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Key words: cat, involuntary movement, transition, interneurons, central pattern generator.
INTRODUCTION
An open question concerning both the locomotion central
pattern generator (CPG) and the scratching CPG in the
cat is whether or not they are separate spinal cord
*Corresponding author. Address: Instituto de Fisiolog a, Benemerita
Universidad Autonoma de Puebla, 14 sur 6301, Col. San Manuel,
A.P. 406, Puebla, Puebla CP 72570, Mexico. Tel: +52-22-22-295500x7326; fax: +52-22-22-33-4511.
E-mail address: eliasmanjarrez@gmail.com (E. Manjarrez).
Abbreviations:
CPG,
central
pattern
generator;
ENG,
electroneurograms; LGS, bilateral gastrocnemius plus soleus; MG,
medial gastrocnemius; PF, pattern formation; RG, rhythm generator;
sCDP, sinusoidal cord dorsum potentials; TA, tibialis anterior.
http://dx.doi.org/10.1016/j.neuroscience.2014.12.038
0306-4522/ 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
156
glycinergic inhibitory interneurons are active during swimming, escape, and struggling (Liao and Fetcho, 2008)
Likewise, by using calcium imaging, it was found that
two dierent sets of interneurons drive swimming and
escape tasks (Ritter et al., 2001).
We found that after the application of curare in the
precollicularpremammillary
decerebrate
cat,
the
spontaneous locomotion is abolished and the scratching
episode was followed by a locomotor-like motor pattern.
In this context, the aim of the present study is to
introduce
the
phenomenon
of
post-scratching
locomotion in the cat and show how it can be employed
as a tool to study neuronal mechanisms underlying the
organization of spinal CPGs. This has allowed us to
provide experimental evidence to support previous
studies showing that both scratching and locomotor
CPGs share common neurons.
EXPERIMENTAL PROCEDURES
Summary
We performed experiments in 16 precollicular
premammillary decerebrate cats. The scratching
episodes were elicited by stimulation of the ipsilateral
pinna. Electrical potentials of exor and extensor nerves
of the hindlimbs as well as from the spinal cord were
recorded during these episodes.
Preparation
We performed experiments in cats (weight range 24 kg),
in accordance with the European Communities Council
Directive of 24 November 1986 (86/609/EEC), the
guidelines contained in the National Institutes of Health
Guide for the Care and Use of Laboratory Animals (8523, revised in 1985) and the Norma Ocial Mexicana
NOM-062-ZOO-1999. Moreover, the Institute of
Physiology from the Benemerita Universidad Autonoma
de Puebla, Mexico, approved the present study. First,
the animals were gently handled and introduced in a
comfortable anesthesia-induction-box at a temperature
of 2229 C and 4070% relative humidity. The
anesthesia was induced with a mix of isourane (34%)
delivered in a mixture of 30% oxygen and 70% nitrous
oxide. The animals did not exhibit discomfort. Second,
when the animals were completely anesthetized we
employed an anesthesia face mask with the same
mixture of isourane (12%), 30% oxygen and 70%
nitrous oxide. Radiant heating and a heating pad were
employed to maintain the temperature of the animals at
about 37 C. Third, the administration of the anesthetic
mixture was maintained by a tracheostomy tube
inserted into the trachea. Fourth, blood pressure was
monitored from the right carotid artery using a
transducer. The radial vein was cannulated to
administer dextran and saline solution as necessary to
maintain blood pressure between 80 and 120 mmHg.
We administered atropine (0.05 mg/kg subcutaneous)
and dexamethasone (2 mg/kg intravenous). A buer
solution of glucose (5%) and bicarbonate (100 mM) was
continuously infused for blood pH maintenance. The
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RESULTS
In 14 of 16 cats, we recorded the electrical activity of the cat
spinal cord from L4 to S1 spinal segments during two motor
events in the same experiment: ctive scratching and postscratching ctive locomotion. Fig. 1 shows a typical
recording from our experiments. The scratching episode
was elicited by stimulation of the ipsilateral pinna. We
observed that after the end of the scratching episode, a
locomotion
episode
starts
(postscratch
ctive
locomotion). Fig. 1 illustrates sinusoidal cord dorsum
potentials (sCDP) during both motor tasks as well as the
patterns of exor and extensor electroneurographic activity
during scratching and locomotion. The lasting ring activity
in the exor (TA) trace during the post-scratching delay in
Fig. 1 was rare to observe among all recordings.
We observed that after the application of curare in
precollicularpremammillary decerebrate cats, the
incidence of spontaneous locomotion (those episodes
which we could not associate with scratching) was
about 2% of all locomotion episodes and was found in 4
of the cats: Experiment A (1/18) (number of nonassociated locomotion episodes/number of postscratching locomotion episodes); Experiment B (3/40);
Experiment C (3/40); Experiment D (1/10).
We also observed spontaneous scratching episodes
without locomotion (about 26.2% of all scratchings),
which mostly occurred at the beginning of the curare
application. After a period of about two or three minutes
we observed a high incidence of post-scratching
locomotion episodes.
In about 73.8% of the trials obtained from 14 cats, the
scratching episode was followed by a locomotor-like
motor pattern. Furthermore, the mean duration of the
scratching and post-scratching locomotion episodes was
8.6 2 s and 18.5 7 s, respectively. The latency from
the end of each scratching episode (magenta arrow in
Fig. 1. Scratching episode followed by a post-scratch locomotion episode. Top trace, sinusoidal cord dorsum potentials (sCDP) recorded from the
L6 spinal cord during both tasks. Middle trace, simultaneous recording of an extensor (Ext) nerve, MG. Bottom trace, simultaneous recording of a
exor nerve (Flex), TA. The rst arrow (green) indicates the beginning of the scratching episode, the second arrow (magenta) indicates its end, and
the third arrow (yellow) indicates the beginning of the post-scratch locomotion episode. Periods used to calculate power spectra are illustrated with
horizontal brackets. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)
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160
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Fig. 4. Lumbo-sacral amplitude distribution of sinusoidal cord dorsum potentials (sCDP) occurring during scratching and post-scratching
locomotion. (A) Diagram of the experimental arrangement, a 32-channel multielectrode was positioned on the dorsal surface of the L4S1 spinal
cord. (B) sCDP amplitude versus recording electrodes 18 and (C) sCDP amplitude versus recording electrodes 2532, positioned on the left and
right sides of the spinal cord, respectively. There is a statistically signicant dierence between the sCDPs amplitudes recorded during scratching
(black triangles) and post-scratching locomotion (white triangles) in electrodes 8 and 32 (Students t-test p < 0.05).
3 for intermediate interneurons. This suggests that activities of the three groups of recorded interneurons are not
proportionally scaled up. Moreover, we computed the ring frequencies of these three groups of neurons during
scratching and post-scratching locomotion; these values
are consistent with the number of spikes per cycle previously analyzed for these neurons. These results suggest
that the change of the motor pattern from scratching to
post-scratching locomotion could involve a circuit reconguration as well; however, this possibility must be carefully taken into account (see Discussion).
DISCUSSION
Interneurons associated with scratching and postscratching locomotion
In the present study we show for the rst time the
occurrence of a phenomenon we call post-scratching
162
Fig. 5. Interneurons ring during scratching and post-scratching locomotion. (A) Simultaneous recordings of the sinusoidal cord dorsum potential
(sCDP), a single exor interneuron (INT) ring during scratching and post-scratching ctive locomotion, and the corresponding MG and TA
electroneurograms. (B) Pooled raster graphs for the rhythmical cycle of 10 single interneurons recorded with the glass micropipette during
scratching and the subsequent post-scratching ctive locomotion. All responses were aligned relative to the end of the scratching episode. Triangles
indicate the beginning of the post-scratching locomotion. The right vertical boxes indicate the phase in which every neuron red: blue for exor, red
for extensor and magenta for Interm (intermediate). All neurons maintain the same phase during both rhythmical activities. (C) Histological location
of interneurons ring rhythmically during both motor tasks. The symbols represent the location of the recorded interneurons illustrated in B. (D) The
same as B, but for 14 other interneurons recorded with the mini matrix Thomas Recording system during scratching and the subsequent postscratching ctive locomotion. All neurons also maintain the same phase during both rhythmical activities. (For interpretation of the references to
color in this gure legend, the reader is referred to the web version of this article.)
163
Fig. 6. Polar plots of the analyzed interneurons. Vectors show the preferred phase of discharge (p < 0.05). The 0 represents the onset of the exor
activity. Data are shown counterclockwise. All neurons red in the same phase during scratching (left panel) and post-scratching locomotion (right
panel). (A) 5/13 interneurons red in the exor phase (indicated by the blue traces). (B) 4/13 interneurons were rhythmically active during the
extensor phase (red trace). (C) 4/13 interneurons were classied as intermediate during both rhythmic tasks (magenta traces indicate the
intermediate). (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)
164
dominant frequency in the power spectrum of the electrical sinusoidal waves recorded during scratching episodes
did not overlap with the dominant frequencies of the spinal
waves recorded during post-scratching locomotion. Moreover, because the frequency of ctive spontaneous locomotion (Trejo et al., 2009) occurs in the same range as
the post-scratching locomotion we suggest that both
motor behaviors and spinal waves could share similar
neuronal circuits.
Speculations in terms of the two-layer CPG
architecture
Regarding the architecture of the CPG, McCrea and
Rybak (2008) proposed a two-level CPG architecture containing a half-center rhythm generator (RG) and a pattern
formation (PF) network. The latter is controlled by the RG
and projects to exor and extensor motoneuron populations. We could suggest that the scratching CPG and the
post-scratching CPG could share the PF circuits but not
the rhythmicity-generating systems in McCrea and
Rybaks hierarchy. This possibility is supported by our
nding that the scratching and the post-scratching CPG
always occurs at dierent frequencies, but the same exor
and extensor nerves (and possibly the same motoneuron
pools) can exhibit either scratching or post-scratching
locomotion. Future experiments conrming that the same
motoneurons can exhibit either scratching or post-scratching locomotion will be necessary to support the hypothesis
that both CPGs could share the same PF circuits.
Hart and Giszter (2010) (see also Hart and Giszter,
2004; Giszter and Hart, 2013) found that some sets of
interneurons in the intermediate nuclei of the spinal cord
may organize individual spinal primitives (i.e., pulsed premotor drives of xed duration that recruit a covarying set
of muscles, yielding particular force patterns). These
authors showed that these interneurons and their primitives are shared across motor behaviors. Our nding
about post-scratching locomotion is consistent with the
Hart and Giszters study. It is tempting to speculate that
both motor behaviors scratching and post-scratching
locomotion may be generated by the same set of interneurons and their primitives. Moreover, in the context of
McCrea and Rybaks CPG architecture, we could suggest
that Hart and Giszters neuronal circuits conform modular drives in the pattern-shaping systems, thus allowing
the transition from scratching to post-scratching
locomotion.
Descending pathways producing scratching and
post-scratching locomotion
We suggest that both rhythms, scratching and postscratching
locomotion,
originate
from
dierent
supraspinal structures. There are several observations
that support this idea. The stimulation of descending
pathways eliciting locomotion (e.g., mesencephalic
locomotor region, MLR) evokes a bilateral coordinated
rhythm in the four limbs; whereas, stimulation of
descending pathways for scratching can evoke
unilateral rhythm in only one hindlimb. It is plausible that
the same supraspinal structures that evoke ctive
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Catedra Marcos Moshinsky (EM), Mexico. AT and JAT acknowledge PhD fellowship support from CONACYT Mexico. BDTV
acknowledge postdoctoral fellowship support from CONACYT
Mexico. AF and EM acknowledge support from Cuerpos
Academicos and PROMEP-PIFI-VIEP-BUAP Mexico. We thank
Pablo Linares for technical assistance. We thank John Reid for
proof reading the English manuscript.
REFERENCES
Berkinblit MB, Deliagina TG, Feldman AG, Gelfan IM, Orlovsky GN
(1978) Generation of scratching. I. Activity of spinal interneurones
during scratching. J Neurophysiol 41:10401057.
Berkowitz A (2001a) Broadly tuned spinal neurons for each form of
ctive scratching in spinal turtles. J Neurophysiol 86:10171025.
Berkowitz A (2001b) Rhythmicity of spinal neurons activated during
each form of ctive scratching in spinal turtles. J Neurophysiol
86:10261036.
Berkowitz A (2002) Both shared and specialized spinal circuitry for
scratching and swimming in turtles. J Comp Physiol [A]
188:225234.
Berkowitz A (2005) Physiology and morphology indicate that
individual spinal interneurons contribute to diverse limb
movements. J Neurophysiol 94:44554470.
Berkowitz A (2008) Physiology and morphology of shared and
specialized spinal interneurons for locomotion and scratching. J
Neurophysiol 99:28872901.
Berkowitz A, Roberts A, Soe SR (2010) Roles for multifunctional and
specialized spinal interneurons during motor pattern generation in
tadpoles, zebrash larvae, and turtles. Front Behav Neurosci 4:36.
Berkowitz A, Hao ZZ (2011) Partly shared spinal cord networks for
locomotion and scratching. Integr Comp Biol 51:890902.
Berkowitz A, Stein PS (1994) Activity of descending propriospinal
axons in the turtle hindlimb enlargement during two forms of ctive
scratching: phase analyses. J Neurosci 14:51055119.
Bernhard CS, Taverner D (1951) The action of D-tubocurine on the
monosynaptic extensor reex. Brit J Pharmacol 6:540.
Bernhard CS, Taverner D, Widen L (1951) Dierences in the action of
tubocurarine and strychnine on the spinal reex excitability of the
cat. Brit J Pharmacol 6:551.
Berridge KC (1989) Progressive degradation of serial grooming
chains by descending decerebration. Behav Brain Res
33:241253.
Biewener AA, Gillis GB (1999) Dynamics of muscle function during
locomotion: accommodating variable conditions. J Exp Biol
202:33873396.
Briggman KL, Kristan Jr WB (2006) Imaging dedicated and
multifunctional neural circuits generating distinct behaviors. J
Neurosci 26:1092510933.
Cohnberg RE (1946) Stimulation of the central nervous system by
curare (intocostrin). J Lab Clin Med 31:866877.
Cuellar CA, Tapia JA, Juarez V, Quevedo J, Linares P, Martinez L,
Manjarrez E (2009) Propagation of sinusoidal electrical waves
along the spinal cord during a ctive motor task. J Neurosci
29:798810.
Cuellar CA, Trejo A, Linares P, Delgado-Lezama, Jimenez-Estrada I,
Abyazova LM, Baltina TV, Manjarrez E (2014) Spinal neurons
bursting in phase with ctive scratching are not related to
spontaneous cord dorsum potentials. Neuroscience 266:6679.
Degtyarenko AM, Simon ES, Norden-Krichmar T, Burke RE (1998)
Modulation of oligosynaptic cutaneous and muscle aerent reex
pathways during ctive locomotion and scratching in the cat. J
Neurophysiol 79:447463.
Deliagina TG, Feldman AG (1981) Activity of renshaw cells during
ctive scratch reex in cat. Exp Brain Res 42:108115.
Deliagina TG, Orlovsky GN (1980) Activity of Ia inhibitory
interneurons during ctitious scratch reex in the cat. Brain Res
193:439447.
Deliagina TG, Orlovsky GN, Pavlova GA (1983) The capacity for
generation of rhythmic oscillations is distributed in the
lumbosacral spinal cord of the cat. Exp Brain Res 53:8190.
166