- Email: [email protected]
A light and electron microscopic study of the development of two regions of the chick forebrain
Developmental Brain Research, 20 (1985) 83-88 Elsevier
83
BRD 50206
A Light and Electron Microscopic Study of the Development of Two Regions of the Chick Forebrain P. BRADLEY
Department of Anatomy, Universityof Newcastle upon Tyne, Newcastleupon Tyne (U.K.) (Accepted December llth, 1984)
Key words: chick - - development - - imprinting - - telencephalon - - quantitative - - synapse
The post hatch development of two regions of the chick telencephalon has been studied using light and electron microscopic quantitative methods. The hyperstriatum ventrale is a region known to be involved in the learning process of imprinting, and hyperstriatum accessorium is a primary telencephalic visual projection area. There were significant changes in synaptic density, neuronal density, glial cell density and in the synapse/neuron ratio in hyperstriatum ventrale. In contrast, hyperstriatum accessorium showed a greater degree of stability. The most rapid changes were seen in the immediate post-hatch period. This coincides with the period during which the critical period for imprinting lies,
INTRODUCTION
METHODS
The newly hatched domestic chick has proved to be a useful experimental animal for studying the neural consequences of learning and visual experience. Recent studies have shown that the intermediate part of the medial hyperstriatum ventrale (IMHV) is involved in the learning process of imprinting 6. In this process young chicks learn the characteristics of a visually conspicuous object by being exposed to it 1. When chicks are trained in this way the mean length of the synaptic apposition zone in the left I M H V is significantly increased 3. It has also been shown that visual experience can affect the responsiveness of neurons in both the hyperstriatum accessorium (HA)5 and the hyperstriatum ventrale 7 to visual stimuli. Unlike I M H V , H A does not appear to be directly implicated in the imprinting process 6. In the present study the development of H A and I M H V has been investigated. Quantitative light and electron microscopical methods have been used to analyse the developmental changes occurring in these regions over a period of time during which the changes related to learning and visual experience have been demonstrated.
We used 27 light-reared 'Ross 1' domestic chicks in this study. Chicks were killed at various intervals after hatching: 5 chicks were killed at each of 0 h, 7 days and 30 days post hatch and 4 chicks were killed at each of 22 h, 48 h and 96 h post hatch. Each chick was anaesthetised by an intraperitoneal injection of 0.3 ml Sagatal anaesthetic (May and Baker, Ltd.), then perfused through the heart with 0.1 M phosphate buffer (pH 7.4) followed by perfusion fixation with 2% glutaraldehyde/2% paraformaldehyde in the same buffer. Each chick was then decapitated, its brain exposed and the head stored in fixative. Two hours later blocks from left and right I M H V and left and right H A were trimmed under stereotaxic control, post fixed in osmium tetroxide, dehydrated, and embedded in Araldite. Two blocks were obtained from each region, giving a total of 8 blocks from each chick. Five semithin (1/xm) toluidine blue stained sections were obtained from each block using a Huxley MkI ultramicrotome. The interval between the sections was at least 15/~m. Cell counts were made from 5 non-overlapping fields on each section. Fields were
Correspondence: P. Bradley, Department of Anatomy, University of Newcastle upon Tyne, Newcastle upon Tyne, U.K.
84 selected by defocussing the microscope prior to moving to a new field. Using a camera lucida attachment all the cells in one field were drawn at a final magnification of 540 x. Cells were only drawn if the nucleus was visible. For each animal at least 500 cells were drawn. From the drawings the numbers of neurons and glial cells were obtained and the diameter of the cell nuclei was measured. The formula Nvn = Ncn/(T + 15) was used to correct the observed cell counts for the effects of section thickness and nuclear diameter; T, section thickness; Ncn observed cell number; Nvn actual cell number. I) is the true mean nuclear diameter and is obtained from the observed mean nuclear diameter, d, by using the formula/3 = 4.d/~8. Neuronal and glial cell densities were expressed as cells/mm 3. In addition to these counts an estimate was made of the numbers of pyknotic neurons in each field. For electron microscopy, ultrathin sections of uniform thickness, as estimated by their refraction colour, were collected on uncoated copper 300-mesh grids and examined using a Jeol 100-S electron microscope. A systematic sampling technique8 was employed to obtain 25 micrographs from each block. The micrographs were printed at a final magnification of 35,400 x and used for quantitative analysis of synaptic morphology. Synapses were identified by the presence of a postsynaptic thickening in conjunction with presynaptic vesicles and classified into those on dendritic shafts and those on dendritic spines. Insufficient profiles were seen on neuronal cell bodies to be quantified. A profile was identified as a dendritic shaft if mitochondria, microtubules or ribosomes were visible. All other postsynaptic profiles were classified as spines. A profile was also identified as a spine if the connecting stalk with a dendrite was visible, or if the spine apparatus was present. A Reichert M O P Digiplan was used to measure the length of the synaptic apposition zone, as defined by the presence of the postsynaptic paramembranous density, and to obtain the numerical densities of shaft and spine synapses. The Schwartz-Saltykov correction procedure was used to produce frequency histograms of synapse size. Point counting techniques were employed to measure the volume density of cell bodies for each set of micrographs and the numerical densities were then corrected so that they were expressed with reference to neuropil. The total numer-
ical density of synapses was then divided by the numerical density of neurons to provide a synapse/neuron ratio for each chick. For a given parameter a mean value was obtained for each chick and for each region. This value combined the data from all the blocks for either I M H V or HA. The mean values were then used for statistical analysis with either standard linear regression tests, analyses of variance, paired or unpaired t-tests. Where a significant variation was revealed by an analysis of variance t-tests were performed on the maximum and minimum points on the curve using residual standard errors. The significance of these t-tests was corrected according to the standard formula for multiple comparisons. All counts were performed without the operator knowing the age of the chick. RESULTS
The curves for neuronal density plotted against age for I M H V and H A are shown in Fig. 1. Neuronal IMHV
110
!°° 100
80
70 '~ ¢J
60
5O 40 30
I 0
I 1
l 2
I 4
I 7
/j
I 30
Age (days) 110 100
HA
90
i
e0
70i
~ 5o 30
I 0
I t
I 2
L, 4 Age (days)
I 7
.
.
i .
.
30-
Means -+ SEM • Neurons ,, Glia
Fig. 1. Plot of mean cell density against age in IMHV and HA.
85 density in both I M H V and H A decreased significantly with age ( I M H V : r = 0.81, P < 0.001, b = 1.53; H A : r = 0.64, P < 0.001, b = 0.82). The rate of decrease in I M H V was significantly higher than that in H A (t = 2.53, P < 0.02). The mean values for I M H V are consistently higher than those for H A . The difference is highly significant (paired t-test; mean difference = 3,751, t = 7.70, P < 0.001). Glial cell density in I M H V showed significant variation with age (analysis of variance, F5,21 = 4.91, P < 0.01). This was evident particularly in a rapid decrease between 0 and 22 h (T = 4.08, P < 0.01). Glial cell density in H A showed no significant variation with age (Fig. 1). The numbers of pyknotic cells observed in both 430
I
H A and I M H V were extremely low at all ages and comprised less than 0.2% of the total neuronal population. The mean length of spine and shaft synaptic apposition zones in I M H V and H A is plotted against age in Fig. 2. Analysis of variance revealed a significant effect of age on the length of shaft synapses in I M H V (F5,21 = 4.78, P < 0.01). This relationship is expressed as an increase in length between 0 and 48 h and a decrease thereafter. No other significant effect of age on synaptic length was detected and this is re-
IMHV SPINES
HA
SHAFT
PINES
SHAFT
IMHV ODAY N'127
N-186
N=IN9
N=138
N=183
N=172
......
i
.2
J" "'\%2..
370
1
DAY ~200
N=l17
340 310 2 DAY
280 ..J
250
I
012
I
I
I
4
"J
"~""--~
Ap (days) 4 DAY
HA
40O
L.< N=173
N=157
N-270
N=275
N=13LI
_~
.-2N5
N=I2N
N'287
N'l~
370 = .o 7 DAY
"~ 310 '~
t
280
250
I 0
I 1
I 2
I 4
I 7 Age (days)
/.,,
I 3O
Means + SEM *- Spines o Shaft
Fig. 2. Mean length of spine and shaft synaptic densities in IMHV and HA plotted against age.
30 FREQUENCyDISTRIBUTION - DIARETER OF SYNAPTICDISKS
Fig. 3 Frequency distribution histograms of shaft and spine lengths. Schwartz-Saltykov corrected data. Values of n refer to the total number of synapses measured in each sample.
86 flected in the relative constancy of the shape of the frequency histograms of synaptic length between 0 and 30 days (Fig. 3). In I M H V there was a significant effect of age on synaptic density (analysis of variance, Fsm = 5.41, P < 0.01) which is seen (Fig. 4) as a steady rise between 0 h and 7 days (r = 0.53, P < 0.02, b = 0.21; t-test 0 h vs 7 days, t = 4.52, P < 0.01) and a decrease thereafter. The rise between 0 h and 7 days appeared to be attributable mainly to a rise over the same period in the density of spine synapses (analysis of variance, Fsm = 5.70, P < 0.01; t-test 0 h vs 7 days, t = 4.13, P < 0.01), since no significant increase in the density of shaft synapses was seen. In H A no significant variation with age was seen in overall synaptic density. This was also true for shaft synapses. However the density of spine synapses showed a significant variation with age (analysis of variance, F5,21 = 4.87, P < 0.01) and a significant increase between 0 h IMHV
5
_J, i3 ¢ |2
I 0
I 1
I 2
I I 4 7 Age (days)
// /,
I 30
HA
i
~ ~,~" ~'-"'--...i.......,,x......_.i ......
o
I 0
I 1
I 2
~
........
I 4 ACe (days)
• .......
,,,, .......
I 7
//
-
,°t
80
i°L 60
40 30
tT 0
t
i
I
I
1
2
4
7
Age (days)
/,I
I 30
Means + SEM • IMHV HA
Fig. 5. Plot of synapse/neuron ratio against age in IMHV a HA. Values expressed in arbitrary units.
and 4 days (t = 3.36, P < 0.01). The density of s 3 apses in H A was significantly higher than that I M H V up to 4 days of age but not thereafter (0 h h, t = 2.71, P < 0.05; 4 days vs 4 days, t = 2.81, P 0.05; 7 days vs 7 days, t = 0.03). The curve of the synapse/neuron ratio against a in both I M H V and H A appeared to increase logaril mically (Fig. 5). This increase was significant wh the data were plotted on a logarithmic scale ( I M H r = 0.58, P < 0.01, b = 0.09; H A ; r = 0.49, P < 0.( b = 0.08). The rate of increase in the synapse/neur, ratio was higher in I M H V than in H A , The ratio w significantly higher in H A than I M H V at 0 h (t 6.01, P < 0.001) but not by 7 days (t = 1.644). DISCUSSION
3
--'r~'~-z
100
I
30
Means -+SEM • Total synaptic density ~r Spine synaptic density o Shaft synaptic density
Fig. 4. Plot of synaptic density against age in IMHV and HA. Values expressed in arbitrary units (synapses/micrograph).
The decrease in neuronal density observed in tt study is a characteristic feature of the developing n( vous system 4. There are 3 main factors which m contribute to this decrease: cell death, glial prolih ation and neuronal growth. The effects of cell dea are difficult to estimate, but at none of the ages e amined was there any indication of sufficient nu~ bers of pyknotic cells to account for the total d crease in cell density. That glial proliferation was r sponsible for at least some of the decline in neuron density with age is shown by the relative constancy glial cell numbers over the same period. If there hl been no glial proliferation it is likely that glial cc
87 density would have fallen at the same rate as neuronal density. The increase in the synapse/neuron ratio suggests either that some growth of dendritic arborisations had taken place to accommodate the greater number of synapses present on each neuron or that the extra synapses were accommodated by an increase in synaptic density on existing dendrites. Both mechanisms would be expected to contribute to some extent to the observed increase in overall synaptic density in I M H V since both the addition of new synapses onto existing dendrites and the growth of new synapse bearing dendrites into existing neuropil will increase the relative density of synapses. However, new dendritic growth would also lead to an increase in the total volume of neuropil and thus synapses present on these new dendrites would contribute less to increases in synaptic density than would the addition of new synapses on to existing dendrites. It is possible that the growth of new dendrites predominates in the later stages of development when, although the synapse/neuron ratio is rising, overall synaptic density is constant or falling; while the addition of new synapses onto existing dendrites may predominate in the immediate posthatch period. There is some evidence that in one of the classes of cell present in IMHV, the pyramidal type cell, there is continued growth of terminal dendrites over the first week posthatch 2. In this case it is likely that the new synapses are formed on spines since spine synapse density shows its greatest rise between 0 h and 4 days after hatching. H A and I M H V both show changes with age. For a number of measures the rate of change in I M H V is greater than that in HA, for example in the decline in neuronal density, in the increase in synapse/neuron ratio and in the increase in total synaptic density. If low synaptic density and high neuronal density can be taken as indicative of a relatively immature neuronal system then I M H V would appear to be more immature than H A at the time of hatching but to have reached approximately the same level of maturity as H A by 30 days posthatch. Such comparisons can
REFERENCES 1 Bateson, P. P. G., The characteristics and context of imprinting, Biol. Rev. , 41 (1966) 177-220. 2 Bradley, P., A Golgi study of the development of the IMHV
however be dangerous since different regions may possess inherently different characteristics. The rise in the synapse/neuron ratio with age for both I M H V and H A is logarithmic. This relationship clearly indicates that the most vigorous changes are those which occur during the immediate posthatch period and that the rate of change slows with age. There is no apparent change with age in the length of synaptic apposition zones in H A or in the length of spine synapse apposition zones in IMHV. The only observed change in apposition zone length was in shaft synapses in IMHV. The significance of these findings is difficult to assess since the effect on mean synaptic length of the growth of existing synapses may be offset by the addition of new synapses with shorter apposition zones. However in these circumstances a broadening of the distribution curve of synapse length would be expected. No such effect was observed and if anything the distributions tend to narrow with age suggesting a stabilisation of synaptic size. Imprinting normally takes place during the first or second days posthatch. At the time of hatching I M H V may be relatively immature when compared with HA. The results of this study show that changes are taking place in both H A and I M H V in the immediate posthatch period. The greatest degree of change is seen in I M H V between 0 h and 4 days of age. During this period there are significant changes in synaptic density, in glial cell density, in the length of shaft synapses and in the synapse/neuron ratio. It may be that the labile nature of this forebrain region is an important factor affecting the speed and efficiency with which young chicks learn. Obviously it is not possible to state that imprinting only takes place because of this lability and in fact it may be argued that the learning processes occur despite the developmental changes going on around them. Nevertheless when analysing behavioural plasticity in newly hatched chicks it is important to be aware of the immature nature of two of the forebrain regions which may be involved.
of the domestic chick, Fidia Res, Ser., Front. Neurosci., Abstr. Bk, 1 (1983) 149-151. 3 Bradley, P. and Horn, G., Imprinting: an electron microscopic study of chick hyperstriatum ventrale, Exp. Brain Res., 41 (1981), 115-120.
88 4 Brizzee, K. R., Vogt, J. and Kharetchko, X., Postnatal changes in glia/neuron index with a comparison of methods of cell enumeration in the white rat. In D. P. Purpura and J. P. Schade (Eds.), Progress in Brain Research, Vol. 4 Elsevier, Amsterdam, (1964) 136-146. 5 Brown, M. and Horn, G. Neuronal plasticity in the chick brain: electrophysiological effects of visual experience on hyperstriatal neurons, Brain Res., 162 (1979), 142-147. 6 Horn, G. Neural mechanisms of learning: an analysis of ira-
printing in the domestic chick, Proc. roy. Soc. B, 213 ( 198l ) 101-137.
7 Payne J. K. and Horn G., Differential effects of exposure to an imprinting stimulus on ~spontaneous' neuronal activity in two regions of the chick brain. Brain Res., in press. 8 James, N. T. Stereology. In G. A. Meek and H. Y. Elder (Eds.), Analytical and Quantitative Methods in Microscopy, Cambridge Univ. Press, London, 1977, pp. 9-28.
83
BRD 50206
A Light and Electron Microscopic Study of the Development of Two Regions of the Chick Forebrain P. BRADLEY
Department of Anatomy, Universityof Newcastle upon Tyne, Newcastleupon Tyne (U.K.) (Accepted December llth, 1984)
Key words: chick - - development - - imprinting - - telencephalon - - quantitative - - synapse
The post hatch development of two regions of the chick telencephalon has been studied using light and electron microscopic quantitative methods. The hyperstriatum ventrale is a region known to be involved in the learning process of imprinting, and hyperstriatum accessorium is a primary telencephalic visual projection area. There were significant changes in synaptic density, neuronal density, glial cell density and in the synapse/neuron ratio in hyperstriatum ventrale. In contrast, hyperstriatum accessorium showed a greater degree of stability. The most rapid changes were seen in the immediate post-hatch period. This coincides with the period during which the critical period for imprinting lies,
INTRODUCTION
METHODS
The newly hatched domestic chick has proved to be a useful experimental animal for studying the neural consequences of learning and visual experience. Recent studies have shown that the intermediate part of the medial hyperstriatum ventrale (IMHV) is involved in the learning process of imprinting 6. In this process young chicks learn the characteristics of a visually conspicuous object by being exposed to it 1. When chicks are trained in this way the mean length of the synaptic apposition zone in the left I M H V is significantly increased 3. It has also been shown that visual experience can affect the responsiveness of neurons in both the hyperstriatum accessorium (HA)5 and the hyperstriatum ventrale 7 to visual stimuli. Unlike I M H V , H A does not appear to be directly implicated in the imprinting process 6. In the present study the development of H A and I M H V has been investigated. Quantitative light and electron microscopical methods have been used to analyse the developmental changes occurring in these regions over a period of time during which the changes related to learning and visual experience have been demonstrated.
We used 27 light-reared 'Ross 1' domestic chicks in this study. Chicks were killed at various intervals after hatching: 5 chicks were killed at each of 0 h, 7 days and 30 days post hatch and 4 chicks were killed at each of 22 h, 48 h and 96 h post hatch. Each chick was anaesthetised by an intraperitoneal injection of 0.3 ml Sagatal anaesthetic (May and Baker, Ltd.), then perfused through the heart with 0.1 M phosphate buffer (pH 7.4) followed by perfusion fixation with 2% glutaraldehyde/2% paraformaldehyde in the same buffer. Each chick was then decapitated, its brain exposed and the head stored in fixative. Two hours later blocks from left and right I M H V and left and right H A were trimmed under stereotaxic control, post fixed in osmium tetroxide, dehydrated, and embedded in Araldite. Two blocks were obtained from each region, giving a total of 8 blocks from each chick. Five semithin (1/xm) toluidine blue stained sections were obtained from each block using a Huxley MkI ultramicrotome. The interval between the sections was at least 15/~m. Cell counts were made from 5 non-overlapping fields on each section. Fields were
Correspondence: P. Bradley, Department of Anatomy, University of Newcastle upon Tyne, Newcastle upon Tyne, U.K.
84 selected by defocussing the microscope prior to moving to a new field. Using a camera lucida attachment all the cells in one field were drawn at a final magnification of 540 x. Cells were only drawn if the nucleus was visible. For each animal at least 500 cells were drawn. From the drawings the numbers of neurons and glial cells were obtained and the diameter of the cell nuclei was measured. The formula Nvn = Ncn/(T + 15) was used to correct the observed cell counts for the effects of section thickness and nuclear diameter; T, section thickness; Ncn observed cell number; Nvn actual cell number. I) is the true mean nuclear diameter and is obtained from the observed mean nuclear diameter, d, by using the formula/3 = 4.d/~8. Neuronal and glial cell densities were expressed as cells/mm 3. In addition to these counts an estimate was made of the numbers of pyknotic neurons in each field. For electron microscopy, ultrathin sections of uniform thickness, as estimated by their refraction colour, were collected on uncoated copper 300-mesh grids and examined using a Jeol 100-S electron microscope. A systematic sampling technique8 was employed to obtain 25 micrographs from each block. The micrographs were printed at a final magnification of 35,400 x and used for quantitative analysis of synaptic morphology. Synapses were identified by the presence of a postsynaptic thickening in conjunction with presynaptic vesicles and classified into those on dendritic shafts and those on dendritic spines. Insufficient profiles were seen on neuronal cell bodies to be quantified. A profile was identified as a dendritic shaft if mitochondria, microtubules or ribosomes were visible. All other postsynaptic profiles were classified as spines. A profile was also identified as a spine if the connecting stalk with a dendrite was visible, or if the spine apparatus was present. A Reichert M O P Digiplan was used to measure the length of the synaptic apposition zone, as defined by the presence of the postsynaptic paramembranous density, and to obtain the numerical densities of shaft and spine synapses. The Schwartz-Saltykov correction procedure was used to produce frequency histograms of synapse size. Point counting techniques were employed to measure the volume density of cell bodies for each set of micrographs and the numerical densities were then corrected so that they were expressed with reference to neuropil. The total numer-
ical density of synapses was then divided by the numerical density of neurons to provide a synapse/neuron ratio for each chick. For a given parameter a mean value was obtained for each chick and for each region. This value combined the data from all the blocks for either I M H V or HA. The mean values were then used for statistical analysis with either standard linear regression tests, analyses of variance, paired or unpaired t-tests. Where a significant variation was revealed by an analysis of variance t-tests were performed on the maximum and minimum points on the curve using residual standard errors. The significance of these t-tests was corrected according to the standard formula for multiple comparisons. All counts were performed without the operator knowing the age of the chick. RESULTS
The curves for neuronal density plotted against age for I M H V and H A are shown in Fig. 1. Neuronal IMHV
110
!°° 100
80
70 '~ ¢J
60
5O 40 30
I 0
I 1
l 2
I 4
I 7
/j
I 30
Age (days) 110 100
HA
90
i
e0
70i
~ 5o 30
I 0
I t
I 2
L, 4 Age (days)
I 7
.
.
i .
.
30-
Means -+ SEM • Neurons ,, Glia
Fig. 1. Plot of mean cell density against age in IMHV and HA.
85 density in both I M H V and H A decreased significantly with age ( I M H V : r = 0.81, P < 0.001, b = 1.53; H A : r = 0.64, P < 0.001, b = 0.82). The rate of decrease in I M H V was significantly higher than that in H A (t = 2.53, P < 0.02). The mean values for I M H V are consistently higher than those for H A . The difference is highly significant (paired t-test; mean difference = 3,751, t = 7.70, P < 0.001). Glial cell density in I M H V showed significant variation with age (analysis of variance, F5,21 = 4.91, P < 0.01). This was evident particularly in a rapid decrease between 0 and 22 h (T = 4.08, P < 0.01). Glial cell density in H A showed no significant variation with age (Fig. 1). The numbers of pyknotic cells observed in both 430
I
H A and I M H V were extremely low at all ages and comprised less than 0.2% of the total neuronal population. The mean length of spine and shaft synaptic apposition zones in I M H V and H A is plotted against age in Fig. 2. Analysis of variance revealed a significant effect of age on the length of shaft synapses in I M H V (F5,21 = 4.78, P < 0.01). This relationship is expressed as an increase in length between 0 and 48 h and a decrease thereafter. No other significant effect of age on synaptic length was detected and this is re-
IMHV SPINES
HA
SHAFT
PINES
SHAFT
IMHV ODAY N'127
N-186
N=IN9
N=138
N=183
N=172
......
i
.2
J" "'\%2..
370
1
DAY ~200
N=l17
340 310 2 DAY
280 ..J
250
I
012
I
I
I
4
"J
"~""--~
Ap (days) 4 DAY
HA
40O
L.< N=173
N=157
N-270
N=275
N=13LI
_~
.-2N5
N=I2N
N'287
N'l~
370 = .o 7 DAY
"~ 310 '~
t
280
250
I 0
I 1
I 2
I 4
I 7 Age (days)
/.,,
I 3O
Means + SEM *- Spines o Shaft
Fig. 2. Mean length of spine and shaft synaptic densities in IMHV and HA plotted against age.
30 FREQUENCyDISTRIBUTION - DIARETER OF SYNAPTICDISKS
Fig. 3 Frequency distribution histograms of shaft and spine lengths. Schwartz-Saltykov corrected data. Values of n refer to the total number of synapses measured in each sample.
86 flected in the relative constancy of the shape of the frequency histograms of synaptic length between 0 and 30 days (Fig. 3). In I M H V there was a significant effect of age on synaptic density (analysis of variance, Fsm = 5.41, P < 0.01) which is seen (Fig. 4) as a steady rise between 0 h and 7 days (r = 0.53, P < 0.02, b = 0.21; t-test 0 h vs 7 days, t = 4.52, P < 0.01) and a decrease thereafter. The rise between 0 h and 7 days appeared to be attributable mainly to a rise over the same period in the density of spine synapses (analysis of variance, Fsm = 5.70, P < 0.01; t-test 0 h vs 7 days, t = 4.13, P < 0.01), since no significant increase in the density of shaft synapses was seen. In H A no significant variation with age was seen in overall synaptic density. This was also true for shaft synapses. However the density of spine synapses showed a significant variation with age (analysis of variance, F5,21 = 4.87, P < 0.01) and a significant increase between 0 h IMHV
5
_J, i3 ¢ |2
I 0
I 1
I 2
I I 4 7 Age (days)
// /,
I 30
HA
i
~ ~,~" ~'-"'--...i.......,,x......_.i ......
o
I 0
I 1
I 2
~
........
I 4 ACe (days)
• .......
,,,, .......
I 7
//
-
,°t
80
i°L 60
40 30
tT 0
t
i
I
I
1
2
4
7
Age (days)
/,I
I 30
Means + SEM • IMHV HA
Fig. 5. Plot of synapse/neuron ratio against age in IMHV a HA. Values expressed in arbitrary units.
and 4 days (t = 3.36, P < 0.01). The density of s 3 apses in H A was significantly higher than that I M H V up to 4 days of age but not thereafter (0 h h, t = 2.71, P < 0.05; 4 days vs 4 days, t = 2.81, P 0.05; 7 days vs 7 days, t = 0.03). The curve of the synapse/neuron ratio against a in both I M H V and H A appeared to increase logaril mically (Fig. 5). This increase was significant wh the data were plotted on a logarithmic scale ( I M H r = 0.58, P < 0.01, b = 0.09; H A ; r = 0.49, P < 0.( b = 0.08). The rate of increase in the synapse/neur, ratio was higher in I M H V than in H A , The ratio w significantly higher in H A than I M H V at 0 h (t 6.01, P < 0.001) but not by 7 days (t = 1.644). DISCUSSION
3
--'r~'~-z
100
I
30
Means -+SEM • Total synaptic density ~r Spine synaptic density o Shaft synaptic density
Fig. 4. Plot of synaptic density against age in IMHV and HA. Values expressed in arbitrary units (synapses/micrograph).
The decrease in neuronal density observed in tt study is a characteristic feature of the developing n( vous system 4. There are 3 main factors which m contribute to this decrease: cell death, glial prolih ation and neuronal growth. The effects of cell dea are difficult to estimate, but at none of the ages e amined was there any indication of sufficient nu~ bers of pyknotic cells to account for the total d crease in cell density. That glial proliferation was r sponsible for at least some of the decline in neuron density with age is shown by the relative constancy glial cell numbers over the same period. If there hl been no glial proliferation it is likely that glial cc
87 density would have fallen at the same rate as neuronal density. The increase in the synapse/neuron ratio suggests either that some growth of dendritic arborisations had taken place to accommodate the greater number of synapses present on each neuron or that the extra synapses were accommodated by an increase in synaptic density on existing dendrites. Both mechanisms would be expected to contribute to some extent to the observed increase in overall synaptic density in I M H V since both the addition of new synapses onto existing dendrites and the growth of new synapse bearing dendrites into existing neuropil will increase the relative density of synapses. However, new dendritic growth would also lead to an increase in the total volume of neuropil and thus synapses present on these new dendrites would contribute less to increases in synaptic density than would the addition of new synapses on to existing dendrites. It is possible that the growth of new dendrites predominates in the later stages of development when, although the synapse/neuron ratio is rising, overall synaptic density is constant or falling; while the addition of new synapses onto existing dendrites may predominate in the immediate posthatch period. There is some evidence that in one of the classes of cell present in IMHV, the pyramidal type cell, there is continued growth of terminal dendrites over the first week posthatch 2. In this case it is likely that the new synapses are formed on spines since spine synapse density shows its greatest rise between 0 h and 4 days after hatching. H A and I M H V both show changes with age. For a number of measures the rate of change in I M H V is greater than that in HA, for example in the decline in neuronal density, in the increase in synapse/neuron ratio and in the increase in total synaptic density. If low synaptic density and high neuronal density can be taken as indicative of a relatively immature neuronal system then I M H V would appear to be more immature than H A at the time of hatching but to have reached approximately the same level of maturity as H A by 30 days posthatch. Such comparisons can
REFERENCES 1 Bateson, P. P. G., The characteristics and context of imprinting, Biol. Rev. , 41 (1966) 177-220. 2 Bradley, P., A Golgi study of the development of the IMHV
however be dangerous since different regions may possess inherently different characteristics. The rise in the synapse/neuron ratio with age for both I M H V and H A is logarithmic. This relationship clearly indicates that the most vigorous changes are those which occur during the immediate posthatch period and that the rate of change slows with age. There is no apparent change with age in the length of synaptic apposition zones in H A or in the length of spine synapse apposition zones in IMHV. The only observed change in apposition zone length was in shaft synapses in IMHV. The significance of these findings is difficult to assess since the effect on mean synaptic length of the growth of existing synapses may be offset by the addition of new synapses with shorter apposition zones. However in these circumstances a broadening of the distribution curve of synapse length would be expected. No such effect was observed and if anything the distributions tend to narrow with age suggesting a stabilisation of synaptic size. Imprinting normally takes place during the first or second days posthatch. At the time of hatching I M H V may be relatively immature when compared with HA. The results of this study show that changes are taking place in both H A and I M H V in the immediate posthatch period. The greatest degree of change is seen in I M H V between 0 h and 4 days of age. During this period there are significant changes in synaptic density, in glial cell density, in the length of shaft synapses and in the synapse/neuron ratio. It may be that the labile nature of this forebrain region is an important factor affecting the speed and efficiency with which young chicks learn. Obviously it is not possible to state that imprinting only takes place because of this lability and in fact it may be argued that the learning processes occur despite the developmental changes going on around them. Nevertheless when analysing behavioural plasticity in newly hatched chicks it is important to be aware of the immature nature of two of the forebrain regions which may be involved.
of the domestic chick, Fidia Res, Ser., Front. Neurosci., Abstr. Bk, 1 (1983) 149-151. 3 Bradley, P. and Horn, G., Imprinting: an electron microscopic study of chick hyperstriatum ventrale, Exp. Brain Res., 41 (1981), 115-120.
88 4 Brizzee, K. R., Vogt, J. and Kharetchko, X., Postnatal changes in glia/neuron index with a comparison of methods of cell enumeration in the white rat. In D. P. Purpura and J. P. Schade (Eds.), Progress in Brain Research, Vol. 4 Elsevier, Amsterdam, (1964) 136-146. 5 Brown, M. and Horn, G. Neuronal plasticity in the chick brain: electrophysiological effects of visual experience on hyperstriatal neurons, Brain Res., 162 (1979), 142-147. 6 Horn, G. Neural mechanisms of learning: an analysis of ira-
printing in the domestic chick, Proc. roy. Soc. B, 213 ( 198l ) 101-137.
7 Payne J. K. and Horn G., Differential effects of exposure to an imprinting stimulus on ~spontaneous' neuronal activity in two regions of the chick brain. Brain Res., in press. 8 James, N. T. Stereology. In G. A. Meek and H. Y. Elder (Eds.), Analytical and Quantitative Methods in Microscopy, Cambridge Univ. Press, London, 1977, pp. 9-28.