each
302
phase the segments were able to regain their caudally-directed metachronal
303
coordination. Thus, we analyzed movements of the leech by characterizing its body
304
form during these two phases (Fig. 4). By colorizing videos of leeches crawling we were
305
able to visualize the progression of the anterior-to-posterior wave of elongation (blue-
306
red = narrower body) and contraction (white-orange = wider body) and thus examine the
307
form of crawling during its recovery (see Methods section for details, Fig. 4A). In sham-
308
operated individuals and pre-transection trials, there was a fluid anterior-to-posterior 12
309
progression of coordinated segmental activity during both phases that propelled the
310
leech forward a considerable distance (Fig. 4B). Here, the notable curvature in the
311
silhouettes was due to the tendency of leeches to crawl thigmotactically around the
312
inside edge of the dish. In sham-operated individuals, just one day following surgery,
313
normal crawling was apparent (Fig. 4B). Figures 4C and 4D show two different
314
transected leeches as crawling was reacquired; note that metachronal body activity was
315
absent or weak at best, as depicted by a lack of blue-red color and its caudally-directed
316
invasion into the body (see Fig. 4C,D, Day 3 and 6 respectively). In fact, only a portion
317
of the leech’s body exhibited a crawl cycle (Fig. 4D, Day 6). Over time, however,
318
crawling did begin to regain its posterior-directed coordinated fluidity, and crawling
319
began to appear more normal. For example, on Day 12 (Fig. 4 C, 3rd-5th silhouette, left
320
to right) the purple-red color can be seen invading the leech in an anteriorly-directed
321
manner, reflecting the narrowing of the body segments as the circular muscles
322
contracted during the crawl elongation phase. In turn, the crawl contraction phase can
323
be appreciated (Fig. 4 C, Day 12, 6th-8th silhouette) by the caudally-directed wave of
324
orange-to-white color that progressively invades the leech body, reflecting the widening
325
(fattening) of each body segment as the longitudinal muscles contracted. By Day 40,
326
both leeches were showing crawl cycles that were similar in form and duration to pre-
327
lesion crawl cycles (Fig. 4C,D). It is noteworthy that individuals showed variability in
328
their recovery (for example, compare Figs. 4C and D on Days 10 and 9), which might
329
facilitate the future detection of factors that promote recovery across individuals. Lastly,
330
transected individuals initially exhibited long duration crawl cycles. For example, on the
331
first day that crawling behavior was expressed we saw an increase in crawl cycle
13
332
duration from the mean pretest value of 7+/-2.43 sec to 21.29+/- 13.16 sec/cycle (n = 9,
333
p0.12).
30
721
Figure 7. Changes in crawling and swimming following complete removal of the
722
subesophageal ganglion (SEG). To rule out axonal reconnection between the
723
proximal and distal severed regions of all cephalic descending axons, we removed the
724
entire SEG in 6 individuals and determined whether crawling could be re-established. A,
725
Diagram depicting SEG removal. B, Representation of the course of recovery for
726
individual animals as described in Fig. 6B. C, Number of crawl or crawl-like cycles
727
occurring within a 6 min trial. C. After Day 10, the decrease in number of crawl cycles
728
was no longer significant (ns, p>0.18, Dunn’s test). D, After Day 30, duration of swim
729
bouts was no longer significantly longer compared to pre-test durations (ns, p>0.09).
730 731
Figure 8. Leeches repeat the recovery process following removal of the ‘lead’
732
ganglion. To ascertain whether or not the ganglion closest to the transection site had
733
become vital for crawl recovery, we tested whether its removal would result in a second
734
loss of crawling behavior with a second round of recovery. A, Diagram depicting the
735
sites of the first and subsequent transections. B, Representation of the course of
736
recovery for each animal as previously described in Fig. 6B. C, Number of crawl or
737
crawl-like cycles occurring within a 6 min trial. At 72 days following the second
738
transection, the number of crawl cycles wais no longer significantly different than pre-
739
test values (ns, p>0.32, SM test). D, Bouts of swimming measured over the course of a
740
single trial for the same 6 individuals tested for crawling. At 8 and 72 days following a
741
second transection, swim bout durations were no longer elevated compared to pre-test
742
values (ns, p>0.08 and p>0.37 respectively, SM test).
743
31
744
Figure 9. Midbody nerve cord transections test possible anatomical constraints
745
on the lead ganglion hypothesis and establish if across transection oscillator
746
coupling is possible. To test whether posteriorly located ganglia were capable of
747
setting up their own crawl wave along the body, nerve cords were transected below M9-
748
10. A, Diagram depicting the midbody transection (left) and representation of the course
749
of recovery for all 6 individuals measured. The blue and pink shadings over the
750
diagram highlight behavioral activity controlled by a partial nerve cord attached to the
751
brain and one that is not, respectively. B, Video image prepared, as in Fig. 4, of a leech
752
crawling at 115 days after transection below M9-10. Note that in the 5th through 7th
753
frames (silhouettes) the anterior half of the leech is entering its crawl elongation phase,
754
while the posterior body half is in a crawl-contraction phase; indicative of 2 independent
755
crawl cycles. C, Number of crawl or crawl-like cycles occurring within a 6 min trial. The
756
blue and pink shading highlights the anterior and posterior body regions; purple denotes
757
the potential for cross-transection activity. Note that no cross-transection body
758
coordination ever returns for crawling (bottom panel). D, Video images of coordinated
759
(sinusoidal) swimming (top clip), and cross-transection uncoordinated swimming
760
(bottom clip). For uncoordinated swimming, the sinusoidal swim wave was not
761
distributed along the entire body; often with the anterior body in a contracted state. E,
762
Bouts of swimming measured over the course of a single 6 min trial for all 6 individuals
763
observed. Swim suppression of the posterior body returned to control levels by Day 10.
764
Cross-transection swim coordination, measured as a whole-body sinusoidal waveform,
765
was not prevented. Note that the time scales on the y axes have been kept the same for
766
purposes of comparison, thus insets (swim, top and bottom) are provided to showcase
32
767
smaller values.
768 769
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966
5
C Transection Brain
No lesion
SEG M1
Ganglia
SEG
M1
98 days following transection
M2 M3
B
Unevoked crawl behavior Shams Transections 60
20 0
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Before 1 day after 40-50 days after transection transection transection
Evoked crawl-like behavior
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D Probability of induced crawl-like activity
Average swim bout duration (s) per 6 minute trial
Swim behavior
Average number of crawl-like cycles per 6 minute trial
A
1.0 0.8 0.6 0.4 0.2 0.0 Before 1 day after 40-50 days after transection transection transection
Figure 1
A Days after transection
No crawl Crawl Only 1 cycle No data
Shams
Transections
Pre 1 2 3 4 5 6 7 8 9 10 13 40-50
Number of crawl or crawl-like cycles in 6 minutes
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Figure 2
A
Deeper water swim behavior
Transections Shams
Swim bout duration (s)
360 300 +
240 +
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Figure 3
Width
Figure 4
Percent of elongations
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Number of active quarters 100
Percent of elongations
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# active quarters 1 2 3 4
80 60 40 20 0 Pre 1 2 3 4 5 6 7 8 9 10 40+ Sham day 1 Test Days after transection
First quarter to elongate A
80
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D
20 0 Pre 1 2 3 4 5 6 7 8 9 10 Test
Days after transection
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40+ Sham day 1
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Percent of elongations
100 80
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60 40
None
20 0 Pre 1 2 3 4 Test
5 6 7 8 9 10
Days after transection
40+ Sham day 1
Figure 5
B
A
M1 removal
Days after transection pre 1 4 8 10 30 115
D
Crawl behavior ns
40
ns
ns
30 ns
20 10 0
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+ + +
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Pre 1 6 8 10 46 115 Test Days after transection
Swim bout duration (s)
Number of crawl-like cycles in 6 minutes
C
No crawl Crawl Only 1 cycle
Swim behavior 360 300 240 ns
180 120 60 0
+
ns
+ +
ns
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Pre 1 6 8 10 46 115 Test Days after transection
Figure 6
B
A
SEG removal Days after transection
pre 1
D
Crawl behavior 30
ns ns
20
10
ns
+
0
Pre Test
+ 1
+
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30
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+ 6
Days after transection
115
Swim bout duration (s)
Number of crawl-like cycles in 6 minutes
C
6
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No crawl Crawl Only 1 cycle
30 115
Swim behavior 360 300 240 180
+
120
+
ns ns
60 0
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+
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Pre 1 6 10 30 115 Test Days after transection
Figure 7
A
B First
No crawl Crawl Only 1 cycle
Second
Days after transection pre
2 4 6 8 10 12 14 76
Time
D
Number of crawl-like cycles in 6 minutes
Crawl Behavior ns
40
+ +
20
0 Pre Test
+ + + + + + + 2
4
6
8
10
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14
72
Days after second transection
Swim bout duration (s)
C
Swim Behavior 360 300
+
+
240
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180
+
120
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60 0
ns
+ ns
+ Pre Test
2
4
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Days after second transection
Figure 8
A
Days after transection pre
1
3
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6
No crawl Crawl Only 1 cycle
7 10 31 115
Anterior
Posterior
B
1.4 cm
D
Coordinated swim
1.1 cm 0.7 cm
Uncoordinated swim
0.4 cm 0.0 cm
3.0 sec
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E
Crawl Anterior to transection
Swim 30
180
Anterior to transection
50 20
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115
Posterior to transection 30 20 10
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Days after transection
Cross transection coordination 30
Swim bout duration per 6 minute trial
Number of crawl-like cycles in 6 minutes
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Pre Test
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Days after transection
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Cross transection coordination ns ns
Days after transection
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0
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ns
ns
1 30
60
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6 10 30 Days after transection
20
20 10
+6
Posterior to transection
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Pre Test
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6
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6
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+ 30
115
+
115
Days after transection
Figure 9
1 2 3 Compensatory Plasticity Restores Locomotion Following Chronic Removal of Descending Projections 4 5 6 7 8 9 10 11 12 13
by
Cynthia M. Harley1, Melissa G. Reilly1, Christopher Stewart3, Chantel Schlegel1, Emma Morley1, Joshua G. Puhl2, Christian Nagel1, Keven M. Crisp3 and Karen A. Mesce1,2
14 15 16
Affiliations: 1Department of Entomology and 2Graduate Program in Neuroscience University of Minnesota, Saint Paul, MN, 55108; 3Department of Biology and Neuroscience Program, Saint Olaf College, Northfield, MN 55057
17
Abbreviated title: Compensatory Plasticity and the Recovery of Locomotion
18 19 20 21 22 23 24
Corresponding author: Dr. Karen A. Mesce, Departments of Entomology and Neuroscience, Graduate Program in Neuroscience, University of Minnesota, 219 Hodson Hall, 1980 Folwell Avenue, St. Paul, MN 55108 Email: [email protected] Phone: (612) 624-3765; (612) 624-3734; Fax: (612) 625-5299
25 26 27 28 29 30 31 32
Number of figures: 9 figures
33
Foundation (IOS-0924155 to K.A.M. and K.M.C.). We are especially grateful to William
34
B. Kristan, Jr. and members of his laboratory for sharing their valuable insights
35
throughout the course of this work. We also thank Irene (Rani) Thomas and Kathleen
36
Klukas for conducting preliminary experiments, which facilitated the studies reported
37
here. We thank Syed Jabbar Haider for his help with behavioral video collection.
Number of pages: Total = 37 pages (32 text pp and 5 ref pp) Number of words: Abstract (233), Introduction (493), Discussion (1,575) Acknowledgements: This work was supported by grants from the National Science
1 Copyright © 2015 by the American Physiological Society.
38 39
Abstract
40
Homeostatic plasticity is an important attribute of neurons and their networks, enabling
41
functional recovery after perturbation. Furthermore, the directed nature of this plasticity
42
may hold a key to the restoration of locomotion after spinal cord injury. Here, we
43
studied the recovery of crawling in the leech, Hirudo verbana, after descending cephalic
44
fibers were surgically separated from crawl central pattern generators shown previously
45
to be regulated by dopamine. We observed that immediately following nerve cord
46
transection leeches were unable to crawl, but remarkably, after a day to weeks, animals
47
began to show elements of crawling and intersegmental coordination. Over a similar
48
time course, excessive swimming due to the loss of descending inhibition returned to
49
control levels. Additionally, removal of the brain did not prevent crawl recovery,
50
indicating that connectivity of severed descending neurons was not essential. Following
51
crawl recovery, a subset of animals received a second transection immediately below
52
the anterior-most ganglion remaining. Similar to their initial transection, a loss of
53
crawling with subsequent recovery was observed. These data, in recovered individuals,
54
support the idea that compensatory plasticity directly below the site of injury is essential
55
for the initiation and coordination of crawling. We maintain that the leech provides a
56
valuable model to understand the neural mechanisms underlying locomotor recovery
57
after injury because of its experimental accessibility, segmental organization and
58
dependence on higher-order control involved in the initiation, modulation and
59
coordination of locomotor behavior.
60
2
61
Introduction
62
Neural circuits are remarkably resilient. Over a permissible range of perturbations, they
63
can become reconfigured to maintain seemingly normal activity (Marder 2011;
64
Turrigiano 2012; 2011). This ability is largely attributed to cellular- and network-level
65
homeostatic processes that can retune a perturbed system and restore its functional
66
state (Marder and Goaillard 2006; Pozo and Goda 2010; Turrigiano 2012). The idea of
67
manipulating state-preserving homeostatic plasticity for restoring locomotor function
68
after spinal cord injury appears promising, especially when repair or regrowth of
69
neuronal connections across sites of injury is not possible (Edgerton et al. 2004;
70
Rossignol and Frigon 2011). While such strategies may hold keys to significant
71
biomedical advances for locomotor recovery, the cellular mechanisms underlying these
72
and most other homeostatic or compensatory processes are just beginning to be
73
understood (Husch et al. 2012; Pozo and Goda 2010; Sakurai and Katz 2009).
74
We propose that the leech serves as a valuable and highly tractable system in
75
which to investigate homeostatic mechanisms contributing to functional locomotor
76
recovery. Invertebrate preparations have long been pivotal in illuminating the cellular
77
bases of rhythmic-pattern generation, its modulation, and stability (Marder et al. 2005;
78
Marder and Calabrese 1996; Selverston 2010). For almost half a century, the medicinal
79
leech, in particular, has served as a model organism to illustrate how neural circuits are
80
organized and modulated to produce locomotor behaviors (Briggman and Kristan 2008;
81
Brodfuehrer et al. 1995; Kristan et al. 2005; Mullins et al. 2011; Puhl and Mesce 2008;
82
2010). Similar to the functions of vertebrate spinal projections (Grillner 2006a; b;
83
Grillner and Wallen 2002; Kiehn 2011), higher-order fibers descending from the leech’s
2
84
cephalic ganglion (i.e., brain) have been known to be vital for the initiation, modulation
85
and coordination of crawling across iterated locomotor pattern generators (Puhl et al.
86
2012). Thus, in this report, we examined whether medicinal leeches (Hirudo verbana)
87
could recover their ability to crawl after their nerve cords were fully transected, while
88
noting whether or not regrowth of descending axons may have contributed to locomotor
89
recovery. By observing leeches for days to months following such transections, we
90
determined whether they could recover their ability to crawl in a coordinated manner,
91
despite the loss of previously essential control elements for that coordination.
92
In mammals, compensatory plasticity is not always beneficial if it becomes
93
undirected during severe supra-spinal injury (Beauparlant et al. 2013); thus discovering
94
therapeutic strategies and how they work to steer beneficial features of plasticity hold
95
promise for promoting adaptive locomotor recovery, especially those related to
96
neuromodulatory intervention (Guertin 2014; Musienko et al. 2011; Sharples et al. 2014).
97
In the leech, we found that the crawl pattern-generating networks distal to the site of
98
injury were able to achieve adaptive solutions for their reconfiguration and
99
intersegmental coordination, independent of descending fibers from the brain becoming
100
reconnected. Understanding how the leech nervous system correctly reconfigures itself
101
post injury, while making occasional misdirected mistakes, will likely provide valuable
102
insights about the cellular substrates of locomotor plasticity across animal species,
103
including humans.
3
104
Materials and Methods
105
Animals and Surgical Procedures
106
Adult medicinal leeches (hermaphrodites), Hirudo verbana (Carena 1820; Siddall et al.
107
2007) were obtained from Niagara Medical Leeches (Westbury NY, USA) and stored at
108
room temperature in a glass terrarium filled with water from a natural well at the
109
University of Minnesota. After surgery, leeches were housed in individual containers (17
110
cm wide x 8 cm tall) with a thin layer of aquarium gravel covering the bottom and well
111
water at 4-5 cm in depth.
112
Leeches chosen for behavioral assays weighed 2.45 +/- 0.48 g. Briefly, the leech
113
CNS consists of: 1) a compound cephalic ganglion comprising the supra-and sub-
114
esophageal (SEG) ganglion (i.e., the brain); 2) the 21 segmentally distributed ganglia
115
(M1-M21) and the connectives that link them together (i.e., the ventral nerve cord); 3)
116
the compound terminal ganglion (‘tail brain’). Prior to surgery, leeches were
117
anesthetized on ice until they were no longer responsive to tactile stimuli. Once
118
anesthetized, they were pinned onto a dissecting tray lined with bees’ wax ventral side
119
up. One thin pin was placed through the leech’s mouth, avoiding damage to the brain,
120
and the other one placed through the caudal sucker. A small vertical incision spanning
121
2-3 annuli was made to access the nerve cord for complete transection. The incision
122
was closed using one or two stitches made with 7-0 or smaller suture thread. A post-
123
mortem examination was conducted in all animals to confirm that the nerve cord
124
(connectives) had remained fully transected (Fig. 1A far right). Herein, we present data
125
from individuals with several different types of manipulations, defined as follows: 1)
126
Transections- A small incision was made 5 annuli below the mouth, which permitted a 4
127
complete transection of the nerve cord between the brain and first ganglion (M1) (see
128
Fig. 1A) or between M1 and the second ganglion (M2). No differences in behavior were
129
observed between these individuals. Three individuals were tested at 40 days and
130
another four tested at 48-50 days following transection; these individuals were
131
combined into a single grouping in the figures labeled as being tested at 40-50 days
132
following transection; 2) M1 removal- M1 was surgically extirpated from the leech (see
133
Fig. 6A); 3) SEG removal- The subesophageal ganglion was removed by surgically
134
detaching it from the supraesophageal ganglion and M1 (see Fig. 7A); 4) Second
135
Transection- Leeches that showed a complete recovery of crawling behavior were
136
anesthetized and transected again at 40-50 days following the first transection (between
137
the brain and M1). The second transection was made between M1 and M2 (see Fig.
138
8A); 5) Midbody- This type of transection was made between M9 and M10 or M10 and
139
M11, resulting in the removal of all descending inputs to the posterior half of the leech
140
while preserving those to the anterior half of the animal (see Fig. 9A inset); 6) Sham-
141
Operated Controls- These surgeries were performed as a control for all of the above
142
conditions, whereby the animal received identical surgical manipulations (e.g., skin and
143
muscles cut and nerve cord tugged), except the nerve cord was not severed.
144 145
Behavioral Arena and Video Acquisition
146
Individuals were tested before and at multiple points following transection. The order in
147
which the tests were presented was randomly chosen. The behavioral arena was a
148
cylindrical acrylic dish, 20.3 cm in diameter by 8 cm in height. It was illuminated with
149
LED lighting to approximately 750 lux. Water within the arena ranged from 19-21˚C. 5
150
Videos were filmed at 38 fps using a DCC1455M high resolution CMOS camera
151
(Thorlabs, Newton NJ, US).
152
Behavior under the following conditions was examined: Deeper water- the arena
153
was filled with well water to a depth of 4 cm. Under this condition a leech was able to
154
swim or crawl. Shallow water- the arena was filled with 15 ml of well water, which was
155
just enough to coat the bottom of the arena with a thin layer of water 1-2 mm high.
156
Under this condition the leech was only able to crawl. Under either condition, the
157
leech’s behavior in the arena was recorded for the 6 minutes immediately following
158
placement of the animal in the arena. Evoked crawling- To initiate crawling, a round no.
159
4 artist’s paintbrush was used for two minutes. The surface of the leech was touched
160
with the brush in an anterior-to-posterior direction to stimulate crawling behavior. In
161
response to this stimulus, a crawl or crawl-like behavior was counted if the leech
162
performed one or more crawl-like cycles (note: an elongation and contraction phase
163
equals one cycle) (Fig. 1D).
164
Behavioral Descriptions and Analysis:
165
Swim and crawl locomotor states were easily separable by visual inspection, defined as
166
follows: Swim- the whole body is flattened and moves with a series of quasi-sinusoidal
167
dorso-ventral movements progressing caudally (Brodfuehrer et al. 1995; Stent et al.
168
1978). During swimming, unlike crawling, neither of the leech’s suckers attaches to the
169
substrate. Crawling and Crawl-like behavior- A defining feature of normal crawling is
170
that during each of its phases—elongation and contraction— the individual body
171
segments are coordinated (Puhl and Mesce 2010). During elongation, each segment
172
sequentially elongates (lengthens) in a stereotypic anterior-to-posterior directed wave. 6
173
During contraction, individual segments contract (shorten) in the same posteriorly-
174
directed manner. Often, there is a delay of a few seconds before the next cycle is
175
initiated. Here, we use the term ‘crawl-like’ to expand the traditional definition of crawling
176
to include some of the atypical locomotor activity we observed during the recovery
177
process. To be counted as crawl-like behavior, animals had to exhibit a body elongation
178
phase followed by a body contraction phase with no body flattening typically associated
179
with swimming. However, the crawl-specific coordination during each elongation or
180
contraction phase (i.e., the caudally-directed metachronal wave) was not a requirement.
181
For example, a reversal in wave direction, a partial wave or an uncoordinated wave
182
would not limit use of the ‘crawl-like’ designator. Using recorded videos, swim and crawl
183
behaviors were quantified by measuring the duration of bouts of swimming or crawling
184
(in deep water) or by counting the number of crawl cycles (in shallow water) within a 6
185
minute (min) test period.
186
Crawl movement analysis: To understand the recovery process better, videos of
187
representative crawl cycles were used to generate a series of frames depicting the
188
kinematics of the elongation and contraction phases. These analyses were based on
189
the fact that, during the elongation and contraction phases, the body narrows and
190
widens respectively—a consequence of hydroskeletal dynamics. Representative crawl
191
phases were divided into 8 images (Fig. 4). The first and last silhouettes indicate the
192
leech’s position at the beginning and end of a crawl cycle. The intervening images were
193
separated by intervals of 15% of the total cycle duration (e.g., about 0.35 s for the pre-
194
lesion series). These images (for example, Fig. 4A i) were then analyzed in ImageJ by
195
applying a threshold filter, which outlined the leech in black against the white
7
196
background (Fig. 4A ii). In those videos with some glare, the silhouettes contained a
197
peppering of open spaces after thresholding (Fig. 4A ii). Such areas were subsequently
198
filled in by using the line-and-fill tools in ImageJ so as not to cause errors during
199
automated thickness (width) colorization. This process allowed for the local thickness
200
plugin to be run. A reference of a 50 pixel-wide line was placed into the image such that
201
all images could be calibrated to the same scale for coloration (Fig. 4A iii). The local
202
thickness algorithm colorized images according to the largest circle that fit into a
203
particular area, i.e., the leech’s silhouette. The local thickness algorithm was run and the
204
image colorized for individual images such that blue-purplish colors represented less
205
wide (narrower) areas of the leech and orange-yellow-white areas represented wider
206
regions of the leech’s body-- again, elements that correspond to the elongation and
207
contraction phases of crawling (Fig. 4B).
208
Coordination analysis: To assess acquisition of the posteriorly-directed
209
coordination of the crawl elongation phase, a given leech was visually divided into four
210
approximately equal sections. When a body quarter was active during elongation, it
211
would appear thinner due to the consequence of circular muscle contractions. For
212
these analyses, up to 25 crawl elongations were measured for each individual in a 6 min
213
window in shallow water, with the activity and order of body quarters in close succession
214
counted. Only the elongation phases were used for analysis because they typically
215
occupied a greater proportion of a given crawl cycle and active body movements were
216
readily observed, assuring a more accurate measure of active body quarters and their
217
coordination. For such analyses we used individuals that were available for all trials (n
218
= 8 for transections; n = 4 for sham-operated controls). 8
219
Statistics and graphing methods for behavioral analyses
220
Unless otherwise noted, the statistical test used was the Skillings-Mack test (SM) that,
221
when significant, was followed by a post-hoc Dunn’s comparison; results are reported
222
from this Dunn’s comparison where applicable (Skillings and Mack 1981). For swim bout
223
durations, this test required that we take the mean of each individual’s swim bout
224
duration and look at its change over time. The statistical test used for data presented in
225
Fig. 1 was the Kolmogorov-Smirnov (K-S) test (Justel et al., 1997), a two sample
226
analysis to compare non-parametric means with small sample sizes. Box Plots—
227
Because many of the data sets included non-normal data and relatively small sample
228
sizes, we used the Tukey box and whisker method of plotting data. In this method, the
229
box surrounds the 25-75 percentiles. A line is present at 50% (the median). The
230
whiskers extend to 1.5 times the interquartile ratio (Tukey 1977). For illustration
231
purposes only, within these plots, the arithmetic mean is marked with a + sign. Outliers
232
are marked by diamonds. Unless otherwise noted, all figures with measures of crawl or
233
swim cycle number, cycle duration, or duration of uninterrupted locomotor cycles (bouts)
234
incorporated the above mentioned box plots.
235
Results
236
The time course and process of crawl recovery after injury
237
The brain (cephalic ganglion) exerts a hierarchical control over behavior in the form of
238
descending axons whose firing rate biases the animal toward (or against) the
239
expression of swimming or crawling (Brodfuehrer and Friesen 1986; Esch et al. 2002;
240
Mesce et al. 2008; Mullins et al. 2012; Puhl et al. 2012). While disconnection of the
9
241
brain has been shown to promote swimming and result in a loss of crawl coordination
242
(Puhl et al. 2012; Puhl and Mesce 2010), these studies have only examined leeches
243
with acute lesions (i.e., within hours of surgery). Here, we examined the recovery of
244
normal function for weeks to months following transection of the nerve cord above M1
245
or M2 (n = 14), and in sham controls where the nerve cord was not severed (n = 7).
246
Consistent with prior studies, transection of the leech nerve cord between the
247
brain and M1 (Fig. 1A) caused a 10 fold increase in the bout duration of swimming (Fig.
248
1B) (n = 14, p0.9, KS test). In contrast, one day
254
after surgery, transected animals showed a significant decrease in the number of crawl-
255
like cycles, with only one transected individual of 14 exhibiting a single spontaneous
256
crawl cycle (Fig. 1C, p0.08, KS test). Sham-operated
10
263
individuals never showed a significant change in crawling (p>0.5, KS test) or swimming
264
behavior (p>0.9, KS test).
265
Having determined that leeches show an initial deficit in crawling after transection,
266
but exhibit the behavior readily at a later date, we examined the time course of recovery.
267
First we wanted to examine when leeches begin to exhibit crawl cycles. These data are
268
indicated in Figure 2A, where each leech is represented by a single row and each day
269
following surgery is represented by a column. Prior to transection all leeches (n= 14)
270
exhibited crawl behavior; however, following transection only a single individual
271
exhibited one crawl cycle before Day 4. In contrast, all sham-operated individuals (n = 7)
272
exhibited crawling the day following their surgery. In transected animals, by 40-50 days
273
following surgery, all 8 leeches tested were able to exhibit multiple crawl cycles (Fig. 2A).
274
To understand better the time course of crawl recovery, the specific number of crawl
275
cycles exhibited was quantified when leeches were placed in either shallow water (Fig.
276
2B) or deeper water conditions (Fig. 3B). In shallow water, transected leeches exhibited
277
a significant decrease in the number of crawl cycles performed following surgery (Fig.
278
2B, p0.95, SM test).
300
While leeches regained an ability to generate crawl-like cycles with two phases
301
(i.e., elongation-contraction) after transection, we wanted to determine if within each
302
phase the segments were able to regain their caudally-directed metachronal
303
coordination. Thus, we analyzed movements of the leech by characterizing its body
304
form during these two phases (Fig. 4). By colorizing videos of leeches crawling we were
305
able to visualize the progression of the anterior-to-posterior wave of elongation (blue-
306
red = narrower body) and contraction (white-orange = wider body) and thus examine the
307
form of crawling during its recovery (see Methods section for details, Fig. 4A). In sham-
308
operated individuals and pre-transection trials, there was a fluid anterior-to-posterior 12
309
progression of coordinated segmental activity during both phases that propelled the
310
leech forward a considerable distance (Fig. 4B). Here, the notable curvature in the
311
silhouettes was due to the tendency of leeches to crawl thigmotactically around the
312
inside edge of the dish. In sham-operated individuals, just one day following surgery,
313
normal crawling was apparent (Fig. 4B). Figures 4C and 4D show two different
314
transected leeches as crawling was reacquired; note that metachronal body activity was
315
absent or weak at best, as depicted by a lack of blue-red color and its caudally-directed
316
invasion into the body (see Fig. 4C,D, Day 3 and 6 respectively). In fact, only a portion
317
of the leech’s body exhibited a crawl cycle (Fig. 4D, Day 6). Over time, however,
318
crawling did begin to regain its posterior-directed coordinated fluidity, and crawling
319
began to appear more normal. For example, on Day 12 (Fig. 4 C, 3rd-5th silhouette, left
320
to right) the purple-red color can be seen invading the leech in an anteriorly-directed
321
manner, reflecting the narrowing of the body segments as the circular muscles
322
contracted during the crawl elongation phase. In turn, the crawl contraction phase can
323
be appreciated (Fig. 4 C, Day 12, 6th-8th silhouette) by the caudally-directed wave of
324
orange-to-white color that progressively invades the leech body, reflecting the widening
325
(fattening) of each body segment as the longitudinal muscles contracted. By Day 40,
326
both leeches were showing crawl cycles that were similar in form and duration to pre-
327
lesion crawl cycles (Fig. 4C,D). It is noteworthy that individuals showed variability in
328
their recovery (for example, compare Figs. 4C and D on Days 10 and 9), which might
329
facilitate the future detection of factors that promote recovery across individuals. Lastly,
330
transected individuals initially exhibited long duration crawl cycles. For example, on the
331
first day that crawling behavior was expressed we saw an increase in crawl cycle
13
332
duration from the mean pretest value of 7+/-2.43 sec to 21.29+/- 13.16 sec/cycle (n = 9,
333
p0.12).
30
721
Figure 7. Changes in crawling and swimming following complete removal of the
722
subesophageal ganglion (SEG). To rule out axonal reconnection between the
723
proximal and distal severed regions of all cephalic descending axons, we removed the
724
entire SEG in 6 individuals and determined whether crawling could be re-established. A,
725
Diagram depicting SEG removal. B, Representation of the course of recovery for
726
individual animals as described in Fig. 6B. C, Number of crawl or crawl-like cycles
727
occurring within a 6 min trial. C. After Day 10, the decrease in number of crawl cycles
728
was no longer significant (ns, p>0.18, Dunn’s test). D, After Day 30, duration of swim
729
bouts was no longer significantly longer compared to pre-test durations (ns, p>0.09).
730 731
Figure 8. Leeches repeat the recovery process following removal of the ‘lead’
732
ganglion. To ascertain whether or not the ganglion closest to the transection site had
733
become vital for crawl recovery, we tested whether its removal would result in a second
734
loss of crawling behavior with a second round of recovery. A, Diagram depicting the
735
sites of the first and subsequent transections. B, Representation of the course of
736
recovery for each animal as previously described in Fig. 6B. C, Number of crawl or
737
crawl-like cycles occurring within a 6 min trial. At 72 days following the second
738
transection, the number of crawl cycles wais no longer significantly different than pre-
739
test values (ns, p>0.32, SM test). D, Bouts of swimming measured over the course of a
740
single trial for the same 6 individuals tested for crawling. At 8 and 72 days following a
741
second transection, swim bout durations were no longer elevated compared to pre-test
742
values (ns, p>0.08 and p>0.37 respectively, SM test).
743
31
744
Figure 9. Midbody nerve cord transections test possible anatomical constraints
745
on the lead ganglion hypothesis and establish if across transection oscillator
746
coupling is possible. To test whether posteriorly located ganglia were capable of
747
setting up their own crawl wave along the body, nerve cords were transected below M9-
748
10. A, Diagram depicting the midbody transection (left) and representation of the course
749
of recovery for all 6 individuals measured. The blue and pink shadings over the
750
diagram highlight behavioral activity controlled by a partial nerve cord attached to the
751
brain and one that is not, respectively. B, Video image prepared, as in Fig. 4, of a leech
752
crawling at 115 days after transection below M9-10. Note that in the 5th through 7th
753
frames (silhouettes) the anterior half of the leech is entering its crawl elongation phase,
754
while the posterior body half is in a crawl-contraction phase; indicative of 2 independent
755
crawl cycles. C, Number of crawl or crawl-like cycles occurring within a 6 min trial. The
756
blue and pink shading highlights the anterior and posterior body regions; purple denotes
757
the potential for cross-transection activity. Note that no cross-transection body
758
coordination ever returns for crawling (bottom panel). D, Video images of coordinated
759
(sinusoidal) swimming (top clip), and cross-transection uncoordinated swimming
760
(bottom clip). For uncoordinated swimming, the sinusoidal swim wave was not
761
distributed along the entire body; often with the anterior body in a contracted state. E,
762
Bouts of swimming measured over the course of a single 6 min trial for all 6 individuals
763
observed. Swim suppression of the posterior body returned to control levels by Day 10.
764
Cross-transection swim coordination, measured as a whole-body sinusoidal waveform,
765
was not prevented. Note that the time scales on the y axes have been kept the same for
766
purposes of comparison, thus insets (swim, top and bottom) are provided to showcase
32
767
smaller values.
768 769
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966
5
C Transection Brain
No lesion
SEG M1
Ganglia
SEG
M1
98 days following transection
M2 M3
B
Unevoked crawl behavior Shams Transections 60
20 0
*
360
120 0 1 day after 40-50 days after Before transection transection transection
Before 1 day after 40-50 days after transection transection transection
Evoked crawl-like behavior
* 240
*
40
D Probability of induced crawl-like activity
Average swim bout duration (s) per 6 minute trial
Swim behavior
Average number of crawl-like cycles per 6 minute trial
A
1.0 0.8 0.6 0.4 0.2 0.0 Before 1 day after 40-50 days after transection transection transection
Figure 1
A Days after transection
No crawl Crawl Only 1 cycle No data
Shams
Transections
Pre 1 2 3 4 5 6 7 8 9 10 13 40-50
Number of crawl or crawl-like cycles in 6 minutes
B
ns
Transections Shams
60
40
+
+
+
ns
+
20
+
+
+
+
+
+
+ +
0
+
Pre 1 Test
+
+ +
+
2
+
3
+
+
4
5
+
6
+
7
+
+
+
8
9
10
13 40-50
Days after transection
Figure 2
A
Deeper water swim behavior
Transections Shams
Swim bout duration (s)
360 300 +
240 +
+
+
+
+ +
180
+
120
+
+
ns
60 0
+
+
+
+
Pre Test
1
2
3
4
+
+
+
+
+
5
+
6
8
7
+
+
9
+ +
10
40-50
Days after transection
B Crawl bout duration (s)
150
Deeper water crawl behavior
120
90 ns
60
30
+
+ +
+
+ +
0 Pre Test
1
+
2
+
+ +
3
+
4
+
+
+
+
5
+
6
+
+
+
7
8
9
+ + +
+
+
10
40-50
Days after transection
Figure 3
Width
Figure 4
Percent of elongations
B
Number of active quarters 100
Percent of elongations
A
100
# active quarters 1 2 3 4
80 60 40 20 0 Pre 1 2 3 4 5 6 7 8 9 10 40+ Sham day 1 Test Days after transection
First quarter to elongate A
80
B
60
C
40
D
20 0 Pre 1 2 3 4 5 6 7 8 9 10 Test
Days after transection
C
40+ Sham day 1
Number of sequentially activated quarters Anterior to posterior 2 3 4
Percent of elongations
100 80
Posterior to anterior 2 3
60 40
None
20 0 Pre 1 2 3 4 Test
5 6 7 8 9 10
Days after transection
40+ Sham day 1
Figure 5
B
A
M1 removal
Days after transection pre 1 4 8 10 30 115
D
Crawl behavior ns
40
ns
ns
30 ns
20 10 0
+
+ + +
+
+ +
Pre 1 6 8 10 46 115 Test Days after transection
Swim bout duration (s)
Number of crawl-like cycles in 6 minutes
C
No crawl Crawl Only 1 cycle
Swim behavior 360 300 240 ns
180 120 60 0
+
ns
+ +
ns
+
+
Pre 1 6 8 10 46 115 Test Days after transection
Figure 6
B
A
SEG removal Days after transection
pre 1
D
Crawl behavior 30
ns ns
20
10
ns
+
0
Pre Test
+ 1
+
+
10
30
+
+ 6
Days after transection
115
Swim bout duration (s)
Number of crawl-like cycles in 6 minutes
C
6
10
No crawl Crawl Only 1 cycle
30 115
Swim behavior 360 300 240 180
+
120
+
ns ns
60 0
+
+
+
+
Pre 1 6 10 30 115 Test Days after transection
Figure 7
A
B First
No crawl Crawl Only 1 cycle
Second
Days after transection pre
2 4 6 8 10 12 14 76
Time
D
Number of crawl-like cycles in 6 minutes
Crawl Behavior ns
40
+ +
20
0 Pre Test
+ + + + + + + 2
4
6
8
10
12
14
72
Days after second transection
Swim bout duration (s)
C
Swim Behavior 360 300
+
+
240
+
180
+
120
+
+
60 0
ns
+ ns
+ Pre Test
2
4
6
8
10
12
14
+
72
Days after second transection
Figure 8
A
Days after transection pre
1
3
4
5
6
No crawl Crawl Only 1 cycle
7 10 31 115
Anterior
Posterior
B
1.4 cm
D
Coordinated swim
1.1 cm 0.7 cm
Uncoordinated swim
0.4 cm 0.0 cm
3.0 sec
C
E
Crawl Anterior to transection
Swim 30
180
Anterior to transection
50 20
120
30
10
+
10 0
60
+
+ +
+
Pre 1 6 10 30 Test Days after transection
115
Posterior to transection 30 20 10
+ *
0
Pre Test
+ 1
*
+ 6
*
*
*
+
+
+
10
30
0
+
+
115
Days after transection
Cross transection coordination 30
Swim bout duration per 6 minute trial
Number of crawl-like cycles in 6 minutes
20
0
+
+
Pre Test
1
10
+
+
0
*
+
60
+
Pre Test
+
0 Pre Test
+ 1
+
6
*
+
10
*
+
30
+
+
+
+
6
10
30
115
Days after transection
180
Cross transection coordination ns ns
Days after transection
115
0
0 Pre Test
+ Pre Test
+ 1
+
*
10
*
+
+
115
ns
ns
1 30
60
*
115
120
+
*
+
30
180
120
+
+
6 10 30 Days after transection
20
20 10
+6
Posterior to transection
*
360
+ +1
Pre Test
*
+
+
1
6
10
+
+
6
+
10
+ 30
+ 30
115
+
115
Days after transection
Figure 9
