THE MAKE-UP OF SPINAL CORD CIRCUITS
WHICH PROCESS INPUTS FROM
THE FEMORAL-SAPHENOUS VEIN
By
BILLY J. YATES
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1986
ACKNOWLEDGEMENTS
performed
small
whom
guidance,
the
assistance
and encouragement of a
Much of the guidance and encouragement
members of my committee: Floyd Thompson, John Munson,
the
Vierck
in this dissertation could not have been
individuals.
of
from
Chuck.
of
without
army
came
presented
studies
The
and
Davenport.
Paul
Special thanks go to Floyd, from
learned many electrophysiological techniques and a great deal
I
philosophy.
I
also could not have completed this work without the
advice
concerning
Parker
Mickle, Paul Reier, John Houle, Celeste Wirsig and Roger Reep.
I
thank
Alan
neurons.
Hedden,
Light
his
for
techniques
that
I
obtained from
suggestions regarding the staining of
wish to express my utmost gratitude to Deb Dalziel, Bill
I
Albis
Josepha
neuroanatomical
Acosta,
Cheong,
Dan
Orlando, Marty Yungmann, Linda Cheshire,
Daftary
Shoobha
and
Ellen
Grygotis
for
superb
technical assistance.
Data
were
SAS
Institute
the
facilities
analyzed
using the Statistical Analysis System (SAS,
Inc., Cary, North Carolina).
of
the
Northeast
Computing was done using
Regional Data Center of the State
University System of Florida.
Financial
Air
Force
Department
the
support
Aerospace
of
was
provided by NIH grant R01 HL 25619, U.S.
Medicine
Neuroscience,
Contract
F33615-82-D-0627
,
the
the Department of Neurological Surgery,
Division of Sponsored Research and the Center for Neurobiological
Sciences.
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS
ii
LIST OF TABLES
v
LIST OF FIGURES
vi
ABSTRACT
viii
CHAPTERS
I
II
III
IV
GENERAL INTRODUCTION
1
TRACING OF AFFERENT PATHWAYS FROM THE FEMORALSAPHENOUS VEIN TO THE DORSAL ROOT GANGLIA USING
TRANSPORT OF HORSERADISH PEROXIDASE
5
Introduction
Methods and Materials
Results
Discussion
19
PROPERTIES OF SPINAL CORD PROCESSING OF FEMORALSAPHENOUS VENOUS AFFERENT INPUT REVEALED BY
ANALYSIS OF EVOKED POTENTIALS
25
Introduction
Methods and Materials
Results
Discussion
25
26
33
41
PHYSIOLOGICAL PROPERTIES OF SINGLE SPINAL CORD
NEURONS ACTIVATED BY STIMULATION OF THE
FEMORAL- SAPHENOUS VEIN
44
Introduction
Methods and Materials
Results
Discussion
44
44
49
65
5
6
9
in
V
VI
THE VARIABILITY IN THE RESPONSES OF NEURONS
LOCATED IN DIFFERENT LAMINAE FOLLOWING
STIMULATION OF THE FEMORAL-SAPHENOUS VEIN
70
Introduction
Methods and Materials
Results
Discussion
70
70
72
92
GENERAL DISCUSSION
101
REFERENCES
106
BIOGRAPHICAL SKETCH
116
iv
LIST OF TABLES
Page
2-1
2-2
4-1
5-1
5-2
Locations of cell bodies in the dorsal root
ganglia giving rise to femoral-saphenous
venous afferents
13
Conduction velocities of primary femoralsaphenous venous afferents
23
Responses of spinal neurons receiving inputs
from the femoral-saphenous vein
53
Spontaneous firing rates of neurons located in
different laminae
78
Locations of neurons receiving convergent inputs
from the femoral-saphenous vein, muscle and skin
..
91
LIST OF FIGURES
Page
2-1
2-2
2-3
3-1
3-2
3-3
4-1
4-2
4-3
5-1
5-2
Cell bodies in the dorsal root ganglia labeled
by the application of HRP to the femoralsaphenous vein
11
Diameters of cell bodies in the dorsal root
ganglia of the kitten giving rise to venous
afferents
16
Diameters of cell bodies in the dorsal root
ganglia of the adult cat giving rise to venous
afferents
...»
18
Minimal spinal cord processing time for inputs
from muscle, skin and the femoral-saphenous vein
..
35
Potentials recorded along a typical electrode
track through L6 following stimulation of the
femoral-saphenous vein
38
The location of the focus of the short latency
negative waves elicited by stimulation of the
femoral-saphenous vein
40
The effects of stimulation of the femoral-saphenous
vein on the excitabilities of interneurons
51
Effects of stimulation of muscle, cutaneous and
venous afferents on the excitability of a single
neuron
59
The response amplitude versus stimulus intensity
relationship for cutaneous afferents
63
Examples of recording sites marked through the
extracellular iontophoresis of HRP
74
Locations of sites at which responses were recorded
from venous afferent-activated interneurons
77
vi
5-3
5-4
5-5
5-6
5-7
Locations of units excited or both excited and
inhibited by stimulation of femoral-saphenous
venous afferents
80
Procedures used to determine whether a unit is
activated at monosynaptic latencies by primary
femoral-saphenous venous afferents
83
Locations of units which were activated at
different minimal latencies following stimulation
of the femoral-saphenous vein
86
Locations of units activated for different
durations following the stimulation of the femoralsaphenous vein
89
Reconstructions of venous afferent-activated
interneurons that were intracellularly stained
using HRP
94
vii
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE MAKE-UP OF SPINAL CORD CIRCUITS
WHICH PROCESS INPUTS FROM
THE FEMORAL-SAPHENOUS VEIN
By
BILLY J. YATES
August 1986
Chairman: Floyd J. Thompson
Major Department: Neuroscience
Several
from
Experiments
(HRP)
from
determine
which
the
vein
afferents
application
vein
the
vein
to the spinal cord and the neurons
process
these
inputs
were
described.
the retrograde transport of horseradish peroxidase
using
to
the
dorsal root ganglia were performed to
number, distribution and sizes of the cell bodies from
the
femoral-saphenous
the
experiments
which
cord
the
afferents which conduct inputs
the
of
femoral-saphenous
the
within
characteristics
showed
project
are
of
afferents
originate.
These
that afferents arising along the entire length of
to
very
few in number.
HRP
venous
localized
spinal
levels and that these
Most of the cell bodies labeled by the
to the femoral-saphenous vein were small in size
viii
(diameter
less than 35 micrometers).
(diameter
greater
estimated
that over 70% of the femoral-saphenous venous afferents are
C
fibers;
micrometers)
50
were
also
noted.
13% were estimated to be A fibers.
least
at
afferents
venous
than
However, some large cell bodies
predicted
were
conduct
to
action
It was
The largest
potentials at
approximately 60 m/sec.
minimal
The
stimulating
large
approximately
primary
afferents
the
as
sites
for
as
inputs from skin; thus, di- or tri-
motoneurons.
and
recording
suggested
afferent
of
same
the
in the femoral-saphenous vein was
arising
fibers
pathways appeared to be the shortest circuits separating the
synaptic
well
cord processing time for inputs elicited by
spinal
input
most
that
unit activity from reconstructed
single
of
the
of
Mappings of field potentials as
first neurons activated by this
found in lamina V.
are
Thus, it is likely that many
large primary femoral-saphenous venous afferents terminate in
the
this region.
Recordings
venous
both
of
were
afferents.
excited
the
and
done from single spinal neurons driven by large
The
predominantly
in
activated
stimulation
Some
of
the
were excited or
inhibited for long durations following stimulation
femoral-saphenous
by
characterized
interneurons
laminae
vein.
V
of
and
These
VII.
Most
neurons
were
located
neurons could also be
large muscle and cutaneous afferents.
neurons were intracellularly stained; the reconstructed
cells had large cell bodies and extensive dendritic fields.
ix
CHAPTER I
GENERAL INTRODUCTION
The
study
pathways
allows
nervous
reflex
which
appears
and coordinates motor behavior.
produced
be
to
circuitry
A spinal
by relatively simple central
is elicited when receptors in the walls of
femoral-saphenous vein are stimulated (Thompson and Barnes, 1979;
Yates, 1983, 1984, 1986; Thompson et al., 1982, 1983).
and
femoral-saphenous
intravenous
afferents
fibers
excited by
following
recordings
low-threshold
stimulation of these afferents showed that they can elicit
simultaneous
(Thompson
et
extensors
are
contraction of flexor and extensor hindlimb muscles
al.,
from
reflexes
include
perfusion pressures as low as 2-3 mm Hg (Thompson et al.,
electrical
arising
venous
Electromyographic
1983).
the
organizes
system
Thompson
The
neural substrate of relatively simple reflex
the
an investigator to gain insight into how the central
system
nervous
the
of
the
motoneurons.
Activation
facilitation
the
reciprocal
generally
muscle
and
of
results
(Sherrington,
of
excited
femoral-saphenous
Stimulation
exteroceptors
is interesting that both flexors and
It
simultaneously
involve
inhibition
1982).
1910;
afferents
inhibition
of
stimulation
by
vein,
other known hindlimb
as
facilitation
cutaneous
of afferents
and
inhibition
afferents
or
of
other
in flexor facilitation and extensor
Lloyd,
typically
agonists
1943a;
Hagbarth,
produces
and
1952).
the reciprocal
antagonists
(Lloyd,
Laporte
1943a;
and
Lloyd,
Matthews,
1952;
The
1969).
unique
segmental
reflex
suggested
to provide a rapid means of controlling the counterpressure
applied
elicited
by
afferent stimulation has been
venous
the high-capacitance intramuscular veins; changes in this
to
counterpressure
potentially
are
important
compensating
in
for
orthostatic blood shifts (Thompson and Yates, 1983, 1984, 1986).
well
is
It
environment,
converge
are
their
the
somatic
those from the femoral-saphenous vein,
reflex
pathways
(Downman,
Evans
1955;
and
Spinal neurons which send
pathway (Rigamonti and Hancock, 1974; Rigamonti et al., 1978),
spinocervical
Foreman
and
pathway
(Rigamonti
spinothalamic
and
Weber,
Rucker
1981;
1980;
Blair
Pacinian
et
are
Barnes,
receptors
that
pulsations
located
1970;
Michelle,
1977) and the
(Hancock
al., 1981,
al., 1975;
et
1984; Milne et al.,
and Holloway, 1982; Ammons et al., 1984; Rucker et al.,
corpuscles
cardiovascular
and
pathways
also receive visceral inputs.
1984),
Perl,
from the internal
inputs
into ascending tracts, including the postsynaptic dorsal
spinoreticular
that
other
1958, 1959, 1960; Evans, 1963).
axons
column
to
that
somatic inputs in the spinal cord on interneurons that
of
McPherson,
addition
in
with
part
established
These inputs arise in part from
sometimes
(Gammon
discharge
in
phase
with
and Bronk, 1935), thermoreceptors
at least in part along blood vessels (Thompson and
Rawson
and
Quick,
1972;
Riedel,
1976),
vascular
that respond to visceral movement or distention (Bessou and
1960;
proprioceptors
Floyd
found
and
Morrison,
1974;
Floyd
et
al.,
1976),
in the intercostal muscles and in the diaphragm
3
(Yamamoto
al., 1960; Remmers and Tsiaras, 1973; Davenport et al.,
et
Marlot
1985;
et
(Komisaruk
and
innervating
blood
Potter
interest
from
Wallman,
al.,
et
and
1983)
cheraoreceptors
(Moore and Moore, 1933; Lim et al., 1962;
Besson
1962;
Henry,
1977;
vessels
compare
to
1985), mechanoreceptors located in the vagina
al.,
cord circuits which process inputs
spinal
the
It would be of great
al., 1972).
et
femoral-saphenous vein with the circuits that process other
the
inputs from the internal environment.
present study examined several of the characteristics of the
The
afferents
spinal
which conduct inputs from the femoral-saphenous vein to the
cord
peroxidase
the
retrograde transport of horseradish
the
number, distribution and sizes of the cell bodies from
these experiments are described in Chapter II.
mappings
were
interneurons
first
and
cord which process these
the
femoral-saphenous venous afferents originate.
the
potential
also
using
within
from the vein to the dorsal root ganglia were performed to
determine
of
neurons
the
Experiments
inputs.
which
and
excited
demonstrated
motoneurons;
different
afferent
altered
discussed
in
stimulation
by
vein
by the venous afferents.
properties
locations
of
to determine the location of the
These mappings
information is presented in Chapter III.
this
are
excitabilities
In addition, field
the shortest pathway between the primary afferents
electrophysiological
interneurons
performed
The results
of
in
the
venous
Chapter
IV.
The
afferent-activated
Interneurons
with
the cord were shown to be affected by venous
in
neurons
different
in
stimulation
the
is
ways; the variability in how the
different
presented
laminae of Rexed were
in Chapter V.
Finally,
Chapter
VI
spinal
considers
cord
femoral-saphenous
circuitry,
the hypothesis that the characteristics of the
circuitry
vein
described
which
are
in
similar
previous
inputs from the internal environment.
processes
to
the
studies,
inputs
from
the
characteristics of the
which
processes
other
CHAPTER II
TRACING OF AFFERENT PATHWAYS FROM THE FEMORAL- SAPHENOUS VEIN TO
THE DORSAL ROOT GANGLIA USING TRANSPORT OF HORSERADISH PEROXIDASE
Introduction
et
mammal,
the
In
1985; McMahon et al., 1985; Norregaard and Moskowitz, 1985),
al.,
extracranial
and
(McMahon
visceral
Morrison,
Bowker,
afferent
demonstrated
walls
trunks
These
large
are
cord
blood vessels receive an
Anatomical
studies
have
also
the hindlimb and into adjacent nerve
of
Hinsey,
further
stimulation
of
1928; Truex, 1936; Polley, 1955).
supported
carefully
elicited
vein
by
isolated
compound
(i)
experiments in which
segments
action
of
the
potentials
from the femoral nerve and dorsal root fibers entering the
lumbar
Yates,
veins
observations
femoral-saphenous
lower
Lim et al., 1962; Floyd and
1984)
innervation.
1926;
recordable
Mathison,
and
(Woollard,
electrical
1960;
presence of large, myelinated fibers coursing along
the
of
Perl,
and
Floyd et al., 1976; Guilbaud et al., 1977; Vance and
Barja
1983;
1985; Norregaard and Moskowitz, 1985),
et al.,
(Bessou
1974;
extensive
the
intracranial (Davis and Dostrovsky, 1985; Keller
spinal
(Thompson
cord
and Barnes, 1979; Thompson and
1986), (ii) evoked field potentials recordable from the lumbar
(Thompson
changes
in
and
Barnes,
1979;
Thompson and Yates, 1986), (iii)
firing patterns recordable from single spinal motoneurons
(Thompson
et
recordable
from
al.,
1982)
hindlimb
and
muscles
(iv)
electromyographic
(Thompson
et
al.,
activity
1982).
The
6
injection
fluid
of
longitudinal
potentials
pulses
into
the
the
vein
wall
stretch
of
the
lumbar
in
femoral-saphenous
similarly
vein
elicited
or
field
cord and activity recordable from
spinal
ventral roots (Thompson et al., 1983).
study
The
gain
presented
concerning
insight
femoral-saphenous
retrograde
venous
transport
femoral-saphenous
determine
vein
this chapter was performed in order to
in
projection
the
afferents
the
to
of
horseradish
to
the
dorsal
pattern
spinal
peroxidase
root
The
cord.
from
(HRP)
ganglia
these
of
used
was
the
to
range of afferent sizes, the relative numbers of these
the
afferents, and the spinal cord segments to which they project.
Methods and Materials
HRP Application Procedures
Experiments
adult
conducted
were
cats (weighing 2.5-3.8 kg).
intraperitoneal
mg/kg)
or
(ip)
injection
intramuscular
an
hydrochloride,
Bristol,
Haver-Lockhart,
0.5
10
kittens
on
(5-6 weeks of age) and
Animals were anesthetized using an
of
sodium pentobarbital (Butler, 38
(im)
injection
mg/kg)
combined
mg/kg).
of
Ketaset
(ketamine
with Rompun (xylazine,
Maintenance
doses
of
sodium
pentobarbital
(12
necessary
maintain areflexia during the time that tissues were in
to
mg/kg)
or
Ketaset
(5
mg/kg)
were delivered as
contact with HRP.
The
expose
kittens
skin
the
and
of
either
of
femoral-saphenous
9
the
vein
hindlimbs was opened ventrally to
and
underlying
muscle.
In
3
adult cats, one to three 10-15 mm segments of the vein
separated from surrounding tissues and placed on rubber dams.
were
segment
of
proximal femoral vein (overlying the vastus medialis
the
adductor
and
femoris
muscles)
application;
other
were
3-6 cm apart.
spaced
were
segments
prevent
small
coursing
in
HRP
covered
spread
tears
with
HRP
sites were located distally and
thick
a
layer
of
petroleum jelly to
Uptake of HRP was promoted by making
there would be damaged.
hrs
application
using
for
in the outer vein wall with sharp forceps so that fibers
3-4
after
prepared
The tissues adjacent to the isolated vein
the tracer.
of
always
was
application
dimethylsulf oxide
2%
A
enzyme
the
applied to the prepared vein segments;
was
removed
from
were irrigated with saline.
sites
wound
was
A 30% solution of Sigma Type VI HRP
clips
the
vein
and
the
Incisions were closed
and
the animals were returned to their cages and
for
these experiments, different HRP application
allowed to recover.
As
sites
control
a
were
used
in
adult cats.
2
In these animals segments of the
femoral-saphenous
vein
described
However, the HRP solution was applied to the muscle
which
been
above.
directly
lay
dissected
accidentally
tracer
separated
from
the adjacent muscle as
underneath the 10-15 mm segment of vein that had
away;
exposed
tissues
these
HRP
to
during
were
the
most
likely
3
2
procedure
was
In one
application sites located 31 mm apart were used; in the
application
distribution
to be
those experiments in which the
was applied to segments of the femoral-saphenous vein.
experiment
other
were
of
sites separated by 20 and 22 mm were used.
cells
compared
in
the
with
dorsal
The
root ganglia labeled by this
that of cells labeled after the enzyme
was applied to the femoral-saphenous vein.
Histological Procedures
After
adult
recovery period of 50-60 hrs in kittens and 90-96 hrs in
a
cats,
transcardially
sodium
0.1%
After
M
perfused
nitrite
chilled
of
animals
the
were
reanesthetized;
were
with 250-500 ml of chilled saline containing
and 1.5 units of heparin per ml followed by
glutaraldehyde/0.6%
1.25%
kittens
paraformaldehyde
2
1
fixative.
an additional 30 min the fixative was replaced with chilled 0.1
phosphate
buffer
transcardially
(pH
perfused;
Adult
7.4).
cats
were
similarly
however, twice the volume of solutions were
used than in the smaller animals.
The
by
spinal cord and lumbosacral dorsal root ganglia were exposed
dorsal
exposed
in 0.1 M phosphate buffer (pH 7.4).
micron
sagittal
Histochemistry
according
xylenes.
the
to
Sections
and
short
eyepiece
give
the
unlabeled
be
using
a
vibratome
(Oxford).
procedures
Mesulam
of
(1982).
Tissues
were
using neutral red, rapidly dehydrated and cleared with
illumination.
to
sections
The tissues were cut into
was performed using the chromagen tetramethylbenzidine
counterstained
noted
L3-S1 dorsal root ganglia (on the side
The
and the L6-S1 spinal cord segments were removed and
HRP)
to
placed
50
laminectomy.
were
Neuronal
oval
(minor)
graticule.
average
cells
cell
using both bright- and dark-field
bodies in the dorsal root ganglia were
in shape (see Figure 2-1).
axes
of
labeled
cells
Both the long (major)
were
measured using an
The mean of these two measurements was taken to
cell
in
observed
diameter.
representative
In
addition,
sections
were
the dimensions of
also measured to
indicate the range of sizes of cell bodies present in the ganglia.
Controls for the Diffusion of HRP
experiments
all
In
scrutinized
were
motoneurons
If
any
In
Thus,
or
absence
of
labeled
an adequate indicator as to whether HRP
be
to
presence
the
into the muscle underlying the femoral-saphenous vein.
labeled
preparation
the spinal cord was
numerous densely labeled motoneuron cell
vein
noted.
diffused
of
which HRP was applied to the muscle underlying
in
appeared
horn
presence of labeled motoneuron cell bodies.
femoral-saphenous
bodies
had
the
experiments
those
the
for
ventral
the
cell
which
in
femoral-saphenous
were
bodies
HRP
vein,
was
the
noted
applied
in
the ventral horn of a
to isolated segments of the
experimental results were discarded and
not analyzed further.
Results
Five
of the 12 experiments (42%) in which HRP was applied to the
femoral-saphenous
neuronal
labeled
Examples
cell
vein
bodies
motoneuron
of
application
cell
were successful; in these experiments labeled
were present in the dorsal root ganglia and no
cell bodies could be detected in the spinal cord.
bodies
in
the dorsal root ganglia labeled by the
of
HRP to the femoral-saphenous vein are shown in Figure
Following
the application of HRP to the femoral-saphenous vein,
2-1.
an
average of 289 +/- 402 (mean +/- S.D.)
dorsal
root
labeled cell bodies in the
ganglia was counted per experiment; an average of
182
confidence intervals presented here also represent mean +/standard deviation.
"X)ther
Examples of cell bodies in the L5 and L6 dorsal
Figure 2-1.
ganglia
labeled
root
by the application of HRP to the
represent
Calibration
bars
50
femoral-saphenous
vein.
micrometers.
Labeled neuronal cell bodies are denoted by
arrows.
11
1
**3
» .
i
"
,
'*
-'.
1
9
^1
-
r
I
V
i
-
•"
•
/.
M
?
-
-
12
+/-
cells
232
portion
was counted per vein segment exposed to HRP.
of Table 2-1 shows the locations of cell bodies in the dorsal
ganglia
root
labeled
by
application
the
of HRP to the vein. In 3
preparations
more
confined
the L6 dorsal root ganglion; in
to
labeled
the
The top
cell
other
than
95%
of the labeled cell bodies counted were
preparation all of the
1
bodies were confined to the L5 dorsal root ganglion; in
preparation
77%
of
the labeled cell bodies counted were
located
in
the
L5
dorsal
located
in
the
L6
ganglion.
counted
were located in the L6 dorsal root ganglion, 21% were located
root
ganglion,
total
In
and
78%
the other 23% were
of the labeled cells
in the L5 ganglion and less than 1% were located at other levels.
numbers
The
ganglia
labeled
and
by
femoral-saphenous
different
cells
by
cell
total
root
(bottom
portion
of
Table
the
application
vein
bodies
had
labeled
of
a
2-1)
were quite
In one experiment 6285 labeled
HRP
muscle underlying the
the
to
The cell bodies
more widespread distribution than did
by HRP application to the vein itself.
In
3.9% of the labeled cells counted were located in the L3 dorsal
ganglion,
located
in
SI
ganglion.
by
the
were
7.3%
the
were
30.5%
vein
bodies in the dorsal root
counted; in the other 6251 were counted.
femoral-saphenous
the
cell
application of HRP to muscle underlying the
the
vein
of
from those described above.
were
labeled
locations
located
in
the
L4
ganglion, 8.6% were
L5
ganglion, 21.2% were located in the L6 ganglion,
located
in the L7 ganglion and 28.5% were located in the
It
is reassuring that most of the cell bodies labeled
application of HRP to muscle underlying the femoral-saphenous
were
located
at
levels
caudal
to
those containing the cell
bodies labeled by HRP application to the vein itself.
13
Table 2-1.
Percentage of cell bodies in different dorsal root
ganglia labeled by the application of HRP to the femoral-saphenous
vein (top) or to the tissues underlying the vein (bottom).
HRP Applied to the Femoral-Saphenous Vein
Experiment #
1
of Sites
to which
# of
Pe rcentage of Labeled Cell Bodies
Counted in Each Gangl: Lon
L3
L4
L5
L6
L7
HRP Was
Applied
Cell
Bodies
Labeled
1
1
391
77
23
2
2
953
3
96
3
2
45
4
3
51
5
3
7
SUMMATION
~~
o
SI
1
100
100
n
100
1447
21
78
«1
HRP Applied to the Muscle Underlying the Femoral-Saphenous Vein
1
2
6251
2
3
6285
12536
SUMMATION
4
10
25
36
25
8
11
7
18
25
32
4
7
9
21
31
29
14
Figure
ganglia
vein
2-2 shows the diameters of cell bodies in the dorsal root
labeled
by
the kitten;
of
the figure also shows the diameters of unlabeled
cells
measured
in
sizes
of
bodies
similar
and
cell
data
adult
animals
bodies
were
noted
<
micrometers).
A
diameter
significantly
ganglia.
adult cats.
Figure 2-3 shows
Data derived from kittens
be
vein
smaller
than
labeled
by
had
average
an
the
those in the adult.
application
diameter
of
of
Cell
HRP to the
+/- 8.6
25.4
35 micrometers);
total
of
only 1.6% were large (diameter > 50
694 unlabeled cells were measured in the
of
larger
0.0001).
30.5
+/-
11.3
Unlabeled cells had an
micrometers;
this
mean
was
than that for labeled cells by Student's t-test
Seventy-three
percent
of
the unlabeled cells were
size (diameter < 35 micrometers); 6.5% were large (diameter
in
micrometers).
> 50
The
relative
labeled
by
the
unlabeled
cell
sizes
the
cation
of
the
for comparison with the labeled cells.
average
small
in
The majority (87.5%) of the labeled cells were small in
(diameter
<
from
to
kitten
the
micrometers.
(p
present
sections to indicate the range of
cats are shown separately because the cell bodies in young
in
kitten
representative
collected
femoral-saphenous
size
application of HRP to the femoral-saphenous
the
in
of
HRP
sizes
of
in
size
bodies in the dorsal root ganglia
application of HRP to the femoral-saphenous vein and
bodies
were similar in the adult cat to the relative
kitten.
Cell
bodies in adults labeled by the appli-
to the femoral-saphenous vein had an average diameter
32.9 +/- 14.0 micrometers.
small
cell
(diameter
<
Most (71.8%) of the labeled cells were
35 micrometers);
only 13.2% were large
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19
(diameter
measured
was
Student's
cells
adults
A total of 1255 unlabeled cells were
comparison
for
with
the
labeled cells.
cells had an average diameter of 41.7 +/- 19.5 micrometers;
mean
this
the
in
Unlabeled
micrometers).
50
>
significantly
t-test
were
small
in
than that for labeled cells by
0.0001). Forty-five percent of the unlabeled
<
(p
larger
size
(diameter
< 35
micrometers); 24.1% were
large (diameter > 50 micrometers).
Both
cats
were
vein;
were
the
cord
was
vein
to
using
Labeled cell
addition,
the
cell
the percentage of large labeled cell
ganglia
bodies.
was
smaller than the percentage of
However, the anterograde transport of
the dorsal root ganglia to afferent terminals in the spinal
far
the
labeling
the labeled cells were small.
on the average, than other cell bodies located
in
in
unlabeled
from
of
smaller,
counted
HRP
small cell bodies in the dorsal root ganglia of
most
ganglia;
bodies
large
and
labeled by the application of HRP to the femoral-saphenous
however,
bodies
in
large
less
extensive than the retrograde transport from the
ganglia,
of
if
intraspinal
either
bright-
it
occurred at all.
portions
of
dark-field
or
afferents
The transganglionic
was never detected
illumination
following
the
application of HRP to the femoral-saphenous vein.
Discussion
Previous
electrical
or
electrophysiological studies in the cat have shown that
mechanical
femoral-saphenous
lumbar
1983,
spinal
1984,
vein
cord
1986;
can
stimulation
elicit
(Thompson
of
isolated
potentials
segments of the
recordable
from the
and Barnes, 1979; Thompson and Yates,
Thompson et al., 1982, 1983).
This study extends
20
these
findings
number,
distribution
femoral-saphenous
of
afferents
these
arising
that
muscle.
had
the
femoral-saphenous
predominantly
labeled
These
in
of
The selective labeling
not
by several lines of evidence.
could be detected in these studies,
diffused
suggesting
from
that
vein to adjacent
the
spread
no
tracer
of
application of HRP to the tissues adjacent to
vein
dorsal
produced
root
labeling
ganglia
caudal
of cell bodies found
those containing
to
also
suggest
that
the
afferents
which
were
labeled
terminated
in
fibers
passage coursing along the vein but arising in muscle.
of
sensory
of the femoral-saphenous vein and were not
It
for all fibers of passage from muscle to be
fibers arising in muscle had picked-up HRP in
if
experiments, the labeling of motoneuron cell bodies should have
In
transported
HRP,
dorsal
root
application
labeled
addition,
distribution
the
ganglia
of
fibers
if
should
tracer
of
of
passage
labeled
from
cell
muscle
had
bodies in the
have been similar to that following the
directly
to
the
muscle.
Since
the cells
by the application of HRP to the femoral-saphenous vein had a
different
skin
unlikely
afferents;
occurred.
origin.
walls
the
very
be
these
No
cell bodies following application of HRP to the vein itself.
data
would
the
of the cell bodies from which these
supported
evidence
that
is
demonstration
the walls of the femoral-saphenous vein in
motoneurons
HRP
Further
occurred
anatomical
sizes
in
was
spinal
of
suggesting
and
an
venous afferents originate.
preparations
labeling
the
providing
by
distribution,
Similarly,
were
labeled
it
is likely that they also had a different
it is unlikely that fibers of passage arising in
in
these
studies.
The
skin
overlying
the
21
femoral-saphenous
vein
in
saphenous
nerve;
this
(Bernhard,
1953;
Crouch,
picked-up
labeled
nerve
cell
bodies
projects
1969).
It
is innervated by the
several
to
spinal
levels
If fibers of passage from skin had
labeled
by
vein
than
bodies
other
hindlimb
cell
dorsal
the
root
ganglia
would have been
also noteworthy that the cell bodies in the dorsal
is
ganglia
in
femoral-saphenous
in
animal
HRP in these experiments, a more widespread distribution of
expected.
root
intact
the
afferents,
the
were
and
application
significantly
in
the
HRP
of
to
the
smaller, on the average,
ganglia.
If
a
general class of
not a specific population, had taken up HRP
these studies, the diameters of the labeled cells should have been
identical to those of other cells in the ganglia.
recent
A
correlate
study
by
Lee
et
al.
(1986) has made it possible to
diameter of a cell body in a dorsal root ganglion with
the
conduction velocity along the peripheral process arising from it.
the
These
investigators
made
intracellular
recordings from single cell
bodies
in the dorsal root ganglia of adult cats using microelectrodes
filled
with an HRP solution, determined the conduction velocity along
peripheral process to the cell body, and then injected the tracer
the
into
the
cell
bodies
neuron so it could later be visualized.
peripheral
at
Cell
to
greater
linear
than
processes
fibers).
reported
less
micrometers
greater
diameter
gave
rise to
than 50 micrometers in diameter were
rise to peripheral processes that carried impulses
than 2.5 m/sec (A fibers).
correlation
in
that carried impulses at less than 2.5 m/sec (C
bodies
give
35
They reported that
between
the
For these neurons there was a
diameter
of
the cell body and the
22
impulse
conduction
velocity;
this
relationship
was defined by the
following equation:
Conduction Velocity = (0.84
However,
of
there
Diameter of Cell Body) - 20.3.
*
was no predictable relationship between the diameters
intermediate-sized
cell
bodies
(35-50
micrometers)
and
the
conduction velocities along their peripheral processes.
Most
HRP
to
(71.8%)
give
of
rise
cell bodies labeled by the application of
in
diameter;
these cell bodies would be expected to
to slowly conducting peripheral processes.
However, 13.2%
the cell bodies labeled by the application of HRP to the
vein were
larger
rise
than 50 micrometers in diameter; these cell bodies should give
rapidly
to
conducting
conduction
velocities
the
also
table
afferents
labeled
based
upon
following
velocities
bodies
vein
of
peripheral
along
shows
the
diameters
cell
of
the
estimated
bodies that were not
application of HRP to the vein.
venous
by
The
predicted conduction velocities of other
The largest
afferents were estimated to have conduction
approximately
labeled
processes.
these processes are shown in Table 2-2;
the
the
femoral-saphenous
be
the
the femoral-saphenous vein of the adult cat were smaller than
micrometers
35
of
60
m/sec.
application
Fifteen percent of the cell
of HRP to the femoral-saphenous
were intermediate in size (35-50 micrometers); no estimate could
made
of
vessels
shown
1983),
other
to
the
conduction velocities along these afferents.
than
the
Blood
femoral-saphenous vein have previously been
be innervated by thermoreceptors (Fruhstorfer and Lindblom,
chemoreceptors
Potter et al., 1962;
(Moore
Besson
and
et al.,
Moore,
1972)
1933;
Lim
and
slowly
et al.,
1962;
conducting
23
Table 2-2.
Estimated conduction velocities along the peripheral
processes of large primary femoral-saphenous venous afferents
and
other large primary sensory neurons determined from the diameters
of
the cell bodies.
Conduction Velocity
Ran S e
Percentage of Cells Falling into the Range
Femoral-Saphenous
Other Afferents
Venous Afferents
20-30 m/sec
47
37
30-40 m/sec
36
32
40-50 m/sec
15
19
50-60 m/sec
2
10
> 60
m/sec
2
24
mechanoreceptors
Floyd
et al.,
also
(Bessou
rapidly
Perl,
1960; Floyd and Morrison, 1974;
It is likely that the femoral-saphenous vein is
1976).
innervated
and
by one or more of these receptor types as well as by
conducting
afferents
that
have
not
been
reported
to be
associated with other vessels.
Only
of
HRP
a small number of afferents were labeled by the application
the
to
segment
femoral-saphenous
exposed
afferents
labeled
A-alpha/beta
enzyme),
the
to
vein
were
large.
femoral-saphenous
(an average of 182 per vein
only a small fraction of the
and
However,
electrical
activation of
venous afferents has been reported to
produce
large field potentials recordable from the cord (Thompson and
Barnes,
1979;
Thompson
electrophysiology
divergence
of
and
be
to
inputs
the
in
cord is substantial.
either
the
1986).
register,
from
spinal
extensive
Yates,
the
it
For
the
would
anatomy
seem
femoral-saphenous
that
and
the
vein to the
Such divergence could be accomplished by
branching
of
the intraspinal portions of the
afferents
or the activation of second-order spinal interneurons that,
in
make
turn,
Visceral
cat
have
contacts
afferent
inputs
previously
been
with a large number of higher-order cells.
into the lower thoracic spinal cord of the
shown
divergence (Cervero et al., 1984).
to
exhibit
a
similar widespread
CHAPTER III
PROPERTIES OF SPINAL CORD PROCESSING OF FEMORAL- SAPHENOUS VENOUS
AFFERENT INPUT REVEALED BY ANALYSIS OF EVOKED POTENTIALS
Introduction
The
study
presented in Chapter II suggested that afferents with
both
large
vein
to the lumbosacral spinal cord of the cat.
the
L6
and
small
segment
afferents.
was
The
of
afferents
spinal
activation
afferents
was
interposed
the
focal
location
input
cord
discussed
motoneurons
this
these
primary
examined
two
link inputs along these
minimum
the
for
chapter
which
The
by
In most preparations
segment
in
interneurons
measured
time required for
femoral-saphenous venous
to estimate the least number of interneurons
primary
the
synaptic
of
project from the femoral-saphenous
motoneurons.
spinal
between
addition,
the
of
the
study
characteristics
with
diameters
the
field
afferents
and
motoneurons.
In
potentials were mapped to determine
interneurons
monosynaptically
excited
by
stimulation of the femoral-saphenous vein.
The
were
field
first
potential
by
potential
fields has since been performed in a number of
of
studies
(Campbell,
et
1945;
Gasser
Austin
and
Graham
(1933); the intracord
and McCouch, 1955; Howland et al.,
Coombs et al., 1956; Fernandez de Molina and Gray, 1957; Willis
al.,
1979)
techniques utilized in this study
described
mapping
1955;
mapping
and
1973;
has
Fu
et al.,
proven
to
1974; Beall et al., 1977; Foreman et al.,
be
an
25
effective
preliminary
method of
26
describing
by
the spatial location of the interneuronal pools, activated
particular
a
stimulus.
recordable
from
excitatory
axo-dendritic
When
the
extracellular
axons
of
horn
most
prominent
field
potential
dorsal horn is the result of ionic movements at
the
synapses
The
and
axo-somatic
synapses (Willis, 1980).
are
activated, positive ionic current leaves the
at
the synapses (sinks) and reappears along the
space
the interneurons (sources).
Because the majority of dorsal
interneurons ventral to the substantia gelatinosa have ventrally
projecting
net
axons (Matsushita, 1969, 1970), the dorsal horn takes on a
negative
charge.
which
The
charge
and
negativity
contains
horn takes on a net positive
of shortest latency is focussed in a region
high
a
ventral
the
density
of
interneurons
monosynaptically
excited by primary afferents (Yates et al., 1982).
Methods and Materials
Surgical Preparation of Animals
Data
were
Intraspinal
the
recorded
potential
from
fields
adult
25
cats
were recorded from
of
7
either
sex.
of these animals;
minimum times between the arrival of the primary afferent volleys
along
muscle,
discharges
Animals
Ayerst
oxygen.
carotid
ligated.
in
were
cutaneous
ventral
the
initially
laboratories)
A
The
venous
roots
was
in
afferents
and
detectable
were determined in the other 18.
anesthetized
vaporized
tracheostomy
arteries
and
with
a
performed
3% Fluothane (halothane,
mixture
and
of nitrous oxide and
either
of
the common
was cannulated; the other common carotid artery was
tracheostomy allowed for artificial ventilation with a
27
Harvard
respiration
measurement
also
arterial
of
cannulated
fluids.
by
pump;
cannulation
of
blood pressure.
the
carotid allowed for
The left cephalic vein was
to allow the intravenous administration of drugs and
Animals were rendered decerebrate at the midcollicular level
transection of the brainstem and aspiration of the portions of the
brain
rostral to this level.
discontinued;
of
the
T10.
this
paralysis
was
(gallamine
segments
at least 4 hours prior to the beginning
session.
were
spinal cord was also transected at
The
immobilized
induced
through
triethiodide,
readministered
and
occurred
recording
Animals
Following decerebration, anesthesia was
when
in
a
spinal
intravenous
Davis-Geek,
unit
(David
injection
mg/kg).
2
of
Kopf);
Flaxedil
Flaxedil
any muscle contractions were noted.
was
Spinal cord
L3-S1 were exposed by laminectomy, the dura mater was opened
pinned
covered
away
with
from
the cord dorsum, and the exposed tissues were
warmed mineral oil. Body and oil pool temperatures were
maintained at 37 degrees C using a heating pad and a heat lamp.
The
expose
of
skin
segment
a
the
vein
gently
accidental
the
was
either
of
the
hindlimbs
was opened dorsally to
of the femoral-saphenous vein.
separated
from
A 10-15 mm segment
surrounding connective tissue and
placed upon a silver bipolar stimulating electrode.
membrane
those
of
was
placed
contact
experiments
beneath
of
in
the stimulated vein segment to prevent
electrode
the
which
A plastic
with surrounding tissues.
In
the minimum time between the arrival of
primary afferent volley and a detectable discharge in the ventral
roots
nerves
was
determined,
a
segment
of
one
or more of the following
was also prepared, in a manner similar to that used to prepare
28
the
vein,
bipolar
for
posterior
tibial
plantaris
nerve
foot
applied
the
to
addition,
the
nerve,
posterior
musculature).
skin
overlying
and
L6
stimulation:
gastrocnemius
deep
or
innervating
electrical
nerve,
nerve
sural
the
nerve,
deep peroneal nerve,
(branch
of
tibial
the
Bipolar stimuli were sometimes also
femoral-saphenous
the
vein.
In
ventral roots were cut at dural entry and
L7
were placed across bipolar hook recording electrodes.
Stimulation Procedures
Bipolar
excite
have
venous
also
afferents
1983,
1984,
stimulating
passing
This
in
the
current
result
and
Thompson
et
that
vein
al.,
Thompson and Yates,
1979;
1982); these studies provided
stimulation
wall.
only excited afferent
Application
immediately
abolished
A number of previous
stimulation to activate the
Barnes,
this
vein
the
electrode
proximal
evoked
the
of
and
ligatures
distal
to
to
the
potentials elicited by
along that vein segment (Thompson and Yates, 1986).
was apparently due to the blockade of impulse conduction
afferents coursing along the vein wall (Gelfan and Tarlov, 1956).
Stimulation
elicited
of
acting
on
Activation
isolated
unique
a
simultaneous
and
in
of
electrical
(Thompson
evidence
arising
portions
utilized
1986;
considerable
fibers
stimulation was used in these experiments to
femoral-saphenous venous afferents.
the
studies
electrical
spinal
excitation
the
of
inhibition
segments
thigh,
of
crus
of
reflex;
the
hindlimb
and
the
foot
femoral-saphenous
stimulation
flexor
and
(Thompson
produced
vein
the
extensor
muscles
et
1982).
al.,
cutaneous afferents produces the reciprocal excitation
of
flexors and extensors; activation of large muscle
29
afferents
also
elicits
hindlimb
muscles
afferents
which
of
Chapter
(see
the
vein
but
arising
chapter
I).
It
thus
is
likely
that the
were excited by the stimulation of isolated segments
femoral-saphenous
the
reciprocal excitation and inhibition of
the
vein belonged to a unique class arising in
wall and were not fibers of passage coursing along the vein
muscle
in
involved
the
or
skin.
The studies presented in the last
application
of HRP to isolated vein segments;
the
procedures used to isolate portions of the femoral-saphenous vein
for
exposure
to
similar.
The
afferents
arising
to
results
isolated
the
tracer
this
and
presented
for
electrical
stimulation are
Chapter II suggested that only
in
in the vein were labeled by the application of HRP
vein segments; thus, muscle and cutaneous afferents
apparently
were
electrical
stimulation
not
coursing along these segments. It is clear that
is a technique which allows for the selective
activation of femoral-saphenous venous afferent fibers.
During
mapped,
experiments
the
stimulated
prepared
in
which
segment
of
intracord
by
a
1
Hz.
for
were
intensity
was
Stimuli were delivered
digital stimulator (Frederick Haer Pulsar 6i).
dorsum
vein
using single-shock square-wave pulses of current, 0.2 msec
duration, repeated at a frequency of
threshold
potentials were
femoral-saphenous
the
in
to
field
eliciting
used
to
field
excite
potentials
the
afferent
Stimuli
3
times
recordable from the cord
fibers.
This stimulus
was chosen because previous studies had shown it sufficient
elicit compound action potentials recordable from the dorsal roots
with
fibers
A-beta
shown
components
in
of
maximal
amplitude.
The
A-delta and C
Chapter II to also arise from the femoral-saphenous
30
vein
were not excited by this stimulus intensity (Thompson and Yates,
thus,
1986);
very
a
synchronous spinal input was elicited.
During
experiments in which the minimal times required for activation of
the
spinal
motoneurons
were
stimulated
at
potentials
recordable
sural
the
Stimuli
intensity
an
nerve
from
were
applied
monosynaptic
measured,
reflexes
vein was
2-3 times that necessary to evoke field
the
cord dorsum.
stimulated
muscle
to
femoral-saphenous
the
at
nerves
The hindlimb skin and
twice cord dorsum threshold.
were
graded
evoke
to
only
recordable from the ventral roots or to elicit
both monosynaptic and polysynaptic reflexes.
Recording Procedures
Intraspinal
were
recorded
glass
except
of
0.5-4.0
from
the
using
across
Intraspinal field potentials
using tungsten microelectrodes insulated with epoxy or
for a 10-20 micrometer tip which had initial impedances
megohms.
Potentials
were
recorded at 0.2
the
and
cord
L6
0.5,
ground;
subsequently
contained
segment.
1.0,
and
1.5
Five tracks were conducted
Penetrations
2.0
large blood vessels.
the
adjacent
preamplifiers
intervals
were optimally made at
mm lateral to midline; slight
in the choice of penetration site were often necessary to
puncturing
margin
ram
dorsum to 4.4 mm in depth; electrodes were positioned
cord
alterations
animal
fields .
a hydraulic microdrive (Narishige).
midline
avoid
potential
reference
(Grass
into
the
to
a
tape
preamplifiers
electrode was positioned in the wound
cord.
with
P511)
Potentials
a
recorder
with
Potentials were referenced to
a
time
were
constant
(Crown-Vetter
bandwidth
of
led
into
A.C.
of 250 msec and
model
A)
D.C. to 15 kHz.
which
The
31
potentials
Ortec
were
displayed on an oscilloscope and were averaged by an
computer.
Records of averaged potentials were made with an X-Y
plotter (Hewlett-Packard).
A
radiofrequency
lesion
was placed at the bottom of each track
to
facilitate the histological localization of the track.
of
lesions
fixative
depths
were
also
The depths
analyzed as a control for tissue shrinkage by
other factors (such as cord dimpling) which might cause
and
measured
from
histological
material
deviate from those
to
measurements read from the vernier of the hydraulic microdrive.
Cord
dorsum
recorded
animal
as
from
were
measurement
of
latency
arrived
at
which
input
the
cord;
several
propagating
the
of
from
microelectrodes.
dorsum
cord
excitability
Molt
et
cord
through
potentials
dorsum
The
studies
have shown that the initial
dorsum potential is
a
compound action
large primary afferent fibers (Austin
In addition, the amplitude
is known to vary in proportion to spinal
(Bernhard and Koll, 1953; Gelfan and Tarlov, 1956;
al., 1978).
a
control
excitability
remained
that
cord
little
Cord
along the fastest-conducting afferents
McCouch, 1955; Howland et al., 1955).
provided
were
in these experiments for two reasons.
at
potential
cord
potentials
dorsum potentials provided a means to determine
cord
spike
of
recorded
recorded
triphasic
and
dorsum
The potentials were led into A.C. preamplifiers, etc,
potentials
for
Cord
L6 using silver ball electrodes and were referenced to
ground.
potentials
the
potentials .
Thus, the measurement of cord dorsum potentials
to
assure
constant
damage
was
that
the
level
of
spinal
cord
throughout the recording session and
produced by electrode penetrations in
32
experiments in which intraspinal field potentials were mapped.
those
Data
recorded from animals in which the cord dorsum potentials varied
in amplitude during the recording session were not analyzed.
Compound
recorded
which
action potentials .
Compound action potentials were
from the L6 and L7 ventral roots during those experiments in
minimal
the
required
time
motoneurons
was
determined.
positioned
in
close
experiments.
for an afferent input to activate
bipolar recording electrodes were
The
proximity
Compound
action
spinal
the
to
potentials
cord
were
led
in
these
into
A.C.
preamplifiers, etc, as described above.
Histological Procedures
At
conclusion
the
animals
were
saline
perfused
followed
fixative.
L6
gelatin-albumin,
cut
microtome
could
easily
recorded
fewer
be
heart
with
1
1
mappings,
of heparinized
of 1.25% glutaraldehyde/1% paraformaldehyde
1
segment
into
stained
the
potential
field
with
40
was
removed,
micrometer
cresyl
quick-embedded
sections
violet.
4
from
in
microscopic
in
with a freezing
Most electrode tracks
reconstructed from histological material; only data
along reconstructed tracks were considered for analysis.
than
recorded
laminae
and
intraspinal
through
by 1.5
The
of
If
tracks were reconstructed in a particular animal, data
that
which
animal
the
examination
were not analyzed. Identification of the
recording sites were located was done through
of
histological
material and by comparison
with published accounts of laminar organization (Rexed, 1952, 1954).
33
Results
Figure
times
3-1
shows
required
muscle,
for
skin,
and
determination
the
afferent
input
latency
represents
intraspinal
activation
femoral-saphenous
time
illustrated
is
portions
in
combination
a
of experiments in which minimal
of spinal motoneurons by inputs from
minimal
this
of
results
the
vein
for
part
were
femoral-saphenous
"A"
of
A
venous
the figure.
conduction
of
examined.
This
along
time
the
of primary afferents, synaptic delay times, and
conduction
time
comparison
of the central delay following venous afferent stimulation
with
delay
the
afferents,
1981),
along
the
processes of spinal neurons.
the
following
synaptology
provides
an
stimulation
which
of
estimate
of
cutaneous
of
is
well
minimal
the
However, a
and
muscle
established (Brown,
number
of
synapses
interposed between the primary afferents and alpha-motoneurons.
minimal
The
volley
in
and
part
between
the arrival of the primary afferent
a detectable discharge in the ventral roots are indicated
"B"
stimulated.
delays
times
of
Figure
3-1.
The abscissa shows the afferent type
Data regarding muscle afferents represent the pooling of
measured
following
posterior
tibial,
plantaris.
The
stimulation
gastrocnemius,
latencies
from
deep
of
the
peroneal,
triphasic
spike
following
nerves:
deep posterior or
to
onset of both
monosynaptic
and polysynaptic reflexes recorded from the ventral root
are
Data regarding cutaneous afferents represent the pooling
of
skin
shown.
delays
measured
overlying
the
following
stimulation of the sural nerve or the
femoral-saphenous
vein.
Latencies reported for
muscle and cutaneous input reflect the processing time by the input
Figure 3-1. Minimal spinal cord processing time for inputs from
(A) The method
muscle, skin and the femoral-saphenous vein.
(B) A
used to determine minimal spinal cord processing time.
comparison of these latencies. Abbreviations: CD, cord dorsum
potential; VR, potential recorded from the ventral roots; TPS,
triphasic spike.
B
V.
A.
L 6' L
7
V.
A.
L 6" L
6
MUSCLE
(po'y«yn)
CUTAN.
MUSCLE
(monosyn)
36
segment
cord
muscle
for
nerves
and
femoral-saphenous
stimulation
separating
ventral
spike
the
combination
multiple
all
Two
afferents
and
L6
central
(L7 for the stimulated
for the skin overlying the
delay
times
following
femoral-saphenous vein are reported: the latency
as
spike
activity
recorded from L6
latency separating the L6 triphasic
the
recorded
and
from the L7 ventral roots.
Thick bars
mean minimal spinal cord processing time; thin bars indicate
error.
significantly
Stars
indicate
values
shown
to
differ
from all others by an analysis of variance procedure in
with
either
a
Waller-Duncan K-ratio T test or Duncan's
range test (alpha=0.05)
elicited
reflex
vein).
well
as
standard
spike
nerve
triphasic
L6
activity
and
sural
the
roots
indicate
the
of
stimulated
the
recorded
others;
muscle
by
The delay separating the triphasic
.
afferent stimulation and the monosynaptic
from the ventral root was significantly shorter than
delay separating the L6 triphasic spike elicited by
the
femoral-saphenous
venous
afferent
stimulation
and
ventral
root
activity recorded from L7 was significantly longer than all others.
Figure
3-2
shows
femoral-saphenous
a
typical
recorded
dorsal
advanced
a
a
gray
waveforms
recorded
following
track
through
L6.
The
potentials were
series of negativities near the cord dorsum or in the
matter
into
of
venous afferent stimulation at various depths along
raicroelectrode
as
series
the
(waveforms
ventral
A-D).
horn,
the
As
the
negative
microelectrode
tip
waves decreased in
amplitude and eventually reversed in polarity (waveforms E and F).
Figure
3-3
shows
the focus of the short latency negative waves
elicited by stimulation of the femoral-saphenous vein.
The dots
WD
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Abo
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M
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l-l
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41
indicate
points
the
lines
80%
or
both
more
to
from
collect
the
location
data;
onset
most
In
the
of
and
orientation of electrode
the time over each diagram indicates
the
experiments
triphasic spike of the cord dorsum
(5 of 7),
the initial negativities
focussed in the neck of the dorsal horn; this region corresponds
Rexed's
initial
lamina V (Rexed, 1952, 1954).
negativities
roughly
axis,
medial
the
indicate
to
potential.
were
maximal could be recorded; the arrows at the top of
of
used
latency
maximal-amplitude potentials were recordable;
surrounding the dots indicate the area in which potentials
diagrams
tracks
where
and
in
In 4 of
experiments, the
7
were also focussed, along the medial-to-lateral
center of the gray matter; however, foci both
the
lateral to this point were also noted.
It is likely that
inconsistencies in results observed in a few experiments were due
more
limitations in the field potential mapping technique than to
to
physiological
failed
pass
to
recording
variability
directly
session,
the
between
through
animals.
potential
a
If
a
microelectrode
generator
during a
spatial location of that potential generator
could appear displaced from its actual location.
Discussion
The
dorsal
latency
and
separating
ventral
roots
femoral-saphenous
venous
femoral-saphenous
vein
latency
noted
msec).
Minimal
way
for
inputs
spinal
from
of
afferents,
(4.4
following
evoked
muscle
L6,
the
following
recordable
input
from
the
segment
for
stimulation
of
the
msec) was approximately the same as the
stimulation
cord
activity
of
cutaneous
afferents
(4.3
processing time, estimated in the same
which
reach alpha-motoneurons through
42
polysynaptic
known
that
muscle
was
fastest
the
only slightly shorter (3.8 msec).
circuits
which
link
It is
cutaneous inputs and
inputs with interneurons and then motoneurons are di- and tri-
synaptic
or
circuits,
(Lloyd, 1943b, 1943c).
tri-
synaptic
These data are consistent with a di-
circuit being the shortest pathway between primary
femoral-saphenous venous afferents and alpha-mo toneurons.
Following
negative
electrical
stimulation of the femoral-saphenous vein,
waves of the cord dorsum potential can be recorded in spinal
animals
for
20 msec or more (Thompson and Yates,
reflect
the
activation
Fernandez
1956;
al.,
Yates
et
al.,
potentials
These waves
dorsal horn interneurons (Coombs et al.,
de Molina and Gray, 1957; Willis et al., 1973; Fu et
Beall
1974;
of
1986).
et
al., 1977; Foreman et al., 1979; Willis, 1980;
1982).
elicited
In
spinal
in
afferent
stimulation
1982).
Thus,
circuits
interconnect
addition,
maximal
animals
ventral
root action
by femoral-saphenous venous
have a duration of 20-25 msec (Thompson et al.,
although
relatively
venous
short
di-
and
tri-
synaptic
afferents and motoneurons, much higher
order circuits also appear to exist.
minimal
The
ventral
longer
latency
activity
root
separating
L6 dorsal root activity and L7
elicited by FVA stimulation was significantly
(by at least 1.7 msec) than other processing times estimated.
Increased
portion
intraspinal
of
conduction
distances
can
account for only a
this
longer
latency;
femoral-saphenous
venous
afferents from L6 to L7 is less than
(Thompson
and
additional
synapse
Yates,
is
1986).
It
interposed
the conduction time along primary
is
likely
between
that
at
1
msec
least
one
femoral-saphenous venous
43
afferents
and L7 motoneurons than is interposed between the afferents
and L6 motoneurons.
A
distribution
produced
in
dorsal
elicited
by
have
shown
horn
can
primary
1957;
intracord
stimulation
by
the
of
horn
fields similar to that
of the femoral-saphenous vein (negativities
and
stimulation
potential
positivities
of
in the ventral horn) is also
other afferent types.
Previous studies
that the negative waves of shortest latency in the dorsal
be attributed to monosynaptic excitation of interneurons by
afferents (Coombs et al., 1956; Fernandez de Molina and Gray,
Willis
et
al.,
1973;
Fu
et
al., 1974; Beall et al., 1977;
Foreman
et
largest
negative waves of shortest latency were recorded from Rexed's
lamina
V,
al.,
it
1979; Willis, 1980; Yates et al., 1982).
Since the
appears that most of the first interneurons excited by
the large venous afferents are found in this region.
CHAPTER IV
PHYSIOLOGICAL PROPERTIES OF SINGLE SPINAL CORD NEURONS
ACTIVATED BY STIMULATION OF THE FEMORAL-SAPHENOUS VEIN
Introduction
The
data presented in Chapter III suggested that at least one or
interneurons
two
femoral-saphenous
concluded
that
afferents
are
chapter
this
following
the
interposed
venous
located
stimulation
large
the
responses
(A-beta)
It
activated
these
by
processed
show
to
by
whether
spinal
large
of single spinal cord neurons
In addition,
inputs from muscle and skin on the
of
afferent-activated interneurons were examined.
conducted
was also
The study discussed in
femoral-saphenous vein.
the
primary
motoneurons.
Rexed's lamina V.
in
of
and
interneurons
convergence
of
between
afferents
first
the
examined
patterns
venous
are
venous
interneurons
afferent
This study was
inputs
were
highly
and, if so, whether these neurons
were modality-specific.
Methods and Materials
Surgical Preparation of Animals
Experiments
cats
with
were
spinal
anesthetized
using
because
a
suggest
whether
performed
cords
excitabilities
of
transected
alpha-chloralose.
comparison
an
on
of
the
data
unanesthetized, decerebrate
19
at
T12
and
5
intact
cats
The two preparations were used
obtained
from
each type would
elicited input had strong or weak effects on the
the
neurons
being studied.
44
It is known that the
45
excitabilities
using
of
alpha-chloralose
preparations.
The
preparations
inputs
are
Monroe,
on
than
excitabilities
influences
the
to
if
1981).
the
input
spinal
decerebrate-spinal
likely
(Alvord
in
in
decerebrate-spinal
these
anesthetized
of descending supraspinal
has
1954;
Balis and
powerful effects on these neurons.
neurons
the
Fuortes,
preparations by a particular input, it
are
preparations,
that
and
spinal neurons are driven in both
If
anesthetized
that
However,
neurons
Brown,
and
likely
is
lower
lower
due
the
1964;
decerebrate
it
are
to the spinal interneurons as well as to direct effects of the
anesthetic
is
spinal interneurons in intact animals anesthetized
input
but
has
activated
by
the
input
in
not anesthetized preparations,
only
weak
influences
on
the
excitabilities of these neurons.
Anesthesia
be
decerebrated
(Merck,
and
experiment;
blood
the intravenous injection of alpha-chloralose
Maintenance
pressure
either
if
doses
the
of the drug (25 mg/kg) were
However, the electrocardiogram
were continuously monitored during the
heart
rate or blood pressure increased,
that the animal was recovering from anesthesia, additional
alpha-chloralose
stabilized
using
in the group of animals which would not
delivered every four hours.
arterial
indicating
induced
mg/kg).
75
routinely
was
was
administered
their original level.
at
until
both of these measurements
The other animals were rendered
decerebrate using the procedures described in Chapter III.
Most
unit
of
the surgical procedures used to prepare cats for single
recordings
were
similar
to those described in Chapter III for
46
preparing
animals
additional
for
stitches
threads.
bladder
to
due
to
minimize
addition
and
the
to provide better mechanical
hammock
was
formed by placing
blood vessels.
In many animals the
so that it would remain empty; this helped
blood pressure and prevented blood pressure surges
bladder.
movements
these
to
dural
pulsating
catheterized
full
a
A
used
Several
way the cord was lifted away from underlying
this
In
stabilize
also
of field potentials.
dura and then by suspending small weights from
the
bodies
was
were
recording.
through
vertebral
recording
the
procedures
stability
the
for
of
A bilateral pneumothorax was also used to
the
spinal
procedures
for
cord
during
respiration.
In
increasing mechanical stability,
holes
were made in the arachnoid and pia overlying the L6 spinal cord
using
sharp
forceps.
This
procedure
permitted
the
insertion of
microelectrodes into the cord without breaking the tips.
Stimulation Procedures
Bipolar
electrical
femoral-saphenous
the
posterior,
Square-wave
excite
lateral
pulses
afferent
frequencies
100
responses
of
Hz
used
to
activate
cutaneous afferents coursing in
was
of
current,
fibers.
1
were
gastrocnemius
0.2
and
hamstring
nerves.
msec in duration, were used to
While
data were being collected, stimulus
Hz were used.
However, stimulation frequencies up
used
to
differentiate
afferent
generated by interneurons (see below).
microelectrode
vein
afferents,
was
sural nerve and muscle afferents running in the posterior tibial,
deep
to
venous
stimulation
penetrations
continuously
were
stimulated
being
at
responses
from
Throughout the time
done, the femoral-saphenous
an
intensity
3
times
that
47
necessary
As
to evoke a field potential recordable from the cord dorsum.
discussed
Chapter
in
maximal
A-beta
volley
A-delta
and
afferents.
an
C
attempt
concerns
to
regarding
intensities
up
potential;
the
stimulus
spinal
the
to
recruit
this
III,
intensity provides a
cord but does not excite the
High current intensities were not used in
small-diameter
the
stimulus
spread.
venous afferents due to
Nerves
were
stimulated
at
to 10 times the threshold for eliciting a cord dorsum
minimal
current
intensity
for
affecting
the
excitability of a unit was also determined.
Recording Procedures
Intracellular
using
glass
micropipette electrodes filled with
tris-buffered
had
initial
HRP
and extracellular single unit recordings were done
M
0.15
impedances
solution
had
Microelectrodes
were
(Narishige).
vein
however,
entry
Electrodes filled with
3
M KC1
of 15-30 megohms; electrodes filled with the
initial
impedances
positioned
dorsal
L6
segments
The
these
were
using
columns
were
should
have
portion
of
microdrive
latter
used
micrometers
should
(pH 7.3).
M KC1 or 8% HRP in
usually
of
a
30-90
hydraulic
megohms.
microdrive
ipsilateral to the stimulated
impaled
by the electrodes;
a few penetrations were also made through the L6 dorsal root
zone.
during
KC1
The
nerve
and
KC1
3
to
depth
penetrations.
record
VII
in
When electrodes filled with
3
M
responses, only units located at 700-1500
were studied.
remained
lamina
was angled sharply towards the midline
At these depths, the electrode tips
the dorsal horn or in the most superficial
(Rexed,
have been encountered.
1952,
1954);
thus, no motoneurons
In addition, the units were tested for
48
their
responses
action
potentials
latency
with
responses
high
frequency
recorded
from
to
high
from
(Yates
were
invariant
were
not
The
reconstruct
show
constant
of
iontophoresis
sites
marked
be
to
filled
characterized
the
of
sites
electrodes
of
recording
recording
are altered by high frequency
with 100 Hz stimulation, these responses
use
staining
extracellular
afferents
If responses recorded from a unit
1982).
latency
in
allowed
intracellular
interneurons
et al.,
analyzed.
solution
primary
is known that
it
stimulation frequencies, whereas the latencies of
recorded
stimulation
stimulation;
the
tracer;
with
either
neuron
or
by
the
by
the
the procedures used to
discussed in Chapter V.
are
the HRP
Only data
recorded from units located outside of lamina IX were analyzed.
All
potentials
were
referenced to animal ground; the reference
electrode
was located in the wound margin adjacent to L6.
were
into
and
led
subsequently
constant
of
bandwidth
microelectrode pre-amplifier (Winston model 1090A)
into
0-2500
of
an
msec,
250
microelectrode
into
a
histogram
unit
generated
poststimulus
of
amplifier
an
(Grass P511) with a time
recorder (Vetter model D) with a 10 db
tape
Hz and oscilloscope.
the tape recorder.
records
A.C.
pre-amplifier
of
Potentials
was
also
The direct output of the
led into an oscilloscope and
Analysis of data was facilitated by the time
Ortec
time
histograms
computer (model 4621); the latter unit
histograms
were
from
generated
the
using
data.
an
X-Y
Hardcopy
plotter
(Hewlett-Packard).
At
small
the
bundles
conclusion
of
the
of
L7
single
dorsal
unit recordings from 11 animals,
rootlets
were cut proximally and
placed
across
posterior
a
silver
bipolar recording electrode.
tibial
nerves
were then stimulated using the same current
intensities
applied
activate interneurons during the single unit
to
recordings.
Potentials
recorder
oscilloscope.
Ortec
and
computer.
sites
These
were
afferents
were
led
into
an
amplifier,
A.C.
tape
Potentials were also averaged using the
In addition, the conduction distances from the nerve
stimulation
data
The sural and
the dorsal root recording site were measured.
to
used
activated
to determine the conduction velocities of the
the minimal current intensities necessary to
by
alter the excitabilities of interneurons.
Results
Responses of Interneurons
enous Venous Afferents
Data
were
recorded
femoral-saphenous
the
Following Stimulation of
the Femoral-Saph-
from 81 neurons activated by stimulation of
vein
decerebrate
in
cats; detailed analyses
were
made
data
were useful in quantitative determinations of such parameters as
the
onset
data
were
of the data recorded from 66 units.
latency
useful
femoral-saphenous
skin.
evoked
of
in
activity in neurons, etc., all of the
determining
venous
While only the latter
afferent
the
patterns
of
convergence of
input with inputs from muscle and
In anesthetized animals, data were recorded from 14 units; the
responses
of
all
of these neurons following femoral venous afferent
stimulation were characterized in detail.
Figure
interneurons
at
an
4-1
illustrates
following
intensity
3
the
types
of
responses recorded from
femoral-saphenous venous afferent stimulation
times
that necessary to evoke field potentials
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spikes
52
recordable
from
recorded
from
neurons
following
out
42
animals.
stimulation
with
of
(64%)
of
other
the
the venous afferent-driven
femoral-saphenous
vein
in
24 neurons (36%) responded to vein
combination
of
spontaneous
the
using
femoral-saphenous
characterized
The
a
below
anesthetized
66
A burst of action potentials was
one
or
more
bursts of action
a period during which the firing rate of the unit was
and
depressed
of
stimulation
decerebrate
potentials
dorsum.
cord
the
rate.
alpha-chloralose
The neurons studied in cats
responded
in
similar
ways to
venous afferent stimulation; half of the 14 neurons
were
excited
by
vein stimulation, and the other half
were both excited and inhibited.
quantitative
The
neurons
activated
summarized
neurons
+/-
in
were
of
stimulation
Table
4-1.
similar
in
the
response properties of these
of the femoral-saphenous vein are
The mean spontaneous firing rates of the
decerebrate and anesthetized animals (15.5
16.7 spikes per second in the former and 14.3 +/- 20.3 spikes per
second
in
the
substantial
a
by
aspects
latter).
ongoing
Clearly,
most
of
the units examined had
activity in the absence of stimulation; however,
number of units showed no recordable spontaneous firing.
units
the
absence
(18%) characterized in decerebrate cats were silent in the
stimulation;
of
exhibited
no
units
important,
will
or
that
is
be
not
Twelve of
5
spontaneous
since
of the units (36%) in anesthetized animals
firing.
The
occurrence
of these silent
a unit which does not fire spontaneously
classified as only being excited by elicited inputs, whether
it
receives
inhibitory inputs.
It is only possible to show
the firing rate of a unit decreases below the spontaneous level
53
Responses of spinal interneurons to electrical stimulation
femoral-saphenous venous afferents, posterior tibial nerve and
the
of
represent mean +/- standard
All confidence intervals
sural nerve.
deviation.
Onset latencies were measured from the onset of the
triphasic spike of the cord dorsum potential. All means except those
for spontaneous firing rate are in msec; mean spontaneous firing rates
are in spikes per sec.
Table
4-1.
Units
Decerebrate Animals
'Burst + Inhibition' Units
13.5 +/- 17.2
(n = 42)
19.1 +/- 15.6
(n = 24)
'Burst'
Spontaneous
firing rate
Onset latency of
activity from
vein stimulation
Duration unit
was excited by
vein stimulation
Duration unit
was inhibited
by vein stimulation
Total duration
excitability
was altered by
vein stimulation
Total duration
excitability
was altered by
tibial nerve
stimulation
Total duration
excitability
was altered by
sural nerve
stimulation
13.5 +/- 19. a
(n = 42)
15.5 +/- 16.7
(n = 66)
5.6 +/- 4.9 a
(n = 24)
10.6 +/- 16.3
(n = 66)
43.3 +/- 72.4
(n = 66)
59.7 +/- 82.7
(n = 24)
34.0 +/- 65.0
(n = 42)
Both Types
Combined
41.3 +/- 26.4
(n = 24)
34.0 +/- 65.0
(n = 42)
102
+/- 186
(n = 11)
42.6 +/- 48.0
(n=10)
a
101
+/- 88.
(n = 24)
89.8 +/- 73.7
(n = 11)
45.0 +/- 30.4
(n=8)
a
58.4 +/- 80.6
(n = 66)
95.8 +/- 138
(n = 22)
43.7 +/- 40.0
(n=18)
^Values for 'burst' and 'burst + inhibition' units were significantly
different by Student's t-test (p < 0.05).
54
Table 4-1
—continued.
Anesthetized Animals
'Burst + Inhibition' Units
'Burst'
Units
4.0 +/(n
6.8 +/(n
9.6
=7)
6.2
=7)
17.4 +/- 19.8
(n
=7)
24.6 +/- 23.4
(n =
7)
4.0 +/(n =
4.2
7)
18.5 +/- 20.8
(n =
7)
Both Types
Combined
14.3 +/- 20.3
(n = 14)
5.4 +/-
5.3
(n = 14)
17.9 +/- 19.4
(n = 14)
43.6 +/- 63.3
(n =
17.4 +/- 19.8
(n
=7)
18.6
(n
=1)
5.1 +/(n
5.9
=3)
7)
62.1 +/- 66.4
(n =
7)
75.1 +/- 81.1
(n =
4)
81.5 +/- 68.6
(n =
5)
38.5 +/- 51.9
(n = 14)
63.8 +/- 64.6
(n =
5)
52.9 +/- 65.3
(n =
8)
55
following
stimulation if the unit fires spontaneously.
units
the
being
with
only
Thus, some of
low spontaneous rate, classified in this study as
a
excited
venous afferent stimulation, may indeed also
by
have been inhibited by this input.
Table
elicited
4-1
also
venous
by
of
variances
these
excitabilities
values
vein
following
the
latencies
of
the responses
stimulation and the durations that the
were altered by this stimulation.
were
neurons
of
femoral-saphenous
msec
units
the
onset
the
afferent
excitabilities
in
shows
enormous.
elicited
The
changes
stimulation
by
in
of
The
the
the
had an average onset latency of 10.6 +/- 16.3
onset
of the triphasic spike of the cord dorsum
potential
in decerebrate animals and 5.4 +/- 5.3 msec in anesthetized
animals.
These
Student's
t-test
action
et
In
and
thus,
represented
to be significantly different by
The
through
triphasic spike is a compound
the
McCouch,
the
largest
1955;
onset
primary
Howland et al., 1955;
latency
measured from the
the onset latency of activity following
cats,
there
was
also a significant difference (p <
in the onset latency of elicited activity in the units excited
venous
afferent
by
this
stimulation
and
the
units
difference
in
the
stimulation
anesthetized cats.
both
excited
and
input; the mean latency was 13.5 +/- 19.6 msec in
former and 5.6 +/- 4.9 msec in the latter.
afferent
active
of femoral-saphenous venous afferent input at the cord.
decerebrate
inhibited
the
1982);
shown
0.039).
<
(Austin
spike
arrival
0.016)
by
al.,
were
propagating
fibers
triphasic
the
(p
potential
afferent
Yates
means
onset
latency
of
activity
A similar significant
elicited
by venous
was not shown between units of the two types in
56
The
excitabilities
femoral-saphenous
vein
were
decerebrate
In
vein
stimulation.
neurons
of
were
mean
The
altered
the
by
units
receiving
modulated
inputs
from
the
for long durations following
duration
excitabilities
the
of
the
vein stimulation was 58.4 +/- 80.6 msec in
and 38.5 +/- 51.9 msec in anesthetized animals.
animals
decerebrate animals there was also a significant difference in the
duration
neurons
units
that
both
excited
by
venous
afferent
stimulation
and
excited and inhibited by this stimulation were affected
by inputs from the femoral-saphenous vein (p < 0.0008).
Convergence of Inputs from the Femoral-Saphenous Vein with Inputs from
Muscle and Skin on Single Spinal Neurons
effects
The
largest
muscle
largest
cutaneous
units
The
the
of
nerve
receiving
studied.
crus
nerve
The
of
also
the
sural nerve, the
hindlimb, on the excitabilities of
from
the
femoral-saphenous
vein
were also
tibial nerve innervates a number of muscles
including
triceps
the
surae,
tibialis
nerve
1954;
innervates
Crouch,
skin overlying the foot and
1969).
In decerebrate animals, 78
(98%) of the venous afferent-activated units tested could
80
activated by stimulation of the tibial nerve at an intensity
be
10
times
threshold
78
(74%)
of
stimulation
dorsum
and
hallucis longus, lumbrical muscles and interosseus
sural
(Jefferson,
hindlirab,
the
foot,
and
muscles.
the
of the posterior tibial nerve, the
of
posterior
flexor
out
of
input
posterior,
ankle
stimulation
of
the
at
an
for eliciting a cord dorsum potential; 58 out of
tested
units
were
also
driven
by
sural nerve
intensity 10 times that necessary to evoke a cord
potential; 56 out of 77 (73%) of the neurons were activated by
57
stimulation
of
(69%)
stimulation
by
tested
nerves.
anesthetized animals,
In
out of 13
9
venous afferent-activated units examined could also be
the
activated
the
both
of
units
of the tibial nerve; 10 out of 13 (77%) of
were also driven by sural nerve stimulation;
7
out
of 13 (54%) could be activated by stimulation of both nerves.
Figure
data
4-2
recorded
shows
from
femoral-saphenous
activity
of
msec
following
The
had
the
single
following
the
unit
sural
tibial
histograms generated from
stimulation
of
the
nerve and the tibial nerve.
The
nerve stimulation had an average onset
this nerve in decerebrate animals and 3.9 +/- 2.8
of
anesthetized
in
stimulation
in
by
time
+/- 10.3 msec following the triphasic spike produced
6.6
stimulation
by
vein,
elicited
latency
a
poststimulus
animals.
average
an
triphasic
The activity elicited by sural nerve
onset
latency
of
8.2
+/-
5.4
msec
spike produced by stimulation of this nerve
decerebrate animals and 4.4 +/- 4.9 msec in anesthetized animals.
venous afferent-activated neurons were excited, inhibited or both
excited
Most
and inhibited by stimulation of the sural and tibial nerves.
these
of
animals
nerve;
were
16
neuron
units
out
(37
of 54 or 69%) studied in decerebrate
both excited and inhibited by stimulation of the tibial
units (30%) were excited by tibial nerve stimulation and
(2%)
was
inhibited
by
stimulation
of
this
nerve.
1
In
contrast,
most of these units (22 out of 37 or 59%) were only excited
by
nerve stimulation in decerebrate cats.
were
(5%)
sural
both
were
excited
Only 13 units (35%)
and inhibited by sural nerve stimulation;
inhibited
anesthetized animals, 40%
by
the
stimulation
of
this
2
nerve.
cells
In
of the tested units were excited by tibial
Poststimulus time histograms showing the effects of
Figure 4-2.
stimulation of the femoral-saphenous vein, posterior tibial nerve
The
and sural nerve on the excitability of a single spinal neuron.
histograms were each generated from 64 consecutive sweeps. The
stimulus was delivered at the onset of the traces.
59
100 msec
25
I
spikes
60
nerve
stimulation,
inhibited.
In
excited
by
excited
and
no
obvious
40% were both excited and inhibited, and 20% were
the
same
sural
animals,
nerve
inhibited
simple
half
stimulation,
relationships
between
of
the
femoral-saphenous
stimulation
of
the
tibial
neurons
of
were
the
sural
To
better
and the other half were both
by the stimulation of this nerve.
stimulation
the
of the units examined were
There were
how a neuron responded to
vein
and how it responded to
and sural nerves.
The excitabilities of
modulated for long durations following stimulation
and
tibial nerves; these durations are shown in Table
4-1.
afferent
input
interneurons,
hamstring
posterior
nerves
the
of
convergence
of
stimulation
lateral
tested
nerve
using
of
gastrocnemius
on
of
muscle
3
muscle nerves— the
nerve
and
the
units in decerebrate cats.
17
current
the
innervates
intensities
the
nerve
innervates
1954;
Crouch,
lateral
nerve
flexors
3
semitendinosus
gastrocnemius
examined
patterns
10
times
deep
The
that
to evoke a field potential recordable from the cord dorsum.
crus,
by
the
stimulated
semimembranosus,
the
effects
nerve—were
hamstring
lateral
the
on femoral-saphenous venous afferent-activated spinal
nerve,
were
necessary
The
determine
and
innervates
gastrocnemius
the
2
of
the
biceps
thigh,
the
femoris;
the
extensors located in the
and the soleus; the deep posterior
a number of muscles located in the foot (Jefferson,
1969).
All of the 17 venous afferent-activated units
could also be driven by stimulation of the deep posterior or
lateral gastrocnemius nerve; 12 of the units could also be driven
stimulation
of
the
hamstring
nerve.
It was clear that muscle
61
inputs
from
extensors
many
parts
of
the
hindlirab and from both flexors and
converged on single interneurons also receiving inputs from
the femoral-saphenous vein.
Following
the
recordings
from
many
interneurons, small bundles of
L7 dorsal rootlets were cut proximally and placed across a silver
bipolar
recording
exposed
to the same current intensities which were necessary to alter
the
electrode.
excitabilities
recorded
from
intensities
of
were
analyzed
excited fibers.
the
A-alpha
and
elicited
Figure
components
of
decerebrate
cats,
were
volleys
units
only
these
stimulus
to determine the conduction velocities of
figure
the
to
the
shows
evoked
sural
the
effects of different stimulus
nerve
dorsal
the
on
root
action
18 out of 29 units (62%) for
(94%)
amplitudes
potential.
of 3
In
which an analysis was
driven by stimulus intensities applied to the tibial nerve
elicited
A-alpha
and
A-beta components in the compound
activated
by
current
for
which
applied
The other 11 units
intensities that produced A-delta
as well as A-alpha and A-beta volleys.
intensities
and
by
potentials recorded from the dorsal roots.
were
(38%)
elicited
potentials,
In most cases, low stimulus intensities elicited
This
applied
action
action
A-delta volleys; the results of a typical study are shown in
4-3.
which
rootlets,
Compound
A-beta volleys, whereas higher stimulus intensities also
intensities
done
interneurons.
dorsal
the
The tibial and sural nerves were then
Sixteen out of the 17
an analysis was done were driven by stimulus
to
the
sural nerve which only elicited A-alpha
A-beta components in the compound action potentials recorded from
the dorsal roots.
Only
1
unit was driven by current intensities that
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64
elicited
in
A-delta
as well as A-alpha and A-beta volleys.
anesthetized animals were quite similar.
for
which
applied
an
to
components
roots.
out
of
activated
that
by current intensities that
volleys as well as A-alpha and A-beta volleys.
units
A-beta
dorsal
still
root
nerve.
for
which an analysis was done were driven by
applied
to the sural nerve which only elicited
components
elicited
Two
in
compound
the
action potentials
The other unit was driven by current
A-delta
well
as
as
A-alpha and A-beta
root
volleys,
increasing
action
in
A-alpha
the
amplitude.
and A-beta volleys were
This was especially true for
potentials elicited by stimulation of the tibial
Thus, in most cases in which both A-delta volleys and volleys
larger
minimally
conclude
afferents
necessary
which
to
fiber
Nonetheless,
afferents
spinal
was
However, at minimal stimulus intensities necessary to evoke
A-delta
unit.
3
unit
from the dorsal roots.
volleys.
along
nerve which only elicited A-alpha and A-beta
A-delta
intensities
dorsal
tibial
other
and
recorded
often
was done were driven by stimulus intensities
intensities
A-alpha
units (86%)
in the compound action potentials recorded from the dorsal
the
stimulus
7
analysis
the
The
produced
Six out of
The results
were
activate
group
it
elicited
was
neuron,
a
carried
clear
by
the
that
stimulus
it
was
intensities
impossible to
input which activated the
large muscle and cutaneous
were included in the population which carried inputs to the
cord that altered the excitabilities of neurons also driven by
inputs from the femoral-saphenous vein.
65
Discussion
Venous Afferent Effects on the Excitability of Spinal Neurons
response
The
properties of the neurons driven by stimulation of
the
femoral-saphenous
for
units activated by electrical stimulation of cutaneous and muscle
These
afferents.
previous
especially
neurons,
studies have shown that most spinal cord
those
spontaneous
substantial
were similar to those reported by others
vein
decerebrate
in
firing
preparations,
However,
rate.
have
a
units which do not
fire
in the absence of stimulation have also been reported (LeBars et
al.,
1975; Light and Durkovic, 1984).
duration
and
afferent
of
activity
stimulation
electrical
following
elicited
stimulation
venous
by
the range of values reported
in
of
neurons
spinal
in
included
are
In addition, the onset latency
large
fibers
in the hindlimb
nerves (Feldman, 1975).
Neurons
stimulation
in
previous
excited
muscle
1969;
either
excited
studies,
spinal
In this study, as well as
interneurons
were
also
shown
to be
and cutaneous afferents (Hongo et al., 1966; Hillman and Wall,
Wagman and Price, 1969; Feldman, 1975).
Units both excited and
by venous afferent stimulation are likely to receive inputs
several
separate
others
for
be
direct
inputs
inputs
both excited and inhibited by
and inhibited by electrical stimulation of
excited
excitation,
due
or
of the femoral-saphenous vein.
both
or
inhibited
through
were
to
from
stimulation.
higher-order
The
pathways,
the inhibition.
responsible
for
the
The excitation could either
primary afferents to the unit or to
from
spinal
inhibition
some
is
neurons
also
driven by venous afferent
likely
due
to inputs from
66
venous
afferent-driven
responsible
neurons
for
that
possibility
responsible
that
is
afferent-induced
responsible
in
neurons.
these
spinal
al.,
afferents
1954,
The
1962).
period
stimulation
in
many
units
the
that
the
were
the
excitation.
due
is
of
the
An
alternate
the
venous
tonically
active
to
presynaptic inhibition of primary
course of 200 msec or more (Eccles et
1963b;
Eccles
inhibition
of
units
duration
Krnjevic, 1959; Wall,
and
produced
by
venous
afferent
had a time course of 50 msec or less; in
was less than 20 msec.
Thus, it is likely
inhibition recorded from most of the units following venous
afferent
primary
most
for
the
time
1963a,
1962,
are likely to be different from the
inhibition
However,
a
however, the neurons
the high spontaneous firing rate observed
for
has
unit;
inhibition
the
presynaptic
afferents
the
to
inhibition
the
are
neurons
stimulation
afferent
due
to
depolarization.
inhibited
stimulation,
is
for
long
postsynaptic
However,
durations
inhibition,
in
and not
the few units that
following
venous
afferent
it is possible that primary afferent depolarization also
contributed
the reduced firing rate.
to
Data recorded from one such
unit are shown in Figure 4-1.
Since
afferent
of
units
manner
the
inputs
pathways,
these
that
units
that
both
excited
and
inhibited by venous
were likely to receive these inputs through a number
the venous afferent influences on the excitabilities of
appear
following
venous
were
to
powerful.
Units that responded in this
venous afferent stimulation were common, suggesting
afferent
excitabilities
be
of
inputs,
spinal
in general, have a powerful impact on
cord neurons.
Units both excited and
67
inhibited
venous
integration
the
of
by
this
and processing of these inputs, although the purpose
integration cannot be determined in studies using electrical
stimulation.
input
afferent inputs also probably are involved with
are
In contrast, units that are only excited by an afferent
more
significantly
duration
likely
to have a role of relaying the input without
processing
after
These latter units may fire for a long
it.
receiving
afferent
the
input,
however,
thereby
prolonging its effects in the nervous system.
Stimulation
on
of
the femoral-saphenous vein had prolonged effects
excitabilities
the
anesthetized
carried
of the units examined in both decerebrate and
animals.
This
further
is
evidence
that
the inputs
by the large femoral-saphenous venous afferents have a potent
influence on the excitabilities of spinal neurons.
Convergence of Inputs on Single Neurons
Stimulation
activated
many
electrical
A-beta
of
spinal
femoral-saphenous
interneurons
stimulation
that
could
venous
also
be driven by
muscle and cutaneous afferents.
of
afferents
Previous
studies
of the properties of single spinal interneurons that received
noxious
inputs
convergence
from
of inputs from muscle and skin occurred on these units as
well
(Pomeranz
al.,
1977;
Guilbaud
al.,
1981;
Blair
Morrison,
1983;
"failed
the internal environment showed that widespread
1982;
et
al.,
1968;
et al., 1977; Foreman and Weber, 1980; Milne et
et
Rucker
al.,
and
Rucker et al., 1984).
to
detect
Hancock et al., 1973, 1975; Gokin et
1981,
1984;
Holloway,
Cervero, 1982; McMahon and
1982;
Takahashi and Yokota,
Pomeranz et al. (1968, p. 528), in fact,
cells which responded only to visceral afferents"
68
in
the
thoracic
venous
afferent
inputs
from
spinal
cord.
inputs
the
were
viscera,
Thus, spinal neurons which processed
similar
in
that
to those that process noxious
both
types of neurons received
widespread convergent inputs from muscle and skin.
Compari son of Responses in Decerebrate and Anesthetized
Animals
The
responses
stimulation
differed
preparations.
lower
of
neurons
somewhat
These
in
following
decerebrate
and
Inputs
more
likely
inputs
elicited
afferent
in anesthetized
by
venous
The onset latency
afferent
stimulation
was
shorter in anesthetized cats than in decerebrate cats.
neurons
to
venous
differences could be explained by a generalized
activity
significantly
to
mediated
through
multi-synaptic connections are
affected by a lowered state of excitability than
be
mediated through connections comprised of only a few synapses.
neurons
affected
mean
spinal
neuronal excitability in the latter animals.
the
Thus,
of
by
onset
anesthetized
activated at long latency would be expected to be more
anesthesia than neurons activated at short latency.
latency
of
elicited
activity
in
the
neurons
The
in
animals would accordingly be expected to be shorter than
in decerebrate animals.
However,
units
In
were similar in both anesthetized and decerebrate preparations.
both
cutaneous
muscle
groups
inputs
and
excitabilities
both
many of the properties of the venous afferent-activated
excited
of
animals
on
single
cutaneous
of
and
these
the
convergence
of venous, muscle and
neurons was common.
inputs
units.
have
powerful
This suggests that
influences
on
the
It was also common for units to be
inhibited following venous afferent stimulation in
69
the
two types of preparations.
As discussed above, it is likely that
these
units which responded to inputs from the femoral-saphenous vein
in
a
complex manner are involved with the integration and processing
of
the
inputs.
Thus,
it
appears
that venous afferent inputs are
highly processed in the spinal cord by interneurons.
CHAPTER V
THE VARIABILITY IN THE RESPONSES OF NEURONS LOCATED IN DIFFERENT
LAMINAE FOLLOWING STIMULATION OF THE FEMORAL-SAPHENOUS VEIN
Introduction
The
respond
of
data
presented
in Chapter IV suggested that spinal neurons
in a wide variety of ways following the electrical activation
A-beta
feraoral-saphenous
venous
were
excited
were
both excited and inhibited.
by
milliseconds
only
at
following
long
affected
vein
of
others
the femoral-saphenous vein; others
Some units were driven within a few
of
the vein; others were driven
excitabilities
milliseconds
were
Some neurons
some
vein
by
modulated
of
for
neurons were
stimulation;
over
100
the
msec.
In
most neurons driven by stimulation of the femoral-saphenous
could
afferents;
of
The
few
a
of
stimulation
latency.
only
excitabilities
addition,
stimulation
afferent fibers.
hindlimb
also
be activated by stimulation of muscle and cutaneous
however, a few units were not activated by the stimulation
nerves.
It
would be of interest to determine whether
the
neurons
the
feraoral-saphenous vein had similar locations in the spinal cord.
that
responded in similar ways following stimulation of
The study discussed in this chapter examined this possibility.
Methods and Materials
Experiments
cats
with
anesthetized
were
spinal
using
performed
cords
on
transected
alpha-chloralose.
70
unanesthetized, decerebrate
12
at
The
T12
and
responses
5
intact
cats
recorded from
71
these
animals
describe
described
stimulation
tibial
nerve
animals
for
responses
Chapter
in
chapter
this
IV;
will
locations of the units with different response patterns
the
following
in
were
of
sural
and
femoral-saphenous
the
nerve.
venous
afferents,
Surgical procedures used to prepare
recordings and the techniques used to record single unit
were described in detail in Chapter IV.
The data described
this chapter were recorded using microelectrodes filled with an 8%
solution of HRP in tris-buffered 0.15 M KC1 (pH 7.3).
After
attempt
was
neuron
a
was
made
successful,
positive-going
Current
was
delivered
been
had
HRP
iontophoretically
was
rectangular
injected
microelectrode
extracellularly
penetrate the element studied.
to
through
characterized
pulses,
through
injected
with
nA
1-3
1
Hz.
electrode for 7.5-15 min and was
the
companion
the
If the attempt
msec in duration, at
500
an
,
bridge
pre-amplifier.
This
unit (Winston BR-1) to the
bridge
unit
had
been
custom-modified
by
high
If the characterized neuron could not be impaled, HRP
was
blank
voltages.
iontophoresed extracellularly using
electrode
parallel
blank
to
above
in
place
observed
tracks
sections
from
at least
1
was
each
electrode
protruded
left
the manufacturer to make it capable of presenting
inserted
track
into
which
nA pulses for 20-30 min.
5
opposite
the
yielded
broken
the
surface of the cord.
through
in
which
the
the
using
scissors
fixation
tissue
side of the cord
Once positioned, the
data.
was
A
so
that
only
1-2 mm
These electrode tips were
process; they produced easily
which
data were recorded.
were
used
to help identify
A rostrocaudal distance of
mm separated sites of HRP iontophoresis.
72
After
animal
least
at
was
an hour following the last marking attempt, the
perfused
heparinized
saline
through
removed
into
and
stored
sections
carotid
artery
with
paraformaldehyde fixative.
overnight
in
the fixative.
2
of
1
The L6 cord was
The tissue was cut
micrometers thick using a vibratome (Oxford).
50
of
1
1
containing 0.1% sodium nitrite followed by
glutaraldehyde/1.2%
2.5%
the
The
sections
were
solution
comprised of 0.05% 3,3* diaminobenzidine tetrahydrochloride
and
incubated
hydrogen
0.1%
sections
onto
then
peroxide
for
in
min at room temperature in a
30
0.1
M
tris buffer at pH 7.6.
The
were then rinsed in 0.1 M phosphate buffer (pH 7.4), mounted
slides,
cleared
in
dried, counterstained using 1% neutral red, dehydrated,
xylene
reconstructions
and
were
coverslipped.
made
of
stained
Two-dimensional camera lucida
neurons
and
extracellular
recording sites.
Pugh
used
and
Stern
(1984) have shown that HRP can be very reliably
to label extracellular single unit recording sites.
histologically
Injection
space,
reconstructed recording sites are shown in Figure 5-1.
site
addition
to
Examples of
diameters
internalization
distinguishable
from
50
to
250 micrometers.
In
visible brown reaction product in the extracellular
the
identification
ranged
of
of
HRP
by
neurons
provided
positive
the center of the injection site which was readily
from damaged vascular or neural elements mechanically
produced by the electrode.
Results
Fifty
sites
afferent-activated
at
which
neurons
responses
were
were
recorded
reconstructed;
36
from
of
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reconstructed
other
14
recording
were
anesthetized
in
decerebrate animals and the
in
animals.
The
locations
of
the
shown
in
Figure
elicited
by
stimulation of the femoral-saphenous
are
activity
sites
5-2.
In
decerebrate cats,
was recorded from Rexed's laminae IV-VIII and X; in anesthetized
venous
cats,
laminae
V
afferent-elicited
neuronal
activity was recorded from
and VI and from the dorsal portion of lamina VII.
Most of
recording sites were located either in lamina V (11 out of 50) or
the
in
were
sites
neuronal
vein
recording
most
the
superficial
portion
lamina
of
VII,
dorsal
to
the
dorsalmost border of lamina VIII (15 out of 50).
Units
firing
rates;
units
both
excited
units
values
shown
are
in Table 5-1.
However, the
of
the
by
vein
two
types
stimulation
were
highest in
The locations of
are shown in Figure 5-3.
by stimulation of the vein.
ventral
the
afferent
units
inhibited
and
Most of the
the dorsal horn (12 out of 20 or 60%) were both excited and
in
inhibited
both
these
in different regions of spinal gray matter.
units
the
the different laminae had similar spontaneous
in
which were excited by venous afferent stimulation and the units
density
in
located
horn
stimulation
located
excited
deeper
and
(22
out
of
In contrast, most of the units
30
or
73%) responded to venous
with only a burst of action potentials.
Only
2
than the dorsalmost border of lamina VIII were
inhibited by venous afferent stimulation; clearly,
units of this latter type were rare deep in the ventral horn.
An
neurons
investigation was also done to determine the locations of the
driven
afferents.
The
at monosynaptic latencies by femoral-saphenous venous
cord
dorsum
potential elicited by venous afferent
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Table 5-1.
Spontaneous firing rates of neurons located in different
laminae.
Confidence intervals represent mean +/- standard deviation.
All spontaneous firing rates are in spikes per second.
Lamina of
Rexed
n
Mean Spontaneou
Firing Rate
IV
2
17.5 +/- 21.1
2.5 - 32.4
V
11
8.8 +/- 11.7
0.0 - 33.3
VI
7
15.6 +/- 12.9
1.1 - 37.2
VII
22
20.8 +/- 21.0
0.0 - 60.0
VIII
5
9.9 +/-
9.6
0.0 - 20.8
X
2
5.0 +/-
7.1
0.0 - 10.0
Range of Spontaneous
Firing Rates
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waves
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action
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of
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potential
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horn
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et
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directly
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to
excited
latency
at
which
by large primary afferent
at the same latency as that of the peak of
wave of the cord dorsum potential, or at shorter
assumed
receive
to
monosynaptic,
or
at
least
inputs from from primary femoral-saphenous venous
Figure 5-4 illustrates these procedures used to determine
units
received
average
latency
dorsum
potential
triphasic
wave
Thus, the latency of the first negative wave of
excited
latencies,
positive
Eccles
being
are
afferents.
The triphasic spike is a compound
activity in the largest afferent fibers
the
potential
Neurons
first
the
spike, a series of negative
of afferent fibers (Austin and McCouch, 1955;
1955;
et al., 1982).
cord
The cord dorsum potential
stimulus, the negative waves are generated by dorsal
presynaptic
Yates
triphasic
positive wave.
interneurons,
et
this latency at which units were
inputs from the vein.
slow
a
estimate
to
of
spike
the
was
in
direct
inputs from the venous afferents.
The
peak of the first negative wave of the cord
2.67
+/-
decerebrate
0.71
msec
animals
and
from
the
onset of the
2.06 +/- 0.85 msec in
anesthetized animals.
Based
on
reconstructed
primary
and
the
these criteria, 8 of the 50 units with recording sites
were
classified
venous afferents;
other
4
4
as
being
directly
excited
by
the
of these units were in anesthetized cats,
were in decerebrate cats.
The locations of these
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were
located
was
lamina
in
V,
1
was located in lamina VI and the other
located much more ventrally in lamina VII.
short
latency
had
a
and
were
the
triphasic
mean
studied
venous afferent stimulation in decerebrate animals
by
activated
in
These units driven at
spontaneous firing rate of 8.2 +/- 7.2 spikes per second
at a mean latency of 1.8 +/- 0.72 msec following
spike
of the cord dorsum potential.
decerebrate
stimulation,
and
excitabilities
The
in part "A" of Figure 5-5; most (6 out of 8 or 75%)
animals
were
excited
by
Two of the units
venous
afferent
the other two were both excited and inhibited.
The
of these units were modulated for 32.6 +/- 44.2 msec.
spontaneous
firing
rates of the units driven by venous afferent
stimulation
at
than
in decerebrate cats; the mean rate was only 0.24 +/- 0.28
those
spikes
per
silent
in
whether
that
second.
Two
of
the units in anesthetized animals were
the absence of stimulation; it was impossible to determine
these
afferent
short latency in anesthetized animals were much lower
units
were
stimulation
both
only
or
excited
excited.
and
inhibited
venous
It is only possible to show
firing rate of a unit decreases below the spontaneous level
the
following
stimulation if the unit fires spontaneously.
units
anesthetized
in
stimulation.
1.06
by
+/-
These
0.55
potential;
the
were both excited and inhibited by vein
cats
four
The other two
units
were activated at a mean latency of
msec following the triphasic spike of the cord dorsum
excitabilities
of the units were modulated by venous
afferents for a mean duration of 20.4 +/- 12.8 msec.
The
locations
stimulation
of
of
the
units
driven at longer latencies by the
the femoral-saphenous vein are shown in parts "B" to
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Figure
"E"
of
the
cord
peak
dorsum
of
units
the
Within
5-5.
msec following the triphasic spike of
3
potential, but at latencies longer than that of the
first negative wave of the cord dorsum potential, a few
located
laminae
in
and
VI
VII
were
activated
vein
by
stimulation.
At slightly longer latencies, many units located in the
ventral
were activated.
also
horn
activated
these
at
longer
neuronal
activity
over
msec
following
the
neurons
activated
at
12
cord;
However, units in the dorsal horn were
produced
latencies.
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onset latency of
by venous afferent stimulation ranged to
arrival
of the afferent volley at the
latencies longer than 12 msec following
the triphasic spike were located both in the dorsal and ventral horn.
Figure
were
5-6
modulated
neurons
were
following
superficial
short
durations
following
activated
means
<
for
lamina
of
of
than
horn
cats
located
in
the
dorsal
horn
and
The
in the
VII were modulated for both long and
However, most of the units located deeper than the
border
(less
decerebrate
msec
portion
dorsal
the
units
of
durations.
dorsalmost
stimulation of the femoral-saphenous vein;
activated from less than 10 msec to over 100 msec.
excitabilities
in
shows the durations that the excitabilities of units
lamina
VIII
50 msec).
and
were
only
activated
for short
The excitabilities of units located
superficial
portions
of
lamina
VII
of
were modulated for a mean duration of 74.8 +/- 86.1
vein
stimulation,
whereas units located deeper were
only an average duration of 26.2 +/- 44.5 msec.
These
were shown to be significantly different by Student's t-test (p
0.035).
Locations of units activated for different durations
Figure 5-6.
of the femoral-saphenous vein.
stimulation
the
following
89
•-DECEREBRATE °-ANESTHETIZED
<10msec
10-49 msec
50-100 msec
>100 msec
90
discussed
As
afferent-driven
tibial
and
from
did
units
could
muscle or skin.
of
venous
the
Table 5-2 shows the locations of the
neurons
that also received convergent inputs
In anesthetized animals most of the units which
not receive convergent inputs from muscle or skin were located in
the
region
latency
by
V,
shortest
which
2
driven
stimulation
by
monosynaptic
unit
cord
dorsum
that
were
was
msec
(3.6
activated
lamina
nerve
latencies
other
driven
four
by
stimulation
driven
the
nerve
sural
were
activated
at
monosynaptic
stimulation
by
vein.
following
latencies
The
other
the
triphasic
of
tibial
the
by
neither
tibial
stimulation
sural
only
of
were
the
two units were activated at 3.6
spike
of
the
cord dorsum
be activated by stimulation of the sural
nerve;
this
unit
was driven at monosynaptic
by stimulation of the femoral-saphenous vein.
units
nerve
one unit studied in decerebrate animals, located in
could
the
stimulation at a fairly short
vein
Two of the 4 units in anesthetized animals
at
Only
by
following the onset of the triphasic spike of the
driven
VII,
or
of
not
msec
potential.
vein.
units in anesthetized animals that were not
potential).
femoral-saphenous
3.8
3
femoral-saphenous
the
of
latencies by stimulation of the femoral-saphenous vein.
other
latency
the
of
contains most of the neurons driven at
stimulation
Accordingly,
and
most
also be activated by stimulation of the
of hindlimb nerves.
afferent-driven
lamina
The
chapter,
last
the
sural nerves; however, a few units were not driven by the
stimulation
venous
in
characterized
in
decerebrate
However, the
cats that were not
nerve stimulation were activated by venous afferent
at
long
latency (11.5 +/- 1.9 msec following the
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92
onset
of
of the triphasic spike of the cord dorsum potential).
the
8
primary
units
classified
femoral-saphenous
receiving
as
venous
monosynaptic
afferents
could
Five out
input from
not be driven by
stimulation of the tibial nerve, the sural nerve or both.
Four
through
of
of
the
two-dimensional
stained.
The
body
other
afferent-activated
neurons
the intracellular iontophoresis of HRP.
locations
cell
venous
the
border
between
Figure 5-7 shows the
of
these
reconstructions
of
the neuronal elements which were
bodies
located
body
stained
bodies
cell
was
cell
cell
were
cell
body
than
25
were
reconstructed;
well
as the
neurons were located in lamina V,
1
the dorsal portion of lamina VII and the
in
laminae
had
2
as
located in the lateral white matter beside the
was
they
of
neurons
and
VI
These cell bodies were large;
VII.
an average diameter of 37.1 +/- 18.2 micrometers.
Only the
located in the lateral white matter had a diameter of less
micrometers.
dendritic
The
these
trees of three of the neurons
dendritic
trees
were
large
and highly
branched.
They
micrometers
and a mean mediolateral spread of 492 +/- 83 micrometers.
had
mean
a
rostrocaudal
spread
of
963 +/- 809
The dendrites also spanned several laminae.
Discussion
This
study
made use of the marking of extracellular single unit
recording
sites
to determine the locations of neurons with different
response
properties
femoral-saphenous
are
generally
bodies
of
the
following
vein.
assumed
units
The
to
the
stimulation
of
the
sites of such extracellular recordings
correspond
characterized
to
the locations of the cell
(Willis and Coggeshall, 1978).
afferent-activated
venous
of
Reconstructions
5-7.
Figure
using HRP. (A) A
stained
intracellularly
were
interneurons that
(B) The
neuron.
stained
a
of
body
cell
the
photomicrograph of
(C)
neurons.
stained
the
of
bodies
cell
the
of
locations
the
of
trees
dendritic
and
bodies
cell
Reconstructions of the
otherwise
Unless
correspond.
(C)
and
The numbers in (B)
neurons.
"1" was
marked, all neuronal processes shown are dendrites. Neuron
reconstructed from somewhat oblique sagittal sections, explaining
The other neurons
transitions in lamination.
the unconventional
were reconstructed from cross sections.
94
c®
'AURA!
WHtTI
MATTit
(J)
©
95
Since
cell bodies are by far the largest neuronal elements from which
action
potentials
extracellular
the
keep
this
However,
region
recorded, it is probable that most stable
will
stable
be
done
in
the
fact
vicinity
of these
recordings from axons at distances far
cell body are sometimes also achieved.
neurons
of
be
recordings
structures.
from
can
It is important to
in mind when drawing conclusions about the locations
based
on
extracellular marking of recording sites.
The
which contains most of the extracellularly characterized units
is
likely
If
a
to also contain most of the cell bodies of these neurons.
recording site is reconstructed outside of this area containing
most
of
conclude
this
other recording sites, it is not prudent to immediately
the
that
the cell body of a neuron of interest is also found at
location.
It is possible that such recording sites which do not
the pattern are locations from which responses were recorded from
fit
axons
Sites
which
at
afferent-activated
lamina
V
cats.
It
of
and
responses
units
were
recorded
in deeper laminae in both decerebrate and anesthetized
is likely that these regions also contain many cell bodies
neurons
receiving
inputs
from
femoral-saphenous
the
no
VIII
or
in the deep portions of lamina VII.
IV,
the
driven
units were found in anesthetized cats
excitabilities
anesthetized
using
decereb rate-spinal
input
venous
were reconstructed predominantly in Rexed's
However,
an
from
in
of
spinal
alpha-chloralose
preparations.
decerebrate-spinal
Thus,
vein.
in lamina
As discussed in Chapter
neurons
are
in
intact
lower
animals
than
in
if neurons are activated by
preparations,
but not anesthetized
96
preparations,
it is likely that the input has only weak influences on
excitabilities
the
neurons
located
these
of
deep
neurons.
the
in
This evidence suggests that
ventral horn receive only weak inputs
from
feraoral-saphenous venous afferents.
very
few
of
VIII.
were
Evidence
which
respond
stimulated
both
provided
found deeper than the dorsalmost border of lamina
presented in Chapter IV suggested that these neurons
in
a
afferents.
excited
region
complex manner receive powerful inputs from the
The observation that very few units which were
inhibited
and
further
support
were
for
found
the
deep
conclusion
in
the ventral horn
that neurons in this
receive only weak inputs from the femoral-saphenous vein.
validity
located
deep
the
in
for
superficially.
that
neurons
VII
ventral
significantly
more
lamina
The
this conclusion is also bolstered by the fact that units
of
stimulation
In
noteworthy that
neurons that were both excited and inhibited by stimulation
vein
the
It is also
horn
were
shorter
driven by venous afferent
durations than units located
The evidence presented in this chapter suggests
located
laminae
in
V,
VI
and the dorsal portion of
receive powerful inputs from the femoral-saphenous vein.
addition,
neurons
located
deeper
in
regions appear to receive
weaker inputs from these afferents.
A
16%)
fairly large percentage of the neurons studied (8 out of 50 or
were
shorter
than
potential.
inputs
activated
from
suggested
that
These
the
that
venous
by
of
the
neurons
afferent
first
were
negative
suggested
venous afferents.
only
a
very
stimulation
at latencies
wave of the cord dorsum
to
receive monosynaptic
The data presented in Chapter II
few
afferents
project
from
the
97
femoral-saphenous
vein
studies
presented
chapter
to
portions
be
of
register,
primary
the
branch
extensively
second
order neurons.
visceral
that
branch
respect,
the
anatomical
would
seem
that
venous
the intraspinal
afferents
must
and make synaptic contacts with a large number of
In contrast, anatomical studies have suggested
great
venous
For
and the studies presented in this
II
it
cord.
femoral-saphenous
afferents
this
to
Chapter
in
in
spinal
the
to
which enter the thoracic spinal cord do not
extent
(Cervero and Connell, 1984).
In this
afferents appear different from other afferents from
the internal environment.
Most
very
of
short
units
the
latencies
predominantly
activated by venous afferent stimulation at
were
lamina
in
latency,
however,
possible
that
were
located
in the neck of the dorsal horn,
Responses from one unit driven at short
V.
recorded from deep within lamina VII.
It is
latter responses were recorded from the axon of
these
an
interneuron
In
any case, these data suggest that most of the interneurons excited
arising from a cell body located in the dorsal horn.
monosynaptically
located
by
large
lamina
in
The
V.
discussed
in
Chapter
activated
at
shortest
femoral-saphenous
III
field
also
latency
potential
suggested
by
venous
the
afferents
mapping
are
experiments
that most of the neurons
large
venous afferents are
located in this region.
Units
located in the dorsal portion of lamina VII were activated
by
venous afferent stimulation at latencies slightly longer than that
of
the first negative wave of the cord dorsum potential.
lamina
VII
neurons
which
were
Many of the
driven only slightly later than the
98
neurons
are
concluded
likely
receive direct or relatively direct inputs from these
to
monosynaptically
these
to receive direct inputs from the venous afferents
lamina
driven
VII
femoral-saphenous
presented
between
venous
is also possible that some of
It
premotor
are
interneurons
afferent-elicited
suggested
III
that
spinal
for
reflex.
the
Data
shortest connection
the
primary venous afferents and alpha-motoneurons is di- or tri-
synaptic.
Studies
intracellular
using
staining
lamina
any,
if
neurons
Chapter
in
neurons.
both
Golgi
the
technique
and
the
neurons with HRP have suggested that few,
of
V neurons send their axons into lamina IX.
However,
many
of these neurons do project to superficial lamina VII.
In turn,
many
neurons in the dorsal portion of lamina VII project to lamina IX
(Matsushita,
suggest
1969,
that
motoneurons
Czarkowska
1970;
shortest
the
al.,
et
These
1976).
data
pathway from primary venous afferents to
may be comprised of a lamina V neuron which projects to a
lamina VII neuron that, in turn, projects to motoneurons.
alternate
An
excited
with
monosynaptically
the
However,
a
1984).
slower
is
primary
dendrites
of
EPSPs
elicited
in
very
are
fast
rise
EPSPs
large,
elicited
motoneurons
typically
by
contacts
4
laminae
in
motoneurons
typically
time,
venous afferents make contacts
by
V or VII.
venous
that
synaptic
the
venous
to 5 msec (Thompson and Yates,
on
distal dendrites have much
afferent-elicited
interactions
of
afferent
to 2 mV in amplitude, and
1
rise times and much smaller amplitudes (Rail, 1970).
likely
through
by
distal
stimulation
have
possibility is that the lamina V neurons which are
lamina
VII
proximal dendrites or cell bodies of motoneurons.
reflex
is
Thus, it
produced
interneurons
with
99
also
is
It
units
were
local
circuits.
likely
tract
that
cells,
in
many of the venous afferent-activated
addition to neurons that form parts of
It has previously been shown that stimulation of the
femoral-saphenous
vein
elicits
field potentials recordable from the
sensorimotor
cerebral
tract
that relay information from the femoral-saphenous venous
cells
afferents
probably
spinothalamic
found
in
send
neurons
Corvaja
et
al.,
projecting
tracts
axons
et al., 1980).
into
the
Most of the
spinoreticular and
The neurons giving rise to these tracts are
ventral
the
(Thompson
their
pathways.
afferent-driven
1973;
cortex
horn,
which
also
contains
many
venous
(Trevino and Carstens, 1975; Trevino et al.,
1977; Fields et al., 1977).
The other major
to the brain from the lumbosacral gray matter, the
postsynaptic
dorsal column and the spinocervical pathways, arise from
cell
mainly
bodies
Craig,
1973;
in
laminae
Brown
1976;
III
and IV (Petit, 1972; Rustioni,
et al., 1976).
These regions contain few
venous afferent-activated units.
Most
of
the neurons (5 out of 8 or 63%) that were classified as
receiving
monosynaptic
afferents
could
nerve,
of
not
be
driven
by
stimulation of the tibial
In contrast, only 6 out of 36 (17%)
units activated at longer latencies by vein stimulation could
also
inputs
also
from primary femoral-saphenous venous
sural nerve or both.
the
the
thus
not
inputs
be driven by stimulation of one of the hindlimb nerves.
It
appears that, in general, neurons receiving the shortest latency
from
convergent
the
inputs
femoral-saphenous
from
vein
are less likely to receive
muscle or skin than are neurons activated by
venous afferents at longer latency.
100
The
intracellularly
interneurons
A
previous
with
that
received
cell
bodies
only
innocuous
but
only
neurons
along
and
using
inputs
only
inputs
is
laminae
staining showed that neurons
V
and VII are wide dynamic
Neurons
inputs (along fine afferents) had small
dendritic spreads; neurons that received
(along large afferents) had large cell bodies
dendritic
afferents
It
afferent-activated
from both large and fine afferents).
noxious
characterized
large
in
extensive
but
small
skin.
intracellular
characteristics
(receive
venous
large cell bodies and extensive dendritic spreads.
study
these
range
had
stained
in
spreads
this
(Ritz and Greenspan, 1985).
study
were
The
shown to receive inputs
arising in the femoral-saphenous vein, muscle
likely
along fine afferents as well.
that many of these neurons receive inputs
CHAPTER VI
GENERAL DISCUSSION
previous
The
chapters have described several characteristics of
the
afferents which conduct inputs from the femoral-saphenous vein to
the
spinal
cord
and the neurons within the cord which process these
inputs.
Many
visceral
afferents.
venous
these
of
afferents
characteristics
However,
appear
some
in
different
are
similar
respects
from
other
to those for
femoral-saphenous
afferents
from the
internal environment.
Several
smaller
have
found
(1984)
the
previous
diameters than somatic afferents.
mean
Cervero et al.
that the application of HRP to visceral nerves entering
thoracic cord labeled a significantly smaller percentage of large
cell
bodies
than
did
sizes
of
in
Similarly,
potentials
nerve)
were
presented
ganglia
had
et
Li
A-alpha/beta
the
action
recorded
dorsal root ganglia (diameter > 40 micrometers)
the
application of HRP to somatic nerves entering the same
the
segments.
vein
studies have suggested that visceral afferents
from
smaller
than
the
saphenous
from
A-delta
and
recorded
(1977) reported that the relative
al.
the
splanchnic
the
relative
nerve
components of the compound
(a
sizes
nerve (a visceral
of these components
cutaneous nerve).
The data
in Chapter II suggested that cell bodies in the dorsal root
labeled
by
the
application of HRP to the femoral-saphenous
significantly smaller mean diameters than other cell bodies
101
102
located
like
in
visceral
comprised
ganglia.
the
Thus, femoral-saphenous venous afferents,
afferents
fibers
of
entering
that
are
thoracic
the
finer,
spinal
cord,
are
on the average, than somatic
afferents.
number
The
cord
has
been
microscopic
the
reported
cord
afferents.
segments;
In fact, wide field electron
300
or
average
of
182
cell
of
cat, the splanchnic nerve, contains only
enters the spinal cord over 12 or more
average
the
is
both
be small.
nerve
This
thus,
segment
the
of
segment
per
to
afferents entering the thoracic spinal
analysis has shown that the major visceral nerve entering
thoracic
3500
visceral
of
less
number
(Kuo
et
of
al.,
afferents entering any one
Similarly, only an
1982).
bodies were labeled in the dorsal root ganglia
the femoral-saphenous vein exposed to HRP.
femoral-saphenous
venous
afferents
and
visceral
Clearly,
afferents
entering the thoracic spinal cord are few in number.
Although
afferents
these
over
drive
are
few in number, stimulation of
produces widespread effects in the spinal cord.
stimulation
example,
afferents
visceral
half
of
of
the
splanchnic
For
nerve has been reported to
dorsal horn neurons in the thoracic spinal cord
(Pomeranz
et al., 1968; Cervero, 1982, 1983a, 1983b).
only
A-beta population of the femoral-saphenous venous afferents
was
the
suggested
spinal
cord
recordable
in
and
from
Stimulation of
Chapter IV to activate many interneurons in the L6
in
this
Chapter
segment.
III
to produce large field potentials
The
central gain for both visceral
afferents and femoral-saphenous venous afferents is very high.
103
high
A
through
two
portions
of
central
means:
that,
higher-order
cells.
gain
extensive
the
or
turn,
in
branching
of
the
intraspinal
the activation of a few second order
make
Several
contacts
studies
afferent
visceral
for
possibility.
for an afferent input can be accomplished
afferents
the
interneurons
central
gain
have
inputs
with a large number of
suggested that the high
is
due
to the latter
one of these studies, the application of HRP to the
In
cut
end of the splanchnic nerve or to a somatic nerve of similar size
was
used
The
labeling
of
that
and
transganglionically visualize the central projections.
to
the cord was much fainter following the application
in
HRP to the visceral nerve; the authors proposed that this suggests
Connell,
Selzer and Spencer (1969) also showed that the
1984).
negative wave of the cord dorsum potential elicited
largest-amplitude
by
have a limited intracord branching (Cervero
afferents
visceral
sympathetic chain stimulation occurred at long latency (20 msec or
stimulation).
more
following
the
were
small
amplitude.
the
activity
neurons
of
are
femoral-saphenous
afferents.
afferents
by
the
neurons,
primary
these data suggest that few
sympathetic afferents at short
In contrast, the data discussed in Chapter V suggested that
neurons
receiving
Since the negative waves are generated by
horn
dorsal
driven
are
latency.
many
in
The short latency negative waves
driven
at
short
latency
by
stimulation of the
vein; 16% of the neurons studied were classified as
monosynaptic
inputs
from
large
femoral-saphenous venous
Although inputs carried along both visceral afferents and
arising
in
the femoral-saphenous vein have a high central
gain, this gain appears to be accomplished through different means in
the two cases.
104
A
number
studies utilizing the transganglionic transport of
of
HRP
have suggested that visceral afferents entering both the thoracic
and
sacral
spinal
Cervero
1983,
1984;
field
potential
recordings
in
laminae
in
I
and V (Kuo et al.,
and Connell, 1984; Kuo and deGroat, 1985).
mappings
presented
in
Chapter
The
III as well as the
suggested
V
that most of the first neurons activated by
large femoral-saphenous venous afferents are located in lamina V.
Thus,
it
this
likely
is
region;
that many primary venous afferents terminate in
laminae containing terminals of visceral afferents
the
femoral-saphenous
overlap.
It
is
terminals
of
the
project
be
terminate
of single unit activity from reconstructed sites discussed
Chapter
the
and
cord
not
afferents
afferents appear to at least partially
known,
however,
venous
fine
to lamina I.
possible
venous
which
afferents.
laminae
contain
the
It is possible that they
Until this information is obtained, it will not
conclude whether the laminae containing terminals of
to
from
viscera
the
and
femoral-saphenous
the
vein
are
identical.
It
converge
al.,
and
well established that inputs from viscera, muscle and skin
is
single spinal neurons (Pomeranz et al., 1968; Hancock et
on
1973,
1975;
Gokin et al., 1977; Guilbaud et al., 1977; Foreman
Weber,
1980;
Milne
Cervero,
Takahashi
Chapter
et
al.,
1981;
Blair
et al., 1981, 1984;
1982; McMahon and Morrison, 1982; Rucker and Holloway, 1982;
Yokota,
and
IV,
it
is
1983;
also
cutaneous
and
Clearly,
femoral-saphenous
venous
Rucker et al., 1984).
common
afferents
for
to
venous
inputs
converge
As discussed in
carried along muscle,
on
afferents,
single
and
neurons.
the
spinal
105
interneurons
much
s
in
common
timulation
similarities
from
the
identical.
that
of
in
process inputs carried along these afferents, have
with
these
visceral
latter
afferents, and the neurons driven by
afferents.
There appear to be marked
how the central nervous system processes many inputs
internal
environment.
Yet,
the
mechanisms
are
not
REFERENCES
Alvord,
E.C.,
and Fuortes, G.F., A comparison of generalized reflex
myoclonic reactions excitable in cats under chloralose anesthesia and
under strychnine, Am. J. Physiol., 176 (1954) 253-261.
Ammons, W.S.,
Blair, R.W.,
and Foreman,
R.D., Greater splanchnic
excitation of primate T1-T5 spinothalamic neurons, J. Neurophysiol.
51 (1984) 592-603.
Austin,
G.
M.
and McCouch,
G.
P.,
Presynaptic component
intermediary cord potential, J. Neurophysiol., 18 (1955) 441-451.
,
of
Balis, G.U.,
and Monroe, R.R.
The pharmacology of chloralose: a
review, Psychopharmacologia, 6 (1964) 1-30.
,
Barja, F., and Mathison, R. , Sensory innervation of the rat portal
vein and the hepatic artery, J.
Auton.
Nerv.
Syst.,
10 (1984)
117-125.
Beall,
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BIOGRAPHICAL SKETCH
Billy
Florida.
nearby
an
Yates
J.
grew
He
Lakeland.
interest
University
undergraduate
laboratory
convinced
and
Mathias.
working
same
in
the
studies,
he
came
year
Thompson
to
the
mammalian
postdoctoral
During his early
work, quite by accident, in the
to
His experiences in this laboratory
in
medicine.
nervous
system
He
also developed an
while
Floyd Thompson in January of 1981.
earned a B.S. degree.
the
in
supervise
research,
he
Department
working with Dr.
cord.
with
of
a
In December of
He immediately entered the
Neuroscience and chose Dr.
dissertation research.
his
developed
spinal
career
the
gain further insight in this field, he began
to
he
program
career
a
order
Dr.
at
career in science would be much more challenging
a
autonomic
the
with
Plant City,
studies
fall of that year.
John Mathias.
than
In
in
1960,
23,
premedical
began
and
Florida
doctoral
this
medicine,
him that
in
October
and attended primary and secondary schools in
up
Dr.
rewarding
interest
the
of
born
He graduated from Lakeland High School in 1978 with
in
of
was
While performing
keen interest in the organization of
He will pursue this interest during his
Dr.
Victor
University in New York, New York.
116
Wilson
at
the
Rockefeller
I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Associate Professor of Neuroscience
1
certify that
I
have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Rl
B.
Munson
rofessor of Neuroscience
I
certify that
I
have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
^kM a
2
Paul W. Davenport
Assistant Professor of Veterinary
Medicine
This dissertation was submitted to the Graduate Faculty of the
College of Medicine and to the Graduate School and was accepted
as partial fulfillment of the requirements for the degree of
Doctor of Philosophy.
August, 1986
^l5ean,
College of Medicine
/w^
mA^t^^ <^^_<>/^oct>t^»
1
Dean, Graduate School
UNIVERSITY OF FLORIDA
3 1262
08557 0058