Brain Research, 171 (1990) 171-175 Elsevier
171
BRES 24272
Increase of dendritic branching of CA3 neurons of hippocampus and self-stimulation areas in subjects experiencing self-stimulation of lateral hypothalamus and substantia nigra-ventral tegmental area P.N. Bindu and T. Desiraju Department of Neurophysiology, National Institute of Mental Health and Neurosciences, Bangalore (India)
(Accepted 29 May 1990) Key words: Neuronal changes in operant behavior; Self-stimulationreward; Plasticity of dendritic branching; Hippocampal CA3 neuron; Lateral hypothalamic neuron; Substantia nigra and ventral tegmental area
Golgi examination of neurons of self-stimulation areas of the lateral hypothalamus and substantia nigra-ventral tegmental areas of adult Wistar rats that had experienced self-stimulation for 10 days revealed a significantlyhigher number of dendritic branching points in the two self-stimulation areas, and also in the hippocampus (CA3 pyramidal neurons) than in inexperienced rats. It has been convincingly established in recent years that dendritic arborizations of neurons can undergo alterations with significant degrees of plasticity during both developing and adult ages under the impact of different conditions of nurture and learning experience2' 4,5,7,9,15. Since the discovery of the intra-cranial selfstimulation operant behavior (SS) about 40 years ago, many studies have been conducted and also several hypotheses have risen and fallen 18 in the attempts to understand the brain-reward mechanisms. But, no study has yet been made on neuronal changes that may be triggered in the brain regions likely to be playing part in the operant behavior, or in the SS electrode sites. Hence the present study was made to see whether any significant changes in dendritic branching occur in the hippocampus, and in the self-stimulation sites of lateral hypothalamus (LH) and substantia nigra-ventral tegmental area (SNV'rA). Adult male Wistar rats of 130-140 days of age were assigned to 3 categories of 20 each: self-stimulation experienced (E), sham control (S), and normal control (N). Each subject of the E and S groups was stereotaxically implanted permanently with a total of 4 bipolar stainless steel electrodes (28 s.g.w.) in the SN-VTA and L H bilaterally. Each subject of the E group was trained for pedal pressing for self-stimulation in a Skinner box set up 13. Each pedal press delivered a stimulus train of sine waves of 50 Hz for 0.25 s. The current intensity was set for each electrode in the rat to elicit the maximum
possible pedal press rate response under that fixed stimulus frequency. This was in the range of 25-75/~A for different electrodes and subjects. The subject was allowed 15 min of daily self-stimulation for each site. Only rats that self-stimulated all the 4 electrodes, at rates above 1000 (per 15 min) at each electrode, were included in the E group. The method of multiple electrode placements with positive responding was standardized over several years, so that a non-responding electrode placement was very rare. The postmortem checking of electrode sites showed that the SS electrode tips were usually in or about the following atlas coordinates12: LH: AP -1.3 mm to -1.8 mm, L 1.8 mm to 2 mm, D 8.5 mm; SN-VTA: A P - 4 . 8 to -5.3 mm, L 1.1 to 1.3 mm, D 8.5 mm. The averages (per 15 min) of the self-stimulation pedal press rates (+ S.D.) of 10 days were: (a) for the 10 rats presented in Fig. 1: L H (left) 1921 + 185, L H (right) 2050 + 192, SN-VTA (left) 2169 + 146, SN-VTA (fight) 2389 + 218; (b) for the 15 rats presented in Fig. 2: L H (left) 1981 + 220, LH (right) 2067 + 217, SN-VTA (left) 2172 + 139, SN-VTA (right) 2338 + 244. The subjects of the S group had the electrodes implanted but were not allowed the experience of SS behavior. Subjects of the N group had no implanted electrodes and lived like the other subjects in the home cages. After 10 days of the daily SS, the subjects of the E group were sacrificed and brain regions of the SS sites (LH, SN-VTA) as well as the lateral parts of hippocampus of both sides were processed according to the rapid
Correspondence: T. Desiraju, Department of Neurophysiology, National Institute of Mental Health and Neurosciences, Bangalore-560029, India.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
172 Golgi method of Stensaas 5'7'9. At the same time, the subjects of the N and S groups were also sacrificed and brain regions similarly processed. Hippocampal tissue was examined in 120-Mm thick sections, but the brainstem and hypothalamic areas had to be examined in 90-Mm thick sections as thicker sections of these regions had a diminished clarity of background. Hence the numbers of zones of 20-/~m spheres assessed were also restricted for these two regions, to minimize any contamination of data due to truncation in cutting. Slides were coded to mask Dendritic
clues about the category of rats to which they belonged. Well impregnated neurons having all dendrites intact without cuts were counted for dendritic branching (bifurcation) points in successive zones of 20-Mm spheres. They were sampled from both sides of the brain since both sides were self-stimulated. Neurons were drawn at 40x magnification with a light microscope with a camera lucida attachment. Data from a total of 300 neurons (100 for each group) are presented in this report. The data were statistically assessed by two-way analysis of variance
branching]
points o f CA 3 p y r a m i d a l
neurons
B. Total counts o f branchin s p o i n t s
A. Numbers of 20 }Jm r a d i a l s p h e r e
Apical in 6 spheres
Basal in 3 spheres
..
..e" "6i ,o"
t J"
~,
¢, •
C. in successive spheres Basal d e n d r i t e s
Apical dendrites
4 x
E
6
x x
x,.._...-- v
'5
E : Experienced S : Sham c o n t r o l N: Normal control
x ~X~ x
n : 10 r a t s
\~
x
E/S
P< 0 . 0 5 F < 0.001
E oZ
1
E N
2
4 Number of s p h e r e
6 (20
0
j
I
2
e~
I
4
pro) f r o m soma
Fig. 1. Assessment of changes in dendritic branching in hippocampal CA3 neurons. A: method of counting number of branch points in apical and basal dendrites in successive zones of 20-#m spheres. B: total number of branching points of all the spheres, counted in 100 neurons from each of the 3 groups of rats (10 rats in each of N, S, E groups). Note significant increase in E group (ANOVA F-test, P < 0.001). C: branching points of the 3 groups of rats represented for successive spheres of counting. Each point on the graph is the mean data of 100 neurons in 10 rats of a group. Note the significant increase in all the spheres in E group. E, self-stimulation experienced group; N, normal control group; S, sham control group, i.e., subject carried implanted electrodes but had not experience of self-stimulation.
173 brain area across groups, to do a balanced statistical assessment. The neurons sampled in LH and SN-VTA were within a distance of about 300/~m from the electrode tip. In hippocampus, the CA3 pyramids were studied. In SNVTA, the majority of neurons are from lateral VTA and medial SN. The cells were of 15-30/~m size, ovoid, polygonal or triangular in shape, and usually gave rise to
( A N O V A , treatment x subjects, data of neurons averaged within subjects) for each of the measurement spheres TM. Although each category had 20 rats, they all were not used in the data contribution to the 3 brain areas assessed (Figs. 1, 2). A few tissues were not well stained and had to be kept aside. Some others, though well stained, have not been included in order to equalize numbers of rats (and total number of neurons) for a given
Dendritic branchin5 points of S N - V T A A. N u m b e r s o f 20 pm r a d i a l
and LH
sphere
SN-VTA n e u r o n
I
jl~ss s
~X~' ~
s
neurons
LH neuron
N
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i
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6.
5
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T o t a l c o u n t s o f branchin 9 p o i n t s
S N - V T A in 4 s p h e r e s
LH in 3 s p h e r e s
10
"0~ 10
8
LE " B
03 E u
¢_
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6
~
4
.o
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E c
2
c
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a.
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SN- VTA
LH
5
5 X
t- {.
~4 ~E
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x
E : Experienced
• E N
S : N :
X
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~- =L 3
= 15 r a t s 100 n e u r o n s
3
o 0
Sham control Normal con'trol
E/S
2 z
P < 0.05 F < 0.001
1
1
1
2
3
4
Number of sphere
C 1 (20)Jm)
2 from
3 soma
Fig. 2. C h a n g e s in dendritic branching in n e u r o n s of substantia nigra-ventrai tegmental area (SN-VTA), and of lateral hypothalamus (LH). Data were o f 100 neurons from 15 rats in each of the 3 groups. Other legends as in Fig. 1. Note significant increase in the first 3 spheres in S N - V T A , and in first 2 spheres in L H neurons.
174 3-5 thick, generally smooth primary dendrites that scarcely gave rise to secondary and tertiary dendritic branches. Dendritic varicosities were not commonly seen. The L H neurons studied were mainly from the middle and anterior areas of hypothalamus, along medial forebrain bundle (MFB), and were small, of a diameter of 10-15 /~m. The cells were fusiform, triangular, or spherical in shape, and usually gave rise to 2-3 long and mainly unbranched dendrites. The general features of the SN and L H neurons observed in this study were comparable to those reported earlier 6'8'1°'~6'17. The CA3 pyramidal neurons of hippocampus (Fig. 1) showed an increase of dendritic branching of both the apical and basal dendrites in the E group in contrast to the other groups. The total of branching points of apical dendrites counted in the 6 20-/~m spheres in the E, S and N groups were 13.35 _+ 2.1, 4.98 _+ 0.6, and 5.2 _+ 0.7 respectively, i.e., an increase in E by 168% over the value of S. For basal dendrites, the dendritic branching points were studied up to 60/~m, and the total values of the E, S and N groups were 10.5 _+ 0.93, 6.85 _+ 0.8, and 6.09 + 0.62 respectively, i.e., an increase in E by 53% over the value of S. A calculation for a corresponding radial sphere zone of 0-60/~m of apical dendrites of E showed an increase of 93% over the value of S. Hence, it appeared that the response of apical dendrites was much greater than that of basal dendrites. The E group SN and LH neurons (Fig. 2) also showed significantly higher dendritic branching than those of the S and N groups. For the SN neurons, the total number of branching points (studied up to 80/~m) for the E, S and N groups were 8.5 _+ 1, 5.3 _+ 0.7, and 5.8 _+ 0.99 respectively, i.e., E was larger than S by 60%. For the LH neurons, the total number of branching points (studied up to 60/~m) for the E, S and N groups were 6.62 _+ 0.75, 4.3 + 0.62 and 4.17 _+ 0.3 respectively, i.e., E was larger than S by 54%. For the LH neurons the increase was significant in the first and second metric spheres, whereas for the SN neurons the increase was significant in the third sphere also. In both the regional neurons, the significant increase was mainly in the perisomatic domains of the dendrites. The branching numbers of neurons of the sham control (S) and normal control (N) groups showed no significant differences in the 3 regions compared. This is the first report revealing changes in dendritic 1 Berger, T.W. and Thompson, R.E, Neuronal plasticity in the limbic system during classical conditioning of the rabbit nictitating membrane response. I. The hippocampus, Brain Research, 145 (1978) 323-346. 2 Chang, EE and Greenough, W.T., Lateralised effects of monocular training on dendritic branching in adult split-brain rats, Brain Research, 232 (1982) 283-292.
branching of CA3 neurons of the hippocampus in subjects that have had the experience of intensely rewarding SS behavior. Hippocampus in the rat has been considered to be playing a significant role in spatial exploration and learning 11. Other studies 1'3'7 have provided evidence for involvement of hippocampal neurons in both classical and operant learning paradigms as well. The results of the present study also establish that the neurons of the SS sites (LH and the SN-VTA) undergo changes following the learning for rewarding stimulation. The increase of dendritic branching of LH and SN neurons of the E group cannot be considered to be due to electrode irritation or implantation injury, since the S group showed no such increase. The increase might be related partly to the physical effect of repeated electrical stimulation, although we could not see evidence for such a cause in a preliminary study conducted on 3 other subjects, that were provided the 'experimenter administered' stimulation through similar electrode placements daily for 5 min during 3 days, sacrificed on the 10th day and examined. The total counts (mean _+ S.D.) of the hippocampal CA3 neurons (n = 25) apical dendrites in the radial zone of 0-120/~m was 5.75 _+ 1.68, of the basal dendrites in the zone of 0-60/~m was 6.9 + 1.93, of the SN neurons (n = 15) dendrites in the zone of 0-80/~m was 6.32 + 1.54, and of the L H neurons (n = 15) dendrites in the zone of 0-60 ~tm was 4.80 + 1.14. These indicated no significant changes in the dendritic branching even in the SN and LH that had a direct exposure to the electrical stimulus, therefore suggesting no major effect of the electrical stimulus per se. Nevertheless, one cannot conclude with confidence that the dendritic changes of the LH and SN were only due to the rewarding experience of the conditioned behavior, but not due to electric stimulation. However, even to note that a significant increase in the dendritic branching due to self-stimulation occurred in not only the stimulated areas of SS but also in hippocampus, is itself an important point, since the neurons of hippocampus have not been previously studied in relation to responding for self-stimulation that is known to be most intensely rewarding, perhaps even more than feeding and sexual behaviors in the rat. Such an intensely rewarding impact of the SS could possibly be the factor that caused the kind of massive increase of dendritic branching of the CA3 hippocampal neurons, particularly of the apical dendrites. 3 Christian, E.P. and Deadwyler, S.A., Behavioral functions and hippocampal cell types: evidence for two non-overlapping populations in the rat, J. Neurophysiol., 55 (1986) 331-348. 4 Greenough, W.T., What's special about development? Thoughts on the bases of experience-sensitivesynaptic plasticity. In W.T. Greenough and J.M. Juraska (Eds.), Developmental Neuropsychobiology, Academic Press, Orlando, 1986, pp. 387-407.
175 5 Gundappa, G. and Desiraju, T., Deviations in brain development of F2 generation caloric undernutrition and scope of their prevention by rehabilitation: alterations in dendritic spine production and pruning of pyramidal neurons of lower laminae of motor cortex and visual cortex, Brain Research, 456 (1988) 205-223. 6 Juraska, J.M., Wilson, C.J. and Groves, P.M., The substantia nigra of the rat: a Golgi study, J. Comp. Neurol., 172 (1977) 565-600. 7 Mahajan, D.S. and Desiraju, T., Alterations of dendritic branching and spine densities of hippocampal CA3 pyramidal neurons induced by operant conditioning in the phase of brain growth spurt, Exp. Neurol., 100 (1988) 1-15. 8 Millhouse, O.E., A Golgi anatomy of the rodent hypothalamus. In P.J. Morgane and J. Panksepp (Eds.), Handbook of the Hypothalamus. Vol. 1. Anatomy of the Hypothalamus, Marcel Dekker, New York, 1979, pp. 221-265. 9 Murthy, S.K. and Desiraju, T., Quantitative assessment of dendritic branching and spine densities of neurons of hippocampal embryonic tissue transplanted into juvenile neocortex, Dev. Brain Res., 46 (1989) 33-45. 10 Oades, R.D. and Halliday, G.M., Ventral tegmental (A10) system: neurobiology. I. Anatomy and connectivity, Brain Res. Rev., 12 (1987) 117-165. 11 O'Keefe, J. and Nadel, L., The Hippocampus as a Cognitive Map, Oxford Univ. Press (Clarendon), London, 1978.
12 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982, 71 pp. 13 Singh, J. and Desiraju, T., Differential effects of opioid peptides administered intracerebrally in loci of self-stimulation reward of lateral hypothalamus and ventral tegmental area-substantia nigra. In R.S. Rapaka and B.N. Dhawan (Eds.), Opioid Peptides: An Update, NIDA Research Monograph 87, US Dept. of Health and Human Services, Washington, D.C., 1988, pp. 180-191. 14 Snedecor, G.W. and Cochran, W.G., Statistical Methods, Oxford and IBH Publishers, New Delhi, 1967. 15 Spinelli, D.N., Jensen, F.E. and Viana Di Prisco, G., Early experience effect on dendritic branching in normally reared kittens, Exp. Neurol., 68 (1980) 1-11. 16 Tepper, J.M., Sawyer, S.F. and Groves, p.M., Electrophysiologically identified nigral dopaminergic neurons intracellularly labeled with HRP: light-microscopic analysis, J. Neurosci., 7 (1987) 2794-2806. 17 Veening, J.G., Swanson, L.W., Cowan, W.M., Nieuwenhuys, R. and Geeraedts, L.M.G., The medial forebrain bundle of the rat. II. An autoradiographic study of the topography qf the major descending and ascending components, J. Comp. Neurol., 206 (1982) 82-108. 18 Wise, R.A. and Rompre, P.P., Brain dopamine and reward, Ann. Rev. Psychol., 40 (1989) 191-225.
171
BRES 24272
Increase of dendritic branching of CA3 neurons of hippocampus and self-stimulation areas in subjects experiencing self-stimulation of lateral hypothalamus and substantia nigra-ventral tegmental area P.N. Bindu and T. Desiraju Department of Neurophysiology, National Institute of Mental Health and Neurosciences, Bangalore (India)
(Accepted 29 May 1990) Key words: Neuronal changes in operant behavior; Self-stimulationreward; Plasticity of dendritic branching; Hippocampal CA3 neuron; Lateral hypothalamic neuron; Substantia nigra and ventral tegmental area
Golgi examination of neurons of self-stimulation areas of the lateral hypothalamus and substantia nigra-ventral tegmental areas of adult Wistar rats that had experienced self-stimulation for 10 days revealed a significantlyhigher number of dendritic branching points in the two self-stimulation areas, and also in the hippocampus (CA3 pyramidal neurons) than in inexperienced rats. It has been convincingly established in recent years that dendritic arborizations of neurons can undergo alterations with significant degrees of plasticity during both developing and adult ages under the impact of different conditions of nurture and learning experience2' 4,5,7,9,15. Since the discovery of the intra-cranial selfstimulation operant behavior (SS) about 40 years ago, many studies have been conducted and also several hypotheses have risen and fallen 18 in the attempts to understand the brain-reward mechanisms. But, no study has yet been made on neuronal changes that may be triggered in the brain regions likely to be playing part in the operant behavior, or in the SS electrode sites. Hence the present study was made to see whether any significant changes in dendritic branching occur in the hippocampus, and in the self-stimulation sites of lateral hypothalamus (LH) and substantia nigra-ventral tegmental area (SNV'rA). Adult male Wistar rats of 130-140 days of age were assigned to 3 categories of 20 each: self-stimulation experienced (E), sham control (S), and normal control (N). Each subject of the E and S groups was stereotaxically implanted permanently with a total of 4 bipolar stainless steel electrodes (28 s.g.w.) in the SN-VTA and L H bilaterally. Each subject of the E group was trained for pedal pressing for self-stimulation in a Skinner box set up 13. Each pedal press delivered a stimulus train of sine waves of 50 Hz for 0.25 s. The current intensity was set for each electrode in the rat to elicit the maximum
possible pedal press rate response under that fixed stimulus frequency. This was in the range of 25-75/~A for different electrodes and subjects. The subject was allowed 15 min of daily self-stimulation for each site. Only rats that self-stimulated all the 4 electrodes, at rates above 1000 (per 15 min) at each electrode, were included in the E group. The method of multiple electrode placements with positive responding was standardized over several years, so that a non-responding electrode placement was very rare. The postmortem checking of electrode sites showed that the SS electrode tips were usually in or about the following atlas coordinates12: LH: AP -1.3 mm to -1.8 mm, L 1.8 mm to 2 mm, D 8.5 mm; SN-VTA: A P - 4 . 8 to -5.3 mm, L 1.1 to 1.3 mm, D 8.5 mm. The averages (per 15 min) of the self-stimulation pedal press rates (+ S.D.) of 10 days were: (a) for the 10 rats presented in Fig. 1: L H (left) 1921 + 185, L H (right) 2050 + 192, SN-VTA (left) 2169 + 146, SN-VTA (fight) 2389 + 218; (b) for the 15 rats presented in Fig. 2: L H (left) 1981 + 220, LH (right) 2067 + 217, SN-VTA (left) 2172 + 139, SN-VTA (right) 2338 + 244. The subjects of the S group had the electrodes implanted but were not allowed the experience of SS behavior. Subjects of the N group had no implanted electrodes and lived like the other subjects in the home cages. After 10 days of the daily SS, the subjects of the E group were sacrificed and brain regions of the SS sites (LH, SN-VTA) as well as the lateral parts of hippocampus of both sides were processed according to the rapid
Correspondence: T. Desiraju, Department of Neurophysiology, National Institute of Mental Health and Neurosciences, Bangalore-560029, India.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
172 Golgi method of Stensaas 5'7'9. At the same time, the subjects of the N and S groups were also sacrificed and brain regions similarly processed. Hippocampal tissue was examined in 120-Mm thick sections, but the brainstem and hypothalamic areas had to be examined in 90-Mm thick sections as thicker sections of these regions had a diminished clarity of background. Hence the numbers of zones of 20-/~m spheres assessed were also restricted for these two regions, to minimize any contamination of data due to truncation in cutting. Slides were coded to mask Dendritic
clues about the category of rats to which they belonged. Well impregnated neurons having all dendrites intact without cuts were counted for dendritic branching (bifurcation) points in successive zones of 20-Mm spheres. They were sampled from both sides of the brain since both sides were self-stimulated. Neurons were drawn at 40x magnification with a light microscope with a camera lucida attachment. Data from a total of 300 neurons (100 for each group) are presented in this report. The data were statistically assessed by two-way analysis of variance
branching]
points o f CA 3 p y r a m i d a l
neurons
B. Total counts o f branchin s p o i n t s
A. Numbers of 20 }Jm r a d i a l s p h e r e
Apical in 6 spheres
Basal in 3 spheres
..
..e" "6i ,o"
t J"
~,
¢, •
C. in successive spheres Basal d e n d r i t e s
Apical dendrites
4 x
E
6
x x
x,.._...-- v
'5
E : Experienced S : Sham c o n t r o l N: Normal control
x ~X~ x
n : 10 r a t s
\~
x
E/S
P< 0 . 0 5 F < 0.001
E oZ
1
E N
2
4 Number of s p h e r e
6 (20
0
j
I
2
e~
I
4
pro) f r o m soma
Fig. 1. Assessment of changes in dendritic branching in hippocampal CA3 neurons. A: method of counting number of branch points in apical and basal dendrites in successive zones of 20-#m spheres. B: total number of branching points of all the spheres, counted in 100 neurons from each of the 3 groups of rats (10 rats in each of N, S, E groups). Note significant increase in E group (ANOVA F-test, P < 0.001). C: branching points of the 3 groups of rats represented for successive spheres of counting. Each point on the graph is the mean data of 100 neurons in 10 rats of a group. Note the significant increase in all the spheres in E group. E, self-stimulation experienced group; N, normal control group; S, sham control group, i.e., subject carried implanted electrodes but had not experience of self-stimulation.
173 brain area across groups, to do a balanced statistical assessment. The neurons sampled in LH and SN-VTA were within a distance of about 300/~m from the electrode tip. In hippocampus, the CA3 pyramids were studied. In SNVTA, the majority of neurons are from lateral VTA and medial SN. The cells were of 15-30/~m size, ovoid, polygonal or triangular in shape, and usually gave rise to
( A N O V A , treatment x subjects, data of neurons averaged within subjects) for each of the measurement spheres TM. Although each category had 20 rats, they all were not used in the data contribution to the 3 brain areas assessed (Figs. 1, 2). A few tissues were not well stained and had to be kept aside. Some others, though well stained, have not been included in order to equalize numbers of rats (and total number of neurons) for a given
Dendritic branchin5 points of S N - V T A A. N u m b e r s o f 20 pm r a d i a l
and LH
sphere
SN-VTA n e u r o n
I
jl~ss s
~X~' ~
s
neurons
LH neuron
N
~L
2; I
",,
i
'~
i
// • . ", . / " I f - - - - " "
%%
e-
.I
'%
~ -
ii /
#s # l
"
6.
5
t,'"
T o t a l c o u n t s o f branchin 9 p o i n t s
S N - V T A in 4 s p h e r e s
LH in 3 s p h e r e s
10
"0~ 10
8
LE " B
03 E u
¢_
"
6
~
4
.o
~
4
E c
2
c
2
~-
0
5-o
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S
E
N S
C. in s u c c e s s i v e E 0
a.
spheres
SN- VTA
LH
5
5 X
t- {.
~4 ~E
E
x
E : Experienced
• E N
S : N :
X
•E 4 S
~- =L 3
= 15 r a t s 100 n e u r o n s
3
o 0
Sham control Normal con'trol
E/S
2 z
P < 0.05 F < 0.001
1
1
1
2
3
4
Number of sphere
C 1 (20)Jm)
2 from
3 soma
Fig. 2. C h a n g e s in dendritic branching in n e u r o n s of substantia nigra-ventrai tegmental area (SN-VTA), and of lateral hypothalamus (LH). Data were o f 100 neurons from 15 rats in each of the 3 groups. Other legends as in Fig. 1. Note significant increase in the first 3 spheres in S N - V T A , and in first 2 spheres in L H neurons.
174 3-5 thick, generally smooth primary dendrites that scarcely gave rise to secondary and tertiary dendritic branches. Dendritic varicosities were not commonly seen. The L H neurons studied were mainly from the middle and anterior areas of hypothalamus, along medial forebrain bundle (MFB), and were small, of a diameter of 10-15 /~m. The cells were fusiform, triangular, or spherical in shape, and usually gave rise to 2-3 long and mainly unbranched dendrites. The general features of the SN and L H neurons observed in this study were comparable to those reported earlier 6'8'1°'~6'17. The CA3 pyramidal neurons of hippocampus (Fig. 1) showed an increase of dendritic branching of both the apical and basal dendrites in the E group in contrast to the other groups. The total of branching points of apical dendrites counted in the 6 20-/~m spheres in the E, S and N groups were 13.35 _+ 2.1, 4.98 _+ 0.6, and 5.2 _+ 0.7 respectively, i.e., an increase in E by 168% over the value of S. For basal dendrites, the dendritic branching points were studied up to 60/~m, and the total values of the E, S and N groups were 10.5 _+ 0.93, 6.85 _+ 0.8, and 6.09 + 0.62 respectively, i.e., an increase in E by 53% over the value of S. A calculation for a corresponding radial sphere zone of 0-60/~m of apical dendrites of E showed an increase of 93% over the value of S. Hence, it appeared that the response of apical dendrites was much greater than that of basal dendrites. The E group SN and LH neurons (Fig. 2) also showed significantly higher dendritic branching than those of the S and N groups. For the SN neurons, the total number of branching points (studied up to 80/~m) for the E, S and N groups were 8.5 _+ 1, 5.3 _+ 0.7, and 5.8 _+ 0.99 respectively, i.e., E was larger than S by 60%. For the LH neurons, the total number of branching points (studied up to 60/~m) for the E, S and N groups were 6.62 _+ 0.75, 4.3 + 0.62 and 4.17 _+ 0.3 respectively, i.e., E was larger than S by 54%. For the LH neurons the increase was significant in the first and second metric spheres, whereas for the SN neurons the increase was significant in the third sphere also. In both the regional neurons, the significant increase was mainly in the perisomatic domains of the dendrites. The branching numbers of neurons of the sham control (S) and normal control (N) groups showed no significant differences in the 3 regions compared. This is the first report revealing changes in dendritic 1 Berger, T.W. and Thompson, R.E, Neuronal plasticity in the limbic system during classical conditioning of the rabbit nictitating membrane response. I. The hippocampus, Brain Research, 145 (1978) 323-346. 2 Chang, EE and Greenough, W.T., Lateralised effects of monocular training on dendritic branching in adult split-brain rats, Brain Research, 232 (1982) 283-292.
branching of CA3 neurons of the hippocampus in subjects that have had the experience of intensely rewarding SS behavior. Hippocampus in the rat has been considered to be playing a significant role in spatial exploration and learning 11. Other studies 1'3'7 have provided evidence for involvement of hippocampal neurons in both classical and operant learning paradigms as well. The results of the present study also establish that the neurons of the SS sites (LH and the SN-VTA) undergo changes following the learning for rewarding stimulation. The increase of dendritic branching of LH and SN neurons of the E group cannot be considered to be due to electrode irritation or implantation injury, since the S group showed no such increase. The increase might be related partly to the physical effect of repeated electrical stimulation, although we could not see evidence for such a cause in a preliminary study conducted on 3 other subjects, that were provided the 'experimenter administered' stimulation through similar electrode placements daily for 5 min during 3 days, sacrificed on the 10th day and examined. The total counts (mean _+ S.D.) of the hippocampal CA3 neurons (n = 25) apical dendrites in the radial zone of 0-120/~m was 5.75 _+ 1.68, of the basal dendrites in the zone of 0-60/~m was 6.9 + 1.93, of the SN neurons (n = 15) dendrites in the zone of 0-80/~m was 6.32 + 1.54, and of the L H neurons (n = 15) dendrites in the zone of 0-60 ~tm was 4.80 + 1.14. These indicated no significant changes in the dendritic branching even in the SN and LH that had a direct exposure to the electrical stimulus, therefore suggesting no major effect of the electrical stimulus per se. Nevertheless, one cannot conclude with confidence that the dendritic changes of the LH and SN were only due to the rewarding experience of the conditioned behavior, but not due to electric stimulation. However, even to note that a significant increase in the dendritic branching due to self-stimulation occurred in not only the stimulated areas of SS but also in hippocampus, is itself an important point, since the neurons of hippocampus have not been previously studied in relation to responding for self-stimulation that is known to be most intensely rewarding, perhaps even more than feeding and sexual behaviors in the rat. Such an intensely rewarding impact of the SS could possibly be the factor that caused the kind of massive increase of dendritic branching of the CA3 hippocampal neurons, particularly of the apical dendrites. 3 Christian, E.P. and Deadwyler, S.A., Behavioral functions and hippocampal cell types: evidence for two non-overlapping populations in the rat, J. Neurophysiol., 55 (1986) 331-348. 4 Greenough, W.T., What's special about development? Thoughts on the bases of experience-sensitivesynaptic plasticity. In W.T. Greenough and J.M. Juraska (Eds.), Developmental Neuropsychobiology, Academic Press, Orlando, 1986, pp. 387-407.
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