- Email: [email protected]
Morphological and electrophysiological properties of ACCx nociceptive neurons in rats
BRAIN RESEARCH ELSEVIER
Brain Research 735 (1996) 83-92
Research report
Morphological and electrophysiological properties of ACCx nociceptive neurons in rats H. Yamamura
a, K . I w a t a b,c,, , Y . T s u b o i
b, K . T o d a d, K . K i t a j i m a c, N . S h i m i z u
J. H i b i y a b, S. F u j i t a b, R . S u m i n o
e, H . N o m u r a
f,
b,c
a Department of Anesthesiology, School of Medicine, Nihon UniL,ersity, 30-10hyaguchi Kamimachi, Itabashi-ku, Tokyo 173, Japan b Department of Physiology, School of Dentistry, Nihon University, Kanda-surugadai, Chiyoda-ku, Tokyo 101, Japan Department of Pathophysiology, School of DentistD,, Nihon Universi~, Kanda-surugadai, Chiyoda-ku, Tokyo 101, Japan d Department of Physiology, Faculty of Dentistry, Tokyo Medical and Dental University, 5-45 Yushima 1-Chome, Bunkyo-ku, Tokyo 113, Japan e Department of Removable partial denture prosthodontics, School ofDentisto', Nihon University, Kanda-surugadai, Chiyoda-ku, Tokyo 101, Japan r Department of Anesthesiology, School ofDentistD', Nihon UniversiO', Kanda-surugadai, Chiyoda-ku, Tokyo 101, Japan Accepted 7 May 1996
Abstract
A total of 33 neurons with cutaneous receptive fields were recorded from the anterior cingulate cortex (ACCx) and successfully injected with neurobiotin. All neurons were in area 24 of the ACCx. Neurons from the ACCx had large receptive fields (RFs), usually bilateral, and some had RFs covering the whole body surface. Most of the neurons were in the deep laminae and had a pyramidal soma with thick apical dendrites and many spines. Thirteen of 33 neurons were classified as pyramidal nociceptive specific (NS) neurons and 12 as noxious-tap neurons, 3 neurons received inhibitory input and were in lamina V. Two non-pyramidal noxious-tap neurons were located in lamina V and 1 pyramidal noxious-tap neuron was located in lamina VI, and 2 pyramidal NS neurons were in lamina III. Axon collaterals of NS neurons were mainly distributed around the soma, whereas those of noxious-tap neurons were also distributed far from the soma. A large number of varicosities were observed on the axon collaterals of both NS and noxious-tap neurons. Our results suggest that NS neurons in the ACCx send information locally to the vicinity of the soma, while noxious-tap neurons send information to a wider area of the ACCx. Keywords: Cingulate cortex; Nociceptive specific neuron; Morphology; Intracellular recording; Neurobiotin
1. I n t r o d u c t i o n
The anterior cingulate cortex (ACCx) is known to be involved in processing aspect of nociception including autonomic regulation, motivation and effect [2,10,14,16,19,21,22]. Vogt et al. [25] have reported that the A C C x is also involved in emotion, maternal behavior, visceromotor control, skeletomotor function and attention to action. Such a large number of functions of A C C x neurons is thought to be modulated by cortico-cortical or subcortico-cingulate neuronal circuits. Previous electrophysiological studies have reported that most neurons in the A C C x respond exclusively to noxious mechanical a n d / o r thermal stimulation of the body and that many also respond to tapping the receptive field [19]. Most of the
* Corresponding author. Fax: + 81 (3) 3219-8341.
nociceptive neurons in the A C C x respond to a variety of noxious stimuli applied to the whole body and many have large receptive fields [19]. Nociceptive A C C x neuron responses were eliminated by lesion of the medial thalamic nuclei [19]. Anatomical tracing studies have shown that medial thalamic neurons have axons projecting to the anterior cingulate cortex [6,18]. These suggest that ACCx neurons receive noxious input from peripheral structures via medial thalamic nuclei [4-6,18]. Furthermore, many clinical reports have shown that cingulumotomy alleviates a patient's affective responses to noxious stimuli [1,3,9]. These results support the idea that the anterior cingulate cortex has an important role in the processing of the affective aspect of nociception. No studies have been done to examine the relationship between the morphological and electrophysiological properties of nociceptive neurons in the ACCx. In the present study intracellular recording and staining methods were used to examine this question.
0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PH S 0 0 0 6 - 8 9 9 3 ( 9 6 ) 0 0 5 6 1 - 6
84
H. Yamamura et al. / Brain Research 735 (1996) 83-92
Table 1 Incidence of nociceptive neurons recorded from ACCx Lamina NS Noxious-tap Inhibitory
2. Materials and methods 2.1. Animal preparation
Experiments were performed on 29 Wistar rats weighing 2 5 0 - 3 5 0 g. The animals were initially anesthetized with thiamylal sodium (50 mg • kg 1, i.p.). During surgery, anesthesia was maintained with halothane ( 2 - 3 % ) . The animals were mounted in a stereotaxic frame, and the left hemisphere of the parietal cortex was exposed. The stereotaxic frame was then removed and the animals' heads were rigidly secured to a frame by means of stainless steel screws attached to the skull and embedded in dental acrylic cement. During recording sessions, the rats were immobilized with pancuronium bromide (1 m g . kg 1. h -~ , i.v.) and ventilated artificially. The anesthesia was maintained at the same depth throughout the experiment by continuous inhalation of halothane ( 1 - 2 % ) and air. Rectal temperature was maintained at 37-39°C by a thermostatically controlled heating pad. The electrocardiogram was monitored continuously.
Ill V VI Total:
pyramidal pyramidal non-pyramidal pyramidal
2 13 2
12
2 1
1 33
NS: nociceptive specific neurons. Noxious-tap: neurons responded to noxious mechanical and tap stimuli, Inhibitory: neurons received inhibitory inputs.
to noxious mechanical stimulation (pinching a n d / o r squeezing) of the skin. Inhibitory neurons decreased their firing frequency following noxious mechanical stimulation of the receptive fields. Neuronal responses were fed to a tape recorder (bandwidth DC to 20 kHz) for subsequent analysis. Intracellular injection was carried out through the neurobiotin-filled electrodes using a depolarizing pulse ( 3 - 5 nA, 300 ms duration, 2 c y c l e / s ) .
2.2. Stimulation ad recording
2.3. Histology
Glass micropipette electrodes were filled with 5% neurobiotin (Vector Lab.) solution with 0.5 M KC1 in phosphate-buffered saline (PBS; pH 8.0). The glass pipette electrodes were advanced through the cortex in 1 Ixm steps, After A C C x neurons had been impaled, the whole body surface was examined carefully for mechanical stimulation-evoked excitation and the neuronal receptive fields were determined. In the present study, mechanical stimulation was introduced for classification of A C C x neurons [7]. Low threshold mechanoreceptive (LTM) neurons responded to light touch or pressure of the skin. Noxious-tap neurons responded to both noxious (pinching a n d / o r squeezing) and innocuous tap stimulation of the skin. Nociceptive specific (NS) neurons responded exclusively
The animals were allowed to survive for 6 - 1 0 h following intracellular injection, and were then deeply anesthetized with pentobarbital sodium and perfused transcardially with 50 ml PBS (pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate buffer. The brain was removed and placed in cold fixative for 4 days, then transferred to cold phosphate-buffered 30% sucrose for 48 h. Serial sections 50 Ixm thick were cut along the path of the electrode penetration. The sections were incubated in peroxidase-conjugated avidin-biotin complex (1 : 100; ABC: Vector Labs.). W e used 3,3'-diaminobenzidinetetra HC1 (DAB; Sigma), nickel-ammonium sulfate and 0.001% hydrogen peroxide in Tris-buffered saline (0.05 M, pH 7.4) to develop the A B C reaction product, a distinctive black
+4.2
+3.6
+3.2
ee%
1ram
L.._~ L
Fig. 1. Schematic drawings of the intracortical distribution of neurons in the ACCx. Sections are arranged rostro-caudally according to the brain maps by Swanson [20]. The number on each section indicates distance from bregma (mm). D: dorsal, L: lateral in this and following figures. Solid circles: nociceptive specific neurons, open circles: noxious-tap neurons, solid triangles: neurons receiving inhibitory input.
H. Yamamura et a l . / Brain Research 735 (1996) 83-92
85
Table 2 Morphological properties of nociceptive neurons in the ACCx (values in mean -- S.E.) Lamina
III V VI
NS
pyramidal pyramidal non-pyramidal pyramidal
Noxious-tap
Area of soma (p~m2)
Number of basal dendrites
Diameter of apical dendrite (Ixm)
138.3 140.8 + 13.6 84.8
2.5 4.6 + 0.5 2
2.0 2.9 + 0.4 2.1
Inhibitory
Area of soma (p~m2)
Number of basal dendrites
Diameter of apical dendrite (p,m)
Area of soma (ixm 2)
Number of basal dendrites
Diameter of apical dendrite (~m)
147.9+ 12.4
5.0 ___0.6
2.6 + 0.3
150.8 33.7
4.5 2
2.1 1.0
97.3
4
0.9
NS: nociceptive specific neurons. Noxious-tap: neurons responded to noxious mechanical and tap stimuli. Inhibitory: neurons received inhibitory inputs.
chromogen. Thionin was used as a counterstain. Precise camera lucida tracings of the stained neurons were drawn at × 400 magnification with a camera lucida drawing tube. A C C x neurons were drawn at × 1000 magnification and the soma size, diameter of apical dendrites, number of basal dendrites and spines were measured. The diameters of apical dendrites were measured at a point 50 Ixm from the soma. The number of spines of the lamina V pyramidal nociceptive neurons was counted over 50 Ixm length of the
apical dendrites from the 50 Ixm distal to the soma, border between laminae III and V, and border between laminae I and II.
2.4. Statistical analysis Statistical analysis was performed by analysis of variance followed by post-hoc Fisher's PLSD or Scheffe's tests. Differences were considered significant at a P value of less than 0.05.
C
A 1 I'11 [
[ It
~,ii i~ i'J
squeezing
l
~111~1
I~,
rl~l,,
,ll,lrqlll
F
iii1~1~!,-i1,--i
squeezing
q,
i
_.3i
I,,,,
l :!'iJlln] 1._
1_ f
squeezing
5
squeezing
D'~..._
squeezing
squeezing
25 mV squeezing
100 i~m
1 sec
#6T1.2443 type: NS
Fig. 2. Sample recordings and reconstruction of a NS neuron with a large receptive field covering the whole body surface. A: intracellular recordings and receptive field. B: camera lucida drawing. Solid triangle indicates the main axon in this and following figures. C: low-magnification drawing of the section where the soma of this NS neuron was located.
86
H. Yamamura et a l . / Brain Research 735 (1996) 83-92
3. Results
distribution of ACCx neurons is illustrated schematically in Fig. 1. As shown in Fig. 1, NS, noxious-tap and inhibitory neurons were intermingled. We did not find any somatotopic arrangement of any type of nociceptive neuron in the ACCx. The morphological properties of NS neurons are summarized in Table 2. Fig. 2 shows the responses and morphology of a NS neuron with a large receptive field which covered the whole body surface. This neuron responded exclusively to squeezing (Fig. 2A). This neuron responded to squeezing of the whole body surface but the right side (contralateral to recording site) of the receptive field was more sensitive. Reconstruction of this NS neuron is illustrated in Fig. 2B. As shown in Fig. 2B and Fig. 3B, NS neurons had apical dendrites with a large number of spines. The main axon (indicated by the solid triangle) ran deep into the subcortical white matter and had many collaterals. Low magnification drawing of this NS neuron is illustrated in Fig. 2C. Most of the nociceptive neurons we recorded from the ACCx sent their apical dendrites to the surface of the medial wall of the ACCx. Fig. 3 shows the responses and morphology of a NS neuron with a receptive field covering the upper half of the body. This NS neuron did not respond to tapping or pressing the receptive field (Fig. 3A), but responded to
A total of 33 neurons with cutaneous receptive fields were intracellularly recorded and successfully injected from the anterior cingulate cortex (ACCx) (Table 1). All ACCx neurons in this study were located in area 24 according to the cytoarchitectonic criteria of Vogt and Peters [24]. Eighteen of 33 neurons in the ACCx were classified as nociceptive specific (NS) neurons according to their responses to mechanical stimulation of the receptive fields. NS neurons responded exclusively to noxious mechanical stimulation (pinching or squeezing) of the receptive fields but not to non-noxious mechanical stimulation such as gentle touching or brushing. Twelve ACCx neurons were classified as noxious-tap neurons which responded to nonnoxious tapping of the receptive fields as well as to noxious mechanical stimulation [7]. These ACCx neurons showed decrement of spontaneous firing frequency following noxious mechanical stimulation of the receptive fields. Most of the nociceptive neurons in the ACCx (30 of 33 nociceptive neurons) had pyramidal soma with many spines on their apical dendrites. Only two neurons were nonpyramidal neurons, which had oval soma without spines on their apical dendrites. As shown in Table 1, most neurons (30 of 33) were distributed in lamina V. The intracortical
A
-~. . . . . . . . 4~a=.,a NIl,I | l b Ju. . . . . .
--[,-.- , ~ , - qnVl-flp-,r-r,T ........ tapping
pressing squeezing
. . . . . . . . . . . ~'.
e
.i. w~
-,
,~rl
IIIII
..=]v.,r-i
I I! ,.~. . . . . .g~
pinching
m V
squeezing
1 sec 1 O0 pm
#32T1-2600
type: N5 (very high threshold)
. ~ /
Fig. 3. Sample recordings of a NS neuron with a receptive field on the upper half of the body surface. A: intracellular recordings. Note that only the squeezing stimulus produced a clear response. B: camera lucida drawing. C: low-magnification drawing of the section where the soma of this NS neuron was located. 1. l I - I I I , V and VI indicate laminae of the ACCx in this and following figures.
H. Yamamura et a l . / Brain Research 735 (1996) 83-92
noxious pinching and squeezing. All NS neurons recorded in the present study had morphological properties like those shown in Fig. 2B and Fig. 3B. The area of the receptive fields of each NS neuron is calculated as a percentage of the whole body surface. The receptive fields of NS and noxious-tap neurons were classified to 4 types as shown in Fig. 4 (A: receptive fields covering bilateral fore- and hind-limbs continuously; B: those covering bilateral upper a n d / o r lower half of the body; C: those covering unilateral fore- and hind-limbs; D: those covering limbs, tail or face). NS and noxious-tap neurons had similar receptive field properties. Noxious-tap neurons responded to both non-noxious tapping and noxious pinching a n d / o r squeezing the receptive fields but did not increase their firing frequency as mechanical stimulus intensity increased. Figs. 5 and 6 illustrate the responses and morphology of two noxious-tap neurons, one with a large receptive field (Fig. 5A) and one with a relatively small receptive field (Fig. 6A). In Fig. 5, tapping, pressing and pinching of the right hind-limb produced phasic responses and squeezing this region, and tail, the left hind-limb, right and left fore-paw produced high
ll A
E
fl
B
F
C
D
G
H
%
C
e~
Fig. 4. Schematic illustrations of receptive fields of NS ( A - D ) and noxious-tap (E-H) neurons.
87
frequency responses (Fig. 5A). The noxious-tap neuron in Fig. 6 also responded to tapping, pressing, pinching and squeezing, but this neuron did not show phasic responses. The morphological properties of noxious-tap neurons are summarized in Table 2. These neurons had a large number of spines on their basal and apical dendrites and many axon collaterals which spread widely into laminae II to VI (Fig. 5B and Fig. 6B). Noxious-tap neurons sent their apical dendrites to the cortical surface of the medial wall of the ACCx, as observed also in NS neurons. Fig. 7 shows high magnification photomicrographs of a pyramidal noxious-tap (A), a pyramidal NS (B), and a non-pyramidal noxious-tap neuron (C). Pyramidal noxioustap neurons have typical pyramidal soma (Fig. 7A-l), apical dendrites (2: middle portion of the apical dendrites; 3: tip of the apical dendrites in Fig. 7A), basal dendrites with a large number of spines (indicated by the arrows in Fig. 7A-1-4) and also many varicosities on their axon collaterals (indicated by the arrows in Fig. 7A-5). Pyramidal NS neurons (Fig. 7B) had similar morphological properties: pyramidal soma (Fig. 7B-l), apical (2: middle portion of the apical dendrites; 3: tip of the apical dendrites), and basal dendrites with many spines (Fig. 7A, B-4). All pyramidal neurons in the present study had many spines on their apical dendrites but they were not distributed in an uniform manner. As illustrated in Fig. 7A, spines were absent near the some (Fig. 7A-2 and -3) but plentiful from the middle to the tip of the apical dendrite. They also had many varicosities on their axon collaterals (Fig. 7B-5), which suggests possible contacts on other neurons distributed in the vicinity of the soma (indicated by the arrows in Fig. 7B-5). Non-pyramidal NS neurons had round soma, many basal dendrites, and many axonal varicosities (Fig. 7C-1 and -2). We recorded only 3 neurons that received inhibitory input from their receptive fields. Two inhibitory neurons were pyramidal and 1 was a non-pyramidal neuron with an oval soma and many basal dendrites. Inhibitory neurons decreased their firing frequency following noxious mechanical stimulation of the whole body surface. An example is shown in Fig. 8A. During squeezing of the bilateral fore- and hind-limbs, dorsal part of the hairy skin and tail, the firing frequency was decreased. Fig. 8B and C illustrate the reconstruction of inhibitory neuron and the intracortical location. Although we recorded 3 inhibitory neurons, only this non-pyramidal neuron was well stained. The number of spines on apical dendrites of pyramidal neurons was counted. Fig. 9 shows the relationship between the number of spines on NS (Fig. 9A) and noxioustap neurons (Fig. 9B) in each lamina. For NS neurons, spines in lamina V (mean ___SE, 4.8 + 4.5, n = 6) were significantly fewer than those in the laminae I (14.3 ___6.5, n = 6, P < 0.05) and I I - I I I (19.5 + 5.2, n = 6, P < 0.05). For noxious-tap neurons, spines in lamina V (12.2 ___3.0, n = 6) were significantly fewer than in laminae I I - I I I (36.7 + 5.2, n = 6, P < 0.05). In all laminae, the number
88
H. Yamamura et al. / Brain Research 735 (1996) 83-92
B
A
\ '\
C
~ ~ 4
5~
tapping
W
squeezing
3
pressing pinching
_J25mv - - '
squeezing
squeezing
sec
#41T1-1675
lO0~m
•
:L
lm'~
Fig. 5. Sample recordings of a noxious-tapneuron with a receptive field on the upper and lower halves of the body surface. A: iotracellularrecordings. Note that tapping, pressing and pinching of the fore paw produced phasic responses, and squeezingcaused a greater response. B: camera lucida drawing. C: low-magnificationdrawing of soma, dendrites, axon and axon collaterals. D: low-magnificationdrawing of the section where the soma of this neuron was located. The arrow in B corresponds to that in C.
of spines of noxious-tap neurons were slightly greater than that of NS neurons.
4. Discussion It has been reported that a large number of neurons in the ACCx receive noxious input from wide areas of the body and that some of them receive input from the entire whole body surface [19,21]. Although most nociceptive neurons in the ACCx have been recognized as NS, i.e., neurons responsive exclusively to noxious mechanical and thermal stimuli, a high proportion of nociceptive neurons in the ACCx also respond to innocuous 'tap' stimuli [19]. Electrophysiological studies show that the response properties of ACCx neurons are similar to those reported for neurons in the parafascicular, centrolateral and submedial nuclei of the thalamus [19]. Neurons in these thalamic nuclei respond primarily to noxious stimuli, have little or no somatotopic organization, and a high proportion of them respond to innocuous 'tap' stimuli [7]. In this study, about 40% of neurons recorded from the ACCx were classified as noxious-tap neurons and remainder were NS
neurons. Intracortical distribution of ACCx neurons in our study was different from that of a previous extracellular recording study [19]. Most of the neurons we found were in lamina V of the ACCx, whereas many were located in lamina III of rabbits [19]. It has been shown that lamina V of the ACCx is thicker than laminae II, III and VI in rats [22,24]. Furthermore, the size of cells in lamina V is larger than this in laminae II, III and VI [24]. Thus, it is likely that there is much higher chance to record intracellular responses from lamina V neurons. These anatomical characteristics of the ACCx and of the intracellular recording technique may produce the intralaminal population differences of nociceptive neurons between our and previous studies. Several morphologically different neurons in the ACCx have been reported in previous anatomical studies [22,24]. Typical pyramidal neurons have morphological characteristics similar to those located in the deep laminae of the primary motor and somatosensory cortices [8,11,12,15,26,27]. In the present study, most of the NS and noxious-tap neurons in lamina V were classified as pyramidal neurons. Pyramidal NS and noxious-tap neurons had relatively large soma and thick apical dendrites with
89
H. Yamamura et a l . / Brain Research 735 (1996) 83-92
many spines. NS and noxious-tap neurons that we recorded from the A C C x had a large number o f axon collaterals spreading around the soma and a large number of axonal varicosities on their axon collaterals. These suggest that nociceptive neurons in the A C C x could mediate recurrent excitation a n d / o r inhibition of other A C C x neurons. Previous anatomical studies have shown that the A C C x (area 24 according to cytoarchitectonical criteria) [13] is innervated by neurons in the medial thalamic nuclei [ 17,23]. Vogt et al. [19] reported that lidocaine injection into medial parts of the thalamus virtually abolished nociceptive responses in all units tested in ACCx. On the other hand, Musil and Olson [13] have studied thalamic and cortico-cortical projections to A C C x and reported that the dominant source of inputs to A C C x might be other cortical areas. It has been reported also that A C C x neurons send their axons to the medial thalamic nuclei [13]. Together with our results, these data suggest that NS neurons in the ACCx receive noxious information via the medial thalamic
A
B
nuclei and other cortical regions and send information to A C C x neurons located in the vicinity of the soma as well as to medial thalamic nuclei. A larger number of axon collaterals with many varicosities were issued by the noxious-tap neurons and these spread over a much wider area than those of NS neurons (Figs. 2, 3, 5 and 6). Axon collaterals of noxious-tap neurons covered a wide area of the A C C x from laminae I to VI (Fig. 5D). This suggests that noxious-tap neurons send informations to the other A C C x neurons located both far and near to their soma. W e have previously reported nociceptive neurons with similar axonal trajectories in lamina IV of the primary somatosensory cortex in cats [12]. W e also observcu , , h i b i t o r y neurons which decreased their firing frequency during noxious mechanical stimulation of the receptive fields. Morphologically, inhibitory neurons were non-pyramidal or small pyramidal neurons. Sikes and Vogt [19] reported inhibitory neurons with long
C
IIIEH| tapping
1
pressing
•
squeezing pinching squeezlng
~J25mV squeezing 2 sec IIGST2-2067
100p.m
Fig. 6. Sample recordings of a noxious-tap neuron with a receptive field on the bilateral hind paws and tail. A: intracellular recordings of noxious-tap neuron. Note that tapping, pressing, pinching and squeezing of the hind paw produced clear tonic responses. B: camera lucida drawing. C: low-magnification drawing of the section where the soma of this neuron was located.
H. Yomamura et al. / Brain Research 735 (1996) 83 92
90
A
B
C
501~m
91
H. Yamamura et al. / Brain Research 735 (1996) 83 92
A ILLIIIIllitlllli! .... I I.I IIilDJlILtl_ "'-'~""
B
...........7 ~ ' " " ~ ' ~ \
squeezing
5
Ill i i i~ll..~]llli.JUJllli__ squeezing
mm
3
squeezing
squeezing
~IIJHJI[H.I~'J.['HlJiJI_.~ squeezing
-~ 7
1 sec
..~_.~
<
#34T2-2836
type: Inhibitory
/
~
lO0~n,
Fig. 8. Sample recordings of an inhibitory neuron. Note decreased firing frequency during squeezing of the whole body surface. A: intracellular recordings. B: camera lucida drawing. C: low-magnification drawing of the section where the soma of this neuron was located.
Number of spines (/50~tm)
Ao I
lO
20
30
,o
5o
I
I
i
I
i
I1-111
Acknowledgements
m V
E -J
B o I
off-responses during and after noxious stimulation. We observed only decreasing firing frequency during noxious stimulation.
10 i
20
30
40
50
i
I
t
i
I1"iJi J Fig. 9. Intracortical distribution of spines (per 50 Ixm of the apical dendrites) in each lamina. A: NS neurons, B: noxious-tap neurons. The number of spines was counted over a 50 Ixm length of the apical dendrites in each lamina. *P < 0.05.
We are grateful to Dr. G.J. Bennett for helpful comments on this manuscript. This study was supported by the Sato Fund, Nihon University School of Dentistry and in part by a Grant-in-Aid for Scientific Research (7457442) from the Japanese Ministry of Education, Science and Culture.
References [1] Amromin, G.D., Cruce, B.L., Felsoory, A. and Todd, E.M., Bilateral stereotaxic cingulotomy following thoracic rhizotomy, cervical cordotomy and thalamotomy in a patient with intractable pain: a clinical pathological study, In B.L. Cruce (Ed.), Pain Research and Treatment, Academic Press, New York, 1975, pp. 227-241.
Fig. 7. High-magnification photomicrographs. A: pyramidal noxious-tap neuron. B: pyramidal NS neuron. C: non-pyramidal NS neuron. 1: view of soma. 2 and 3: view of middle portion of the apical dendrite and the tip of the apical dendrite, respectively in A and B (and basal dendrites in C). 4: view of the basal dendrites. 5: axon collaterals. C: view of the non-pyramidal NS neuron. White arrows in 2, 3 and 4 indicate spines. Arrows in A-5, B-5 and C-2 indicate axonal varicosities.
92
H. Yamamura et al./Brain Research 735 (1996) 83 92
[2] Baleydier, C. and Mauguiere, F., The duality of the cingulate gyrus in monkey: neuroanatomical study and functional hypothesis, Brain, 103 (1980) 525-554. [3] Ballantine, H.T., Bouckoms, A.J., Thomas, E.K. and Giriunas, I.E., Treatment of psychiatric illness by stereotaxic cingulotomy, Biol. Psychiatry, 22 (1987) 807-817. [4] Craig, A.D., Jr., Wiegand, S.J. and Price, J.L., The thalamo-cortical projection of the nucleus submedius in the cat, J. Comp. Neurol., 206 (1982) 28 48. [5] Domesick, B.D., Projections from the cingulate cortex in the rat, Brain Res., 12 (1969) 296-320. [6] Domesick, V.B., Thalamic relationships of the medial cortex in the rat, Brain Behav. E~'ol., 6 (1972) 457-483. [7] Dong, W.K., Ryu, H. and Wagman, I.H., Nociceptive responses of neurons in medial thalamus and their relationship to spinothalamic pathways, J. Neurophysiol., 41 (1978) 1592-1613. [8] Feldman, M.L., Morphology of the neocortical pyramidal neuron. In A. Peter and E.G. Jones (Eds.), Cerebral Cortex, Plenum, New York, 1984, pp. 123-200. [9] Foltz, E.L. and White, L.E., Pain 'relief' by frontal cingulumotomy, J. Neurosurg., 19 (1962) 89-100. [10] Gabriel, M., Kubota, Y., Sparenborg, S., Straube, K. and Vogk B.A., Effects of cingulate cortical lesions on avoidance learning and training-induced unit activity in rabbits, Exp. Brain Res., 86 (1991) 585-600. [11] Ghosh, S., Fyffe, R.E. and Porter, R,, Morphology of neurons in Area 4"y of the cat's cortex studied with intracellular injection of HRP, J. Comp. NeuroL, 269 (1988) 290-312. [12] Iwata, K. Tsuboi, Y., Yagi, J., Kitajima, K. and Sumino, R., Morphology of primary somatosensory cortical neurons receiving input form the tooth pulp, J. Neurophysiol., 72 (1994) 831-846. [13] Musil, S.Y. and Olson, C.R., Organization of cortical and subcortical projections to anterior cingulate cortex in the cat, J. Comp. Neurol., 272 (1988) 203-218. [14] Neafsey, E.J., Prefrontal cortical control of the autonomic nervous system: anatomical and physiological observations, Prog. Brain Res., 85 (1990) 147-166.
[15] Peters, A. and Jones, E.G., Cellular components of the cerebral cortex. In A. Peter and E.G. Jones (Eds.), Cerebral Cortex, Plenum, New York, 1984, pp. 107-200. [16] Raynolds, D.V., Surgery in the rat during electrical analgesia induced by focal brain stimulation, Science, 164 (1969) 444-445. [17] Robertson, R.T. and Kaitz, S.S., Thalamic connections with limbic cortex. I. Thalamocortical projections, J. Comp. Neurol., 195 (1981) 501-525. [18] Shibata, H., Efferent projections from the anterior thalamic nuclei to the cingulate cortex in the rat, J. Comp. Neurol., 330 (1993) 533-542. [19] Sikes, R.W. and Vogt, B.A., Nociceptive neurons in area 24 of rabbit cingulate cortex, J. Neurophysiol., 68 (1992) 1720-1732. [20] Swanson, L.W., Brain Maps: Structure of the Rat Brain, Elsevier, Amsterdam, 1992. [21] Toda, K., Anterior cingulate-induced inhibition of activities of descending periaqueductat gray matter neurons in rats, Pain Res., 7 (1992) 71-79. [22] Vogt, B.A., Cingulate cortex. In A. Peter and E.G. Jones (Eds.), Cerebral Cortex, Plenum, New York, 1985, pp. 89-149. [23] Vogt, B.A., Rosene, D.L. and Pandya, D.N., Thalamic and cortical afferents differentiate anterior from posterior cingulate cortex in the monkey, Science, 204 (1979) 205-207. [24] Vogt, B.A. and Peters, A., Form and distribution of neurons in rat cingulate cortex: area 32, 24, and 29, J. Comp. Neurol., 195 (1981) 603-625. [25] Vogt, B.A., Finch, D.M. and Olson, C.R., Functional heterogeneity in cingulate cortex: the anterior executive and posterior valuative regions, Cerebral Cortex, 2 (1992) 435-443. [26] Yamamoto, T., Samejima, A. and Oka, H., Short latency activation of local circuit neurons in the cat somatosensory cortex, Brain Res., 461 (1988) 199-2038. [27] Yamamoto, T., Samejima, A. and Oka, H., The mode of synaptic activation of pyramidal neurons in the cat primary somatosensory cortex: an intracellular HRP study, Exp. Brain Res., 80 (1990) 12 22.
Brain Research 735 (1996) 83-92
Research report
Morphological and electrophysiological properties of ACCx nociceptive neurons in rats H. Yamamura
a, K . I w a t a b,c,, , Y . T s u b o i
b, K . T o d a d, K . K i t a j i m a c, N . S h i m i z u
J. H i b i y a b, S. F u j i t a b, R . S u m i n o
e, H . N o m u r a
f,
b,c
a Department of Anesthesiology, School of Medicine, Nihon UniL,ersity, 30-10hyaguchi Kamimachi, Itabashi-ku, Tokyo 173, Japan b Department of Physiology, School of Dentistry, Nihon University, Kanda-surugadai, Chiyoda-ku, Tokyo 101, Japan Department of Pathophysiology, School of DentistD,, Nihon Universi~, Kanda-surugadai, Chiyoda-ku, Tokyo 101, Japan d Department of Physiology, Faculty of Dentistry, Tokyo Medical and Dental University, 5-45 Yushima 1-Chome, Bunkyo-ku, Tokyo 113, Japan e Department of Removable partial denture prosthodontics, School ofDentisto', Nihon University, Kanda-surugadai, Chiyoda-ku, Tokyo 101, Japan r Department of Anesthesiology, School ofDentistD', Nihon UniversiO', Kanda-surugadai, Chiyoda-ku, Tokyo 101, Japan Accepted 7 May 1996
Abstract
A total of 33 neurons with cutaneous receptive fields were recorded from the anterior cingulate cortex (ACCx) and successfully injected with neurobiotin. All neurons were in area 24 of the ACCx. Neurons from the ACCx had large receptive fields (RFs), usually bilateral, and some had RFs covering the whole body surface. Most of the neurons were in the deep laminae and had a pyramidal soma with thick apical dendrites and many spines. Thirteen of 33 neurons were classified as pyramidal nociceptive specific (NS) neurons and 12 as noxious-tap neurons, 3 neurons received inhibitory input and were in lamina V. Two non-pyramidal noxious-tap neurons were located in lamina V and 1 pyramidal noxious-tap neuron was located in lamina VI, and 2 pyramidal NS neurons were in lamina III. Axon collaterals of NS neurons were mainly distributed around the soma, whereas those of noxious-tap neurons were also distributed far from the soma. A large number of varicosities were observed on the axon collaterals of both NS and noxious-tap neurons. Our results suggest that NS neurons in the ACCx send information locally to the vicinity of the soma, while noxious-tap neurons send information to a wider area of the ACCx. Keywords: Cingulate cortex; Nociceptive specific neuron; Morphology; Intracellular recording; Neurobiotin
1. I n t r o d u c t i o n
The anterior cingulate cortex (ACCx) is known to be involved in processing aspect of nociception including autonomic regulation, motivation and effect [2,10,14,16,19,21,22]. Vogt et al. [25] have reported that the A C C x is also involved in emotion, maternal behavior, visceromotor control, skeletomotor function and attention to action. Such a large number of functions of A C C x neurons is thought to be modulated by cortico-cortical or subcortico-cingulate neuronal circuits. Previous electrophysiological studies have reported that most neurons in the A C C x respond exclusively to noxious mechanical a n d / o r thermal stimulation of the body and that many also respond to tapping the receptive field [19]. Most of the
* Corresponding author. Fax: + 81 (3) 3219-8341.
nociceptive neurons in the A C C x respond to a variety of noxious stimuli applied to the whole body and many have large receptive fields [19]. Nociceptive A C C x neuron responses were eliminated by lesion of the medial thalamic nuclei [19]. Anatomical tracing studies have shown that medial thalamic neurons have axons projecting to the anterior cingulate cortex [6,18]. These suggest that ACCx neurons receive noxious input from peripheral structures via medial thalamic nuclei [4-6,18]. Furthermore, many clinical reports have shown that cingulumotomy alleviates a patient's affective responses to noxious stimuli [1,3,9]. These results support the idea that the anterior cingulate cortex has an important role in the processing of the affective aspect of nociception. No studies have been done to examine the relationship between the morphological and electrophysiological properties of nociceptive neurons in the ACCx. In the present study intracellular recording and staining methods were used to examine this question.
0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PH S 0 0 0 6 - 8 9 9 3 ( 9 6 ) 0 0 5 6 1 - 6
84
H. Yamamura et al. / Brain Research 735 (1996) 83-92
Table 1 Incidence of nociceptive neurons recorded from ACCx Lamina NS Noxious-tap Inhibitory
2. Materials and methods 2.1. Animal preparation
Experiments were performed on 29 Wistar rats weighing 2 5 0 - 3 5 0 g. The animals were initially anesthetized with thiamylal sodium (50 mg • kg 1, i.p.). During surgery, anesthesia was maintained with halothane ( 2 - 3 % ) . The animals were mounted in a stereotaxic frame, and the left hemisphere of the parietal cortex was exposed. The stereotaxic frame was then removed and the animals' heads were rigidly secured to a frame by means of stainless steel screws attached to the skull and embedded in dental acrylic cement. During recording sessions, the rats were immobilized with pancuronium bromide (1 m g . kg 1. h -~ , i.v.) and ventilated artificially. The anesthesia was maintained at the same depth throughout the experiment by continuous inhalation of halothane ( 1 - 2 % ) and air. Rectal temperature was maintained at 37-39°C by a thermostatically controlled heating pad. The electrocardiogram was monitored continuously.
Ill V VI Total:
pyramidal pyramidal non-pyramidal pyramidal
2 13 2
12
2 1
1 33
NS: nociceptive specific neurons. Noxious-tap: neurons responded to noxious mechanical and tap stimuli, Inhibitory: neurons received inhibitory inputs.
to noxious mechanical stimulation (pinching a n d / o r squeezing) of the skin. Inhibitory neurons decreased their firing frequency following noxious mechanical stimulation of the receptive fields. Neuronal responses were fed to a tape recorder (bandwidth DC to 20 kHz) for subsequent analysis. Intracellular injection was carried out through the neurobiotin-filled electrodes using a depolarizing pulse ( 3 - 5 nA, 300 ms duration, 2 c y c l e / s ) .
2.2. Stimulation ad recording
2.3. Histology
Glass micropipette electrodes were filled with 5% neurobiotin (Vector Lab.) solution with 0.5 M KC1 in phosphate-buffered saline (PBS; pH 8.0). The glass pipette electrodes were advanced through the cortex in 1 Ixm steps, After A C C x neurons had been impaled, the whole body surface was examined carefully for mechanical stimulation-evoked excitation and the neuronal receptive fields were determined. In the present study, mechanical stimulation was introduced for classification of A C C x neurons [7]. Low threshold mechanoreceptive (LTM) neurons responded to light touch or pressure of the skin. Noxious-tap neurons responded to both noxious (pinching a n d / o r squeezing) and innocuous tap stimulation of the skin. Nociceptive specific (NS) neurons responded exclusively
The animals were allowed to survive for 6 - 1 0 h following intracellular injection, and were then deeply anesthetized with pentobarbital sodium and perfused transcardially with 50 ml PBS (pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate buffer. The brain was removed and placed in cold fixative for 4 days, then transferred to cold phosphate-buffered 30% sucrose for 48 h. Serial sections 50 Ixm thick were cut along the path of the electrode penetration. The sections were incubated in peroxidase-conjugated avidin-biotin complex (1 : 100; ABC: Vector Labs.). W e used 3,3'-diaminobenzidinetetra HC1 (DAB; Sigma), nickel-ammonium sulfate and 0.001% hydrogen peroxide in Tris-buffered saline (0.05 M, pH 7.4) to develop the A B C reaction product, a distinctive black
+4.2
+3.6
+3.2
ee%
1ram
L.._~ L
Fig. 1. Schematic drawings of the intracortical distribution of neurons in the ACCx. Sections are arranged rostro-caudally according to the brain maps by Swanson [20]. The number on each section indicates distance from bregma (mm). D: dorsal, L: lateral in this and following figures. Solid circles: nociceptive specific neurons, open circles: noxious-tap neurons, solid triangles: neurons receiving inhibitory input.
H. Yamamura et a l . / Brain Research 735 (1996) 83-92
85
Table 2 Morphological properties of nociceptive neurons in the ACCx (values in mean -- S.E.) Lamina
III V VI
NS
pyramidal pyramidal non-pyramidal pyramidal
Noxious-tap
Area of soma (p~m2)
Number of basal dendrites
Diameter of apical dendrite (Ixm)
138.3 140.8 + 13.6 84.8
2.5 4.6 + 0.5 2
2.0 2.9 + 0.4 2.1
Inhibitory
Area of soma (p~m2)
Number of basal dendrites
Diameter of apical dendrite (p,m)
Area of soma (ixm 2)
Number of basal dendrites
Diameter of apical dendrite (~m)
147.9+ 12.4
5.0 ___0.6
2.6 + 0.3
150.8 33.7
4.5 2
2.1 1.0
97.3
4
0.9
NS: nociceptive specific neurons. Noxious-tap: neurons responded to noxious mechanical and tap stimuli. Inhibitory: neurons received inhibitory inputs.
chromogen. Thionin was used as a counterstain. Precise camera lucida tracings of the stained neurons were drawn at × 400 magnification with a camera lucida drawing tube. A C C x neurons were drawn at × 1000 magnification and the soma size, diameter of apical dendrites, number of basal dendrites and spines were measured. The diameters of apical dendrites were measured at a point 50 Ixm from the soma. The number of spines of the lamina V pyramidal nociceptive neurons was counted over 50 Ixm length of the
apical dendrites from the 50 Ixm distal to the soma, border between laminae III and V, and border between laminae I and II.
2.4. Statistical analysis Statistical analysis was performed by analysis of variance followed by post-hoc Fisher's PLSD or Scheffe's tests. Differences were considered significant at a P value of less than 0.05.
C
A 1 I'11 [
[ It
~,ii i~ i'J
squeezing
l
~111~1
I~,
rl~l,,
,ll,lrqlll
F
iii1~1~!,-i1,--i
squeezing
q,
i
_.3i
I,,,,
l :!'iJlln] 1._
1_ f
squeezing
5
squeezing
D'~..._
squeezing
squeezing
25 mV squeezing
100 i~m
1 sec
#6T1.2443 type: NS
Fig. 2. Sample recordings and reconstruction of a NS neuron with a large receptive field covering the whole body surface. A: intracellular recordings and receptive field. B: camera lucida drawing. Solid triangle indicates the main axon in this and following figures. C: low-magnification drawing of the section where the soma of this NS neuron was located.
86
H. Yamamura et a l . / Brain Research 735 (1996) 83-92
3. Results
distribution of ACCx neurons is illustrated schematically in Fig. 1. As shown in Fig. 1, NS, noxious-tap and inhibitory neurons were intermingled. We did not find any somatotopic arrangement of any type of nociceptive neuron in the ACCx. The morphological properties of NS neurons are summarized in Table 2. Fig. 2 shows the responses and morphology of a NS neuron with a large receptive field which covered the whole body surface. This neuron responded exclusively to squeezing (Fig. 2A). This neuron responded to squeezing of the whole body surface but the right side (contralateral to recording site) of the receptive field was more sensitive. Reconstruction of this NS neuron is illustrated in Fig. 2B. As shown in Fig. 2B and Fig. 3B, NS neurons had apical dendrites with a large number of spines. The main axon (indicated by the solid triangle) ran deep into the subcortical white matter and had many collaterals. Low magnification drawing of this NS neuron is illustrated in Fig. 2C. Most of the nociceptive neurons we recorded from the ACCx sent their apical dendrites to the surface of the medial wall of the ACCx. Fig. 3 shows the responses and morphology of a NS neuron with a receptive field covering the upper half of the body. This NS neuron did not respond to tapping or pressing the receptive field (Fig. 3A), but responded to
A total of 33 neurons with cutaneous receptive fields were intracellularly recorded and successfully injected from the anterior cingulate cortex (ACCx) (Table 1). All ACCx neurons in this study were located in area 24 according to the cytoarchitectonic criteria of Vogt and Peters [24]. Eighteen of 33 neurons in the ACCx were classified as nociceptive specific (NS) neurons according to their responses to mechanical stimulation of the receptive fields. NS neurons responded exclusively to noxious mechanical stimulation (pinching or squeezing) of the receptive fields but not to non-noxious mechanical stimulation such as gentle touching or brushing. Twelve ACCx neurons were classified as noxious-tap neurons which responded to nonnoxious tapping of the receptive fields as well as to noxious mechanical stimulation [7]. These ACCx neurons showed decrement of spontaneous firing frequency following noxious mechanical stimulation of the receptive fields. Most of the nociceptive neurons in the ACCx (30 of 33 nociceptive neurons) had pyramidal soma with many spines on their apical dendrites. Only two neurons were nonpyramidal neurons, which had oval soma without spines on their apical dendrites. As shown in Table 1, most neurons (30 of 33) were distributed in lamina V. The intracortical
A
-~. . . . . . . . 4~a=.,a NIl,I | l b Ju. . . . . .
--[,-.- , ~ , - qnVl-flp-,r-r,T ........ tapping
pressing squeezing
. . . . . . . . . . . ~'.
e
.i. w~
-,
,~rl
IIIII
..=]v.,r-i
I I! ,.~. . . . . .g~
pinching
m V
squeezing
1 sec 1 O0 pm
#32T1-2600
type: N5 (very high threshold)
. ~ /
Fig. 3. Sample recordings of a NS neuron with a receptive field on the upper half of the body surface. A: intracellular recordings. Note that only the squeezing stimulus produced a clear response. B: camera lucida drawing. C: low-magnification drawing of the section where the soma of this NS neuron was located. 1. l I - I I I , V and VI indicate laminae of the ACCx in this and following figures.
H. Yamamura et a l . / Brain Research 735 (1996) 83-92
noxious pinching and squeezing. All NS neurons recorded in the present study had morphological properties like those shown in Fig. 2B and Fig. 3B. The area of the receptive fields of each NS neuron is calculated as a percentage of the whole body surface. The receptive fields of NS and noxious-tap neurons were classified to 4 types as shown in Fig. 4 (A: receptive fields covering bilateral fore- and hind-limbs continuously; B: those covering bilateral upper a n d / o r lower half of the body; C: those covering unilateral fore- and hind-limbs; D: those covering limbs, tail or face). NS and noxious-tap neurons had similar receptive field properties. Noxious-tap neurons responded to both non-noxious tapping and noxious pinching a n d / o r squeezing the receptive fields but did not increase their firing frequency as mechanical stimulus intensity increased. Figs. 5 and 6 illustrate the responses and morphology of two noxious-tap neurons, one with a large receptive field (Fig. 5A) and one with a relatively small receptive field (Fig. 6A). In Fig. 5, tapping, pressing and pinching of the right hind-limb produced phasic responses and squeezing this region, and tail, the left hind-limb, right and left fore-paw produced high
ll A
E
fl
B
F
C
D
G
H
%
C
e~
Fig. 4. Schematic illustrations of receptive fields of NS ( A - D ) and noxious-tap (E-H) neurons.
87
frequency responses (Fig. 5A). The noxious-tap neuron in Fig. 6 also responded to tapping, pressing, pinching and squeezing, but this neuron did not show phasic responses. The morphological properties of noxious-tap neurons are summarized in Table 2. These neurons had a large number of spines on their basal and apical dendrites and many axon collaterals which spread widely into laminae II to VI (Fig. 5B and Fig. 6B). Noxious-tap neurons sent their apical dendrites to the cortical surface of the medial wall of the ACCx, as observed also in NS neurons. Fig. 7 shows high magnification photomicrographs of a pyramidal noxious-tap (A), a pyramidal NS (B), and a non-pyramidal noxious-tap neuron (C). Pyramidal noxioustap neurons have typical pyramidal soma (Fig. 7A-l), apical dendrites (2: middle portion of the apical dendrites; 3: tip of the apical dendrites in Fig. 7A), basal dendrites with a large number of spines (indicated by the arrows in Fig. 7A-1-4) and also many varicosities on their axon collaterals (indicated by the arrows in Fig. 7A-5). Pyramidal NS neurons (Fig. 7B) had similar morphological properties: pyramidal soma (Fig. 7B-l), apical (2: middle portion of the apical dendrites; 3: tip of the apical dendrites), and basal dendrites with many spines (Fig. 7A, B-4). All pyramidal neurons in the present study had many spines on their apical dendrites but they were not distributed in an uniform manner. As illustrated in Fig. 7A, spines were absent near the some (Fig. 7A-2 and -3) but plentiful from the middle to the tip of the apical dendrite. They also had many varicosities on their axon collaterals (Fig. 7B-5), which suggests possible contacts on other neurons distributed in the vicinity of the soma (indicated by the arrows in Fig. 7B-5). Non-pyramidal NS neurons had round soma, many basal dendrites, and many axonal varicosities (Fig. 7C-1 and -2). We recorded only 3 neurons that received inhibitory input from their receptive fields. Two inhibitory neurons were pyramidal and 1 was a non-pyramidal neuron with an oval soma and many basal dendrites. Inhibitory neurons decreased their firing frequency following noxious mechanical stimulation of the whole body surface. An example is shown in Fig. 8A. During squeezing of the bilateral fore- and hind-limbs, dorsal part of the hairy skin and tail, the firing frequency was decreased. Fig. 8B and C illustrate the reconstruction of inhibitory neuron and the intracortical location. Although we recorded 3 inhibitory neurons, only this non-pyramidal neuron was well stained. The number of spines on apical dendrites of pyramidal neurons was counted. Fig. 9 shows the relationship between the number of spines on NS (Fig. 9A) and noxioustap neurons (Fig. 9B) in each lamina. For NS neurons, spines in lamina V (mean ___SE, 4.8 + 4.5, n = 6) were significantly fewer than those in the laminae I (14.3 ___6.5, n = 6, P < 0.05) and I I - I I I (19.5 + 5.2, n = 6, P < 0.05). For noxious-tap neurons, spines in lamina V (12.2 ___3.0, n = 6) were significantly fewer than in laminae I I - I I I (36.7 + 5.2, n = 6, P < 0.05). In all laminae, the number
88
H. Yamamura et al. / Brain Research 735 (1996) 83-92
B
A
\ '\
C
~ ~ 4
5~
tapping
W
squeezing
3
pressing pinching
_J25mv - - '
squeezing
squeezing
sec
#41T1-1675
lO0~m
•
:L
lm'~
Fig. 5. Sample recordings of a noxious-tapneuron with a receptive field on the upper and lower halves of the body surface. A: iotracellularrecordings. Note that tapping, pressing and pinching of the fore paw produced phasic responses, and squeezingcaused a greater response. B: camera lucida drawing. C: low-magnificationdrawing of soma, dendrites, axon and axon collaterals. D: low-magnificationdrawing of the section where the soma of this neuron was located. The arrow in B corresponds to that in C.
of spines of noxious-tap neurons were slightly greater than that of NS neurons.
4. Discussion It has been reported that a large number of neurons in the ACCx receive noxious input from wide areas of the body and that some of them receive input from the entire whole body surface [19,21]. Although most nociceptive neurons in the ACCx have been recognized as NS, i.e., neurons responsive exclusively to noxious mechanical and thermal stimuli, a high proportion of nociceptive neurons in the ACCx also respond to innocuous 'tap' stimuli [19]. Electrophysiological studies show that the response properties of ACCx neurons are similar to those reported for neurons in the parafascicular, centrolateral and submedial nuclei of the thalamus [19]. Neurons in these thalamic nuclei respond primarily to noxious stimuli, have little or no somatotopic organization, and a high proportion of them respond to innocuous 'tap' stimuli [7]. In this study, about 40% of neurons recorded from the ACCx were classified as noxious-tap neurons and remainder were NS
neurons. Intracortical distribution of ACCx neurons in our study was different from that of a previous extracellular recording study [19]. Most of the neurons we found were in lamina V of the ACCx, whereas many were located in lamina III of rabbits [19]. It has been shown that lamina V of the ACCx is thicker than laminae II, III and VI in rats [22,24]. Furthermore, the size of cells in lamina V is larger than this in laminae II, III and VI [24]. Thus, it is likely that there is much higher chance to record intracellular responses from lamina V neurons. These anatomical characteristics of the ACCx and of the intracellular recording technique may produce the intralaminal population differences of nociceptive neurons between our and previous studies. Several morphologically different neurons in the ACCx have been reported in previous anatomical studies [22,24]. Typical pyramidal neurons have morphological characteristics similar to those located in the deep laminae of the primary motor and somatosensory cortices [8,11,12,15,26,27]. In the present study, most of the NS and noxious-tap neurons in lamina V were classified as pyramidal neurons. Pyramidal NS and noxious-tap neurons had relatively large soma and thick apical dendrites with
89
H. Yamamura et a l . / Brain Research 735 (1996) 83-92
many spines. NS and noxious-tap neurons that we recorded from the A C C x had a large number o f axon collaterals spreading around the soma and a large number of axonal varicosities on their axon collaterals. These suggest that nociceptive neurons in the A C C x could mediate recurrent excitation a n d / o r inhibition of other A C C x neurons. Previous anatomical studies have shown that the A C C x (area 24 according to cytoarchitectonical criteria) [13] is innervated by neurons in the medial thalamic nuclei [ 17,23]. Vogt et al. [19] reported that lidocaine injection into medial parts of the thalamus virtually abolished nociceptive responses in all units tested in ACCx. On the other hand, Musil and Olson [13] have studied thalamic and cortico-cortical projections to A C C x and reported that the dominant source of inputs to A C C x might be other cortical areas. It has been reported also that A C C x neurons send their axons to the medial thalamic nuclei [13]. Together with our results, these data suggest that NS neurons in the ACCx receive noxious information via the medial thalamic
A
B
nuclei and other cortical regions and send information to A C C x neurons located in the vicinity of the soma as well as to medial thalamic nuclei. A larger number of axon collaterals with many varicosities were issued by the noxious-tap neurons and these spread over a much wider area than those of NS neurons (Figs. 2, 3, 5 and 6). Axon collaterals of noxious-tap neurons covered a wide area of the A C C x from laminae I to VI (Fig. 5D). This suggests that noxious-tap neurons send informations to the other A C C x neurons located both far and near to their soma. W e have previously reported nociceptive neurons with similar axonal trajectories in lamina IV of the primary somatosensory cortex in cats [12]. W e also observcu , , h i b i t o r y neurons which decreased their firing frequency during noxious mechanical stimulation of the receptive fields. Morphologically, inhibitory neurons were non-pyramidal or small pyramidal neurons. Sikes and Vogt [19] reported inhibitory neurons with long
C
IIIEH| tapping
1
pressing
•
squeezing pinching squeezlng
~J25mV squeezing 2 sec IIGST2-2067
100p.m
Fig. 6. Sample recordings of a noxious-tap neuron with a receptive field on the bilateral hind paws and tail. A: intracellular recordings of noxious-tap neuron. Note that tapping, pressing, pinching and squeezing of the hind paw produced clear tonic responses. B: camera lucida drawing. C: low-magnification drawing of the section where the soma of this neuron was located.
H. Yomamura et al. / Brain Research 735 (1996) 83 92
90
A
B
C
501~m
91
H. Yamamura et al. / Brain Research 735 (1996) 83 92
A ILLIIIIllitlllli! .... I I.I IIilDJlILtl_ "'-'~""
B
...........7 ~ ' " " ~ ' ~ \
squeezing
5
Ill i i i~ll..~]llli.JUJllli__ squeezing
mm
3
squeezing
squeezing
~IIJHJI[H.I~'J.['HlJiJI_.~ squeezing
-~ 7
1 sec
..~_.~
<
#34T2-2836
type: Inhibitory
/
~
lO0~n,
Fig. 8. Sample recordings of an inhibitory neuron. Note decreased firing frequency during squeezing of the whole body surface. A: intracellular recordings. B: camera lucida drawing. C: low-magnification drawing of the section where the soma of this neuron was located.
Number of spines (/50~tm)
Ao I
lO
20
30
,o
5o
I
I
i
I
i
I1-111
Acknowledgements
m V
E -J
B o I
off-responses during and after noxious stimulation. We observed only decreasing firing frequency during noxious stimulation.
10 i
20
30
40
50
i
I
t
i
I1"iJi J Fig. 9. Intracortical distribution of spines (per 50 Ixm of the apical dendrites) in each lamina. A: NS neurons, B: noxious-tap neurons. The number of spines was counted over a 50 Ixm length of the apical dendrites in each lamina. *P < 0.05.
We are grateful to Dr. G.J. Bennett for helpful comments on this manuscript. This study was supported by the Sato Fund, Nihon University School of Dentistry and in part by a Grant-in-Aid for Scientific Research (7457442) from the Japanese Ministry of Education, Science and Culture.
References [1] Amromin, G.D., Cruce, B.L., Felsoory, A. and Todd, E.M., Bilateral stereotaxic cingulotomy following thoracic rhizotomy, cervical cordotomy and thalamotomy in a patient with intractable pain: a clinical pathological study, In B.L. Cruce (Ed.), Pain Research and Treatment, Academic Press, New York, 1975, pp. 227-241.
Fig. 7. High-magnification photomicrographs. A: pyramidal noxious-tap neuron. B: pyramidal NS neuron. C: non-pyramidal NS neuron. 1: view of soma. 2 and 3: view of middle portion of the apical dendrite and the tip of the apical dendrite, respectively in A and B (and basal dendrites in C). 4: view of the basal dendrites. 5: axon collaterals. C: view of the non-pyramidal NS neuron. White arrows in 2, 3 and 4 indicate spines. Arrows in A-5, B-5 and C-2 indicate axonal varicosities.
92
H. Yamamura et al./Brain Research 735 (1996) 83 92
[2] Baleydier, C. and Mauguiere, F., The duality of the cingulate gyrus in monkey: neuroanatomical study and functional hypothesis, Brain, 103 (1980) 525-554. [3] Ballantine, H.T., Bouckoms, A.J., Thomas, E.K. and Giriunas, I.E., Treatment of psychiatric illness by stereotaxic cingulotomy, Biol. Psychiatry, 22 (1987) 807-817. [4] Craig, A.D., Jr., Wiegand, S.J. and Price, J.L., The thalamo-cortical projection of the nucleus submedius in the cat, J. Comp. Neurol., 206 (1982) 28 48. [5] Domesick, B.D., Projections from the cingulate cortex in the rat, Brain Res., 12 (1969) 296-320. [6] Domesick, V.B., Thalamic relationships of the medial cortex in the rat, Brain Behav. E~'ol., 6 (1972) 457-483. [7] Dong, W.K., Ryu, H. and Wagman, I.H., Nociceptive responses of neurons in medial thalamus and their relationship to spinothalamic pathways, J. Neurophysiol., 41 (1978) 1592-1613. [8] Feldman, M.L., Morphology of the neocortical pyramidal neuron. In A. Peter and E.G. Jones (Eds.), Cerebral Cortex, Plenum, New York, 1984, pp. 123-200. [9] Foltz, E.L. and White, L.E., Pain 'relief' by frontal cingulumotomy, J. Neurosurg., 19 (1962) 89-100. [10] Gabriel, M., Kubota, Y., Sparenborg, S., Straube, K. and Vogk B.A., Effects of cingulate cortical lesions on avoidance learning and training-induced unit activity in rabbits, Exp. Brain Res., 86 (1991) 585-600. [11] Ghosh, S., Fyffe, R.E. and Porter, R,, Morphology of neurons in Area 4"y of the cat's cortex studied with intracellular injection of HRP, J. Comp. NeuroL, 269 (1988) 290-312. [12] Iwata, K. Tsuboi, Y., Yagi, J., Kitajima, K. and Sumino, R., Morphology of primary somatosensory cortical neurons receiving input form the tooth pulp, J. Neurophysiol., 72 (1994) 831-846. [13] Musil, S.Y. and Olson, C.R., Organization of cortical and subcortical projections to anterior cingulate cortex in the cat, J. Comp. Neurol., 272 (1988) 203-218. [14] Neafsey, E.J., Prefrontal cortical control of the autonomic nervous system: anatomical and physiological observations, Prog. Brain Res., 85 (1990) 147-166.
[15] Peters, A. and Jones, E.G., Cellular components of the cerebral cortex. In A. Peter and E.G. Jones (Eds.), Cerebral Cortex, Plenum, New York, 1984, pp. 107-200. [16] Raynolds, D.V., Surgery in the rat during electrical analgesia induced by focal brain stimulation, Science, 164 (1969) 444-445. [17] Robertson, R.T. and Kaitz, S.S., Thalamic connections with limbic cortex. I. Thalamocortical projections, J. Comp. Neurol., 195 (1981) 501-525. [18] Shibata, H., Efferent projections from the anterior thalamic nuclei to the cingulate cortex in the rat, J. Comp. Neurol., 330 (1993) 533-542. [19] Sikes, R.W. and Vogt, B.A., Nociceptive neurons in area 24 of rabbit cingulate cortex, J. Neurophysiol., 68 (1992) 1720-1732. [20] Swanson, L.W., Brain Maps: Structure of the Rat Brain, Elsevier, Amsterdam, 1992. [21] Toda, K., Anterior cingulate-induced inhibition of activities of descending periaqueductat gray matter neurons in rats, Pain Res., 7 (1992) 71-79. [22] Vogt, B.A., Cingulate cortex. In A. Peter and E.G. Jones (Eds.), Cerebral Cortex, Plenum, New York, 1985, pp. 89-149. [23] Vogt, B.A., Rosene, D.L. and Pandya, D.N., Thalamic and cortical afferents differentiate anterior from posterior cingulate cortex in the monkey, Science, 204 (1979) 205-207. [24] Vogt, B.A. and Peters, A., Form and distribution of neurons in rat cingulate cortex: area 32, 24, and 29, J. Comp. Neurol., 195 (1981) 603-625. [25] Vogt, B.A., Finch, D.M. and Olson, C.R., Functional heterogeneity in cingulate cortex: the anterior executive and posterior valuative regions, Cerebral Cortex, 2 (1992) 435-443. [26] Yamamoto, T., Samejima, A. and Oka, H., Short latency activation of local circuit neurons in the cat somatosensory cortex, Brain Res., 461 (1988) 199-2038. [27] Yamamoto, T., Samejima, A. and Oka, H., The mode of synaptic activation of pyramidal neurons in the cat primary somatosensory cortex: an intracellular HRP study, Exp. Brain Res., 80 (1990) 12 22.