Acute stress‐induced neuronal plasticity in the corticoid complex of 15‐day‐old chick, Gallus domesticus

Abstract

Several studies conducted on chicken have shown that a single stress exposure may impair or improve memory as well as learning processes. However, to date, stress effects on neuronal morphology are poorly investigated wherefore it was of interest to evaluate this further in chicks. Thus, the present study aims to investigate the role of single acute stress (AS) of 24 h food and water deprivation in neuronal plasticity in terms of spine density of the corticoid complex (CC) in 15‐day‐old chick, Gallus domesticus, by using three neurohistological techniques: Cresyl Violet, Golgi Colonnier, and Golgi Cox technique. The dorsolateral surface of the cerebral hemisphere is occupied by CC which can be differentiated into two subfields: an intermediate corticoid (CI) subfield (arranged in layers) and a dorsolateral corticoid (CDL) subfield. Based on different criteria such as soma shape, dendritic branching pattern, and dendritic spine density, two main moderately spinous groups of neuronal cells were observed in the CC, namely, projection neurons (comprising of multipolar and pyramidal neurons) and stellate neurons. In the present study, the stellate neurons have shown a significant decrease as well as an increase in their spine density in both CI and CDL subfields, whereas the multipolar neurons had shown a significant increase in their spine density in the CDL region only. The present study shows that AS induces neuronal plasticity in terms of spine density in both CI and CDL neurons. The morphological changes in the form of decreased dendritic branches due to stress have been observed in the CI region in comparison to CDL region, which could be linked to more effect of stress in this region. The avian CDL corresponds to the entorhinal cortex of mammals on the basis of neuronal morphology and bidirectional connections between adjacent areas. The projection neurons increase their branches and also their spine number to cope with the stress effects, while the stellate neurons show contrasting effect in their spine density. Therefore, this study will establish that slight modifications in natural stimuli or environmental changes faced by the animal may affect their dorsolateral forebrain which shows neuronal plasticity that help in the development of an adaptive capacity of the animal to survive under changing environmental conditions.

In present study, we have investigated the role of single acute stress (AS) in inducing the neuronal plasticity in the corticoid complex (CC) of 15 day’s old chick, Gallus domesticus by using Cresyl Violet, Golgi Colonnier and Golgi Cox methods. Our study revealed the CC is present at the dorsolateral surface towards caudal end of the cerebrum and contains highly spinous multipolar, pyramidal and stellate neurons. All the neurons show remarkable fluctuations in their morphological features most especially in spine density and dendritic branching pattern as a result of AS effects that could have important outcomes for functions of the CC in emotion, cognition and memory.

1. INTRODUCTION

The avian hippocampal formation (HF) is supposed to be homologous to the mammalian hippocampus, being involved in memory formation, particularly spatial memory (Atoji et al., 2002; Colombo & Broadbent, 2000; Cosme et al., 2003; Lee et al., 1998; Sherry et al., 1992; Shiflett et al., 2004). The caudal pole of the avian HF is considered to be homologous to the ventral pole of the rodent hippocampus (Gualtieri et al., 2019). The corticoid complex (CC) of birds has been supposed to participate in spatial memory (Atoji & Wild, 2005; Colombo et al., 2001). The CC of birds is present at the dorsolateral surface of the telencephalic pallium which can be divided into two subregions: an intermediate corticoid (CI) area adjacent to the parahippocampal area (APH) and a dorsolateral corticoid (CDL) area (Montagnese et al., 1996; Srivastava, Chand, et al., 2009; Srivastava et al., 2014). At the caudal most level the CI and hippocampal complex (HCC) progressively disappear, while the APH remains visible and limited laterally by the CDL in finches (Montagnese et al., 1996; Srivastava, Chand, et al., 2009) and Indian house crow (Srivastava et al., 2014).

The CI could constitute a zone of transition between the HA and HCC based on neuronal types (Montagnese et al., 1996; Srivastava, Chand, et al., 2009; Srivastava et al., 2014) and the distribution of neurotransmitters (Krebs et al., 1991), neuropeptides (Erichsen et al., 1991), zinc, and calbindin (Montagnese, Geneser, et al., 1993; Montagnese, Krebs, et al., 1993). The morphological characteristics of neuronal types have been studied by different researchers in the CC of the strawberry finch (Srivastava, Chand, et al., 2009), Indian house crow (Srivastava et al., 2014), and hippocampal and CC of the zebra finch (Montagnese et al., 1996). All of these studies have shown the presence of dominant multipolar neurons, pyramidal neurons, and stellate neurons in both food storing and nonfood storing birds (Montagnese et al., 1996; Srivastava, Chand, et al., 2009; Srivastava et al., 2014). On the basis of afferent and efferent connections of CDL in Pigeon, it is suggested that the CDL region of birds has been considered as a part of the limbic system (Atoji & Wild, 2005) and it may be comparable with the mammalian entorhinal cortex as it is included in the limbic system (Atoji & Wild, 2005). The CI and CDL in birds work as interface between adjacent regions of hippocampus, whereas the same function is carried out in the mammals by the entorhinal cortex between neocortex and HF (Kerr et al., 2007).

The whole‐brain possess a remarkable ability to undergo functionally relevant adaptations following external and/or internal stimuli like stress and is generally referred to as neural plasticity (Chattarji et al., 2015; Drevets, 2004; McEwen, 2007). The stress induces structural remodeling of neuronal architecture for the adaptation of stressor commonly by strengthening the synaptic transmission (Krugers et al., 2010) as failing to do so may contribute to the onset and recurrence of mood disorders like depression and anxiety (McEwen, 2007; Southwick & Charney, 2012). Stress induced by adverse experiences may lead to acute as well as chronic changes at multiple levels of neural organization such as neuronal replacement, dendritic remodeling, and synapse turnover (Buwalda et al., 2005; Chattarji et al., 2015; Hammels et al., 2015; McEwen, 1998). Acute stress (AS) exposure modifies memory processes and exerts beneficial effects on memory acquisition (Joëls et al., 2006) as well as impairs memory retrieval (de Quervain et al., 1998). Under normal circumstances, AS does not lead to a pathological state but may influence particular brain circuits by modulating synaptic plasticity, which finally influences the animal's behavior (Aguayo et al., 2018). Even a single AS exposure causes increased spine density and heightened anxiety, without changing dendritic arborization (Mitra et al., 2005).

In the adult brain, axons and dendrites remain relatively stable, while dendritic spines appear to be the primary site of structural plasticity (Holtmaat & Svoboda, 2009). Neuroplasticity due to stress is an adaptive response to different stimuli that comprises multiple interacting mediators that influence the structural and functional plasticity of the brain (McEwen & Gianaros, 2010). In adult chickens, the AS resulted in several physiological changes, such as a significant change in body weight (Puvadolpirod & Thaxton, 2000; Shini et al., 2009) and increased feather pecking (Ellethey et al., 2001). Increase in spatial learning function of CC as an effect of stress has also been seen in chickens (Goerlich et al., 2012).

The information about AS effects on dendritic spine density needs to be explored because these areas of the limbic system comparatively received less attention than the hippocampal area. Birds have been used as models for learning and memory and the neural basis of cognition for decades (Emery, 2006). Domestic chicks are the primary model for studying the development and neurobiology of learning and memory (Nakamori et al., 2013). In chickens, the stress effects are poorly investigated wherefore it was of interest to evaluate this further. The present study aims to investigate the role of single AS in inducing the neuronal plasticity in terms of dendritic spine density in both CI and CDL subfields of CC of 15‐day‐old stressed chick and its comparison with the neuronal spine densities with the CC of nonstressed chick, along with the brief important background to neuronal classes, dendritic spines for better understanding.

2. MATERIALS AND METHODS

2.1. Experimental animals

Fourteen chicks, Gallus domesticus, of 20‐cm mean size and having an average weight of 105 g, were used in the present study. The length from the tip of the beak to the endpoint of the tail feather was measured. They were purchased from nearby PAHARI Poultry House (A government department), Hawalbag, Almora. The chicks were brought to the laboratory on the 14th day of their hatching and maintained in the laboratory condition prior to the experiments for 24 h to release stress due to their transport. After 24 h, chicks were divided into two groups of eight (Group 1, Nonstress) and six (Group 2, AS). The chicks of Group 1 were maintained for the next 24 h in the laboratory under normal environmental conditions with free access to food and water (Nonstress, NS) while the chicks of Group 2 were maintained in similar environmental condition without food and water (AS). The present research work was approved by IAEC with protocol no. KUDOPS/106, and all the experimental procedures were carried out according to the guidelines of the Animal Ethics Committee of the Kumaun University, Nainital, Uttarakhand, India.

2.2. Nissl staining

After 24 h, two chicks of group 1 (NS) were sacrificed for Cresyl Violet (Nissl Stain) Method to study the cytoarchitectonic subdivisions of the CC. Chicks were deeply anaesthetized by administering a lethal dose of ketamine. Their heads were decapitated, and the brain was immediately taken out from the skull and postfixed in 10% formalin solution at 4°C. After 24 h of fixation, the brains were rinsed briefly with double distilled water (2–3 min) and then dehydrated in an increasing series of alcohol (30%, 50%, 70%, 90%, and 100% ethanol) for 15–30 min in each, cleared in xylene (15–20 min), and finally embedded in fresh filtered molten paraffin wax (m.p. 56–58°C). Serial sections (10 μm thick) were cut with the aid of a rotary microtome, and the sections were placed onto Mayer's Albumen Glycerol coated glass slides for spreading, dried in dark at room temperature (RT) for overnight and also inside an incubator at 37°C for 20 min. The slides were deparaffinized and cleared in xylene for 15 min. The sections were dehydrated in descending grades of alcohols (100%, 90%, 70%, 50%, and 30%) for 10 min in each and then stained with Cresyl‐violet stain for 2–3 min. Additionally, the sections were dehydrated in an increasing series of alcohol (30%, 50%, 70%, 90%, and 100% ethanol) for 5 min in each, cleared in xylene (5 min), and finally mounted on slides using DPX mounting medium. The slides were allowed to dry overnight at RT in dark, labeled, and studied under a light microscope as well as by bright field microscopy.

2.3. Golgi Colonnier method (Blaesing et al., 2001)

Golgi method stains a limited number of cells at random in their entirety (1%–5% approximately). For the Golgi Colonnier method six chicks, three from group 1 (NS) and three from group 2 (AS) were deeply anaesthetized by administering a lethal dose of ketamine. The brain was immediately removed from the skull and immersed in the 2% paraformaldehyde in 0.1‐M phosphate buffer (pH 7.4) for 24 h at 4°C. After 24 h of fixation, the brains were rinsed briefly with double distilled water (2–3 min) and the two cerebral hemispheres were separated from each other, prechromed in 2.5% potassium dichromate (2 × 60 min), and transferred to 5% glutaraldehyde (v/v) and 2% potassium dichromate (w/v) solution at 4°C for 3 days for chroming. For impregnation, each brain was transferred to silver nitrate solution 0.75% (w/v) at 4°C for 2 days after prewashing in the same solution. Both the chroming and impregnation steps were repeated two times. Each brain was washed two to three times in distilled water between each chroming and impregnation steps. After the completion of the second and final impregnation, each brain was dehydrated in ascending grades of alcohol (30 min in each), cleared in xylene (10 min), and embedded in paraffin for sectioning. The brain sections of 120 μm thick were cut with the microtome, and the sections were deparaffinized and mounted with DPX for inspection.

2.4. Golgi–Cox staining

The six chicks were studied by using Golgi–Cox method three from group 1 (NS) and three from group 2 (AS). After 24 h, all the birds of both groups were sacrificed with an overdose of ketamine. The brain was immediately removed from the skull and immersed in the 4% paraformaldehyde in 0.1‐M phosphate buffer (pH 7.4) for 30 min at RT. After 30 min the brain was transferred into the filtered Golgi–Cox solution (Levine et al., 2013) and stored in dark for 24 h at RT. The next day, the solution was changed and the brains were stored in dark for 14 days at RT. After impregnation, each brain was washed and stored in 1% potassium dichromate solution in dark for 24 h. The next day, the brain was washed two to three times in distilled water and dehydrated in ascending grades of alcohol, cleared in xylene, and embedded in paraffin for sectioning. The brain sections of 120 μm thick were cut with the microtome, and the sections were deparaffinized in xylene and dehydrated in descending grades of alcohol. Sections were then placed in 1% potassium dichromate, 28% ammonia solution, and 1% sodium thiosulfate for 5 min in each. After this, the sections were dehydrated, cleared in xylene, and mounted in D.P.X.

2.5. Microscopic analysis

The microphotographs of the whole cresyl violet stained sections were taken from the original permanent slides with the help of a computer‐aided microscope (stereo‐zoom‐S9i) at a magnification of 40x (4x × 10x), and the boundaries of the whole sections, as well as the desired area (CC), were traced for the drawing of stacked serial sections. Sections were studied under a light microscope, and selected neurons were photographed with a computer‐aided microscope (Leica). Microphotographs depicting Golgi stained neurons of the desired region were taken from the original permanent slides at a particular focus at which the soma was clearly visible. Camera lucida drawings of all the selected neurons were drawn very carefully and precisely from original slides with the help of a camera lucida attached to a simple light microscope at 400x (40x × 10x) primary magnification at various focal planes in the desired region. Microphotographs of selected dendritic segments that did not leave the plane of focus of Golgi stained neurons were taken from the original permanent slides by using a computer‐aided microscope at 400x (40x × 10x) magnification. All the drawings were scanned by using a scanner and corrected with the help of Adobe Photoshop 7.0 computer software inside the computer. All types of microphotographs as well as their drawings were scaled by using paint software on computer before forming and labeling the photo‐plates. The drawings of the synaptic distributions represent actual observations. After examining, the slides were subsequently stored in slide boxes in the dark at RT for long time usage.

2.6. Neurohistological data analysis

The total number of neuron (×) was found out by counting the neurons directly from Golgi impregnated slides under the simple light compound microscope at 400x (40x × 10x) primary magnification and then their percentage was calculated. Various neuronal morphological features, namely, dendritic field, soma diameter, dendritic diameter, spine length, spine head diameter, spine density, axonal length, and projection, were calculated with the help of a computer‐aided microscope at 400x (40x × 10x) magnification. The numbers of dendritic branches touching the 25‐, 50‐, and 100‐µm radius circles from soma center were counted, and axonal lengths were measured directly from camera lucida drawing of the neurons.

To calculate spine density, numbers of dendritic spines (N) were counted per 25 µm of a dendritic segment in 10 segments from each type of neuron from CI and CDL regions. Only dendrites that are lying in a plane, parallel to the section, were used for analysis. The dendritic diameter was taken as the average of three points along the shaft segment under analysis, and the dendritic radius (Dr) was calculated as half of the dendritic diameter. The perpendicular linear distance from the dendritic shaft surface to the longest dendritic spine was taken as spine length (Sl), and the mean of three spine head diameter (Sd) was also measured. The number of visible spines is represented as spine density 1, which underestimates the exact number of spines (Horner & Arbuthnott, 1991), whereas spine density 2 provides a more accurate estimate of the true spine density because it also includes an estimate of spines present on the other side of the dendritic circumference that is obscured in the spine density 1 calculation (Srivastava et al., 2014). Feldman and Peters gave a mathematical formula to calculate spine density (Feldman & Peters, 1979). Spine density 1 = n/Dl and spine density 2 = N/Dl, where n is the number of visible spines, N the corrected spine numbers, and Dl the dendritic length over which spines were counted. The N is calculated as the following equation:

$$N=\frac{n\pi [{(\text{Dr}+\text{Sl})}^2‐{(\text{Dr}+\text{Sd})}^2]}{\left[\frac{\theta }{90}\pi {(\text{Dr}+\text{Sl})}^2\right]‐2[(\text{Dr}+\text{S1})\sin \theta (\text{Dr}+\text{Sd})]}$$

where n is the number of visible spines, Dr the radius of the dendrite, Sd the spine head diameter, Sl the spine length, and $\theta$ the central angle.

2.7. Statistical analysis

The comparative percentage of increased as well as decreased neuronal dendritic spine density was analyzed by using a Student's paired t‐test (with Welch's correction). A minimum criterion of probability level p < 0.05 was accepted as indicative of significant difference. Results were presented as the mean ± standard error of the mean. All the statistical analyses of neurons were performed using Microsoft Excel, Graph Pad Prism, and Adobe Photoshop softwares.

3. RESULTS

3.1. Cytoarchitectonic subdivisions of CC

The present study, based on neurohistological techniques (Nissl‐staining, Golgi‐impregnation), focuses on the cyto‐architecture of the CC in the Chick, G. domesticus. The cresyl violet stained sections revealed that the dorsolateral surface of the cerebral pallium is occupied by the dorsolateral forebrain which is also termed as CC, after the replacement of hyperpallium apicale (HA) (Figure 1). In CC two subfields have been recognized: a wide region present adjacent to the HF is referred to as CI area (Figures 1 and 2a) and laterally placed a thin, superficial, narrow strip‐like structure is called the CDL area (Figures 1 and 2b). At the caudal end, only the CDL and APH have been observed as the CI progressively disappears (Figure 1). In CI, three dorsolaterally arranged layers have been identified, based on the neuronal cyton or soma size and distribution (Figure 2c,e). The outermost layer that is present towards the piamatter (dorsal surface) of the cerebral hemisphere is referred to as Layer‐I which contains less densely packed, small‐sized, purple‐blue colored somas (Figure 2c), whereas the middle Layer‐II is present in the center of the CI region and contains moderately packed small to large‐sized, blue colored somas (Figure 2c). The innermost layer that is present beneath the Layer‐II, towards the ventricle, and contains less densely packed small‐ to medium‐sized purple‐blue somas of neurons is called Layer‐III (Figure 2c). The CDL contains uniformly distributed, small‐ to medium‐sized moderately packed, purple‐blue colored neuronal somas arranged in the narrow striped structure without any layer pattern as observed in CI (Figure 2d,f).

FIGURE 1.

Drawing of stacked serial cresyl violet stained sections (from back to front sections A–O) of the dorsolateral corticoid complex (CC) of 15‐day‐old chick, Gallus domesticus, showing the position of the hippocampal formation (HF), the intermediate corticoid subfield (CI), and the dorsolateral corticoid subfield (CDL) at various levels (from B to K). At the caudal most level, the corticoid complex (CC) is represented by the CDL only and the HF by the parahippocampal area (APH) only (from L to O). Below each section, the distance from the previous section is given from rostral to caudal end. The last section (section A) is 3960 μm posterior from the rostral end of the cerebral hemisphere. Scale bar = 2 μm

FIGURE 2.

Microphotographs showing the Cresyl Violet stained sections (from a to f) of the dorsolateral corticoid complex of 15‐days old chick, Gallus domesticus, (a, c, e) intermediate corticoid subfield, (b, d, f) showing the dorsolateral corticoid subfield (CDL) at different magnification levels, (c) showing the Layer‐I, Layer‐II, and Layer‐III of CI subfield. (e, f) Microphotographs representing the uniformly distributed cells having soma of variable sizes. Arrow‐1 indicates the small‐sized soma, arrow‐2 indicates the medium‐sized soma, and arrow‐3 indicates the large‐sized soma in both CI and CDL. V—ventricle, pia dorsal surface of the cerebral hemisphere, NC—Neocortex. (a, b) 40x (4x × 10x), (c, d) 100x (10x × 10x), (e, f) 400x (40x × 10x) with scale bars 500, 200, and 50 μm, respectively

3.2. Neuronal classes of CC

After the complete analysis of results obtained from Golgi stained sections, the author observed two major spinous neuronal groups, namely, projection neurons (consisting of highly spinous multipolar and pyramidal neurons) and moderately spinous stellate neurons, based on various neuronal features, namely, soma diameter, dendritic field and diameter, dendritic spine density, length and head diameter, dendritic branching points, axonal length, and projection, in both the CI and CDL subfields of both nonstressed and stressed 15‐day‐old chick (Tables 1, 2, 3; Figures 3, 4, 5, 6, 7 and 9, 10, 11, 12, 13, 14). Different neuronal characteristics such as soma diameter, dendritic field, and number of dendritic branches at the 25‐, 50‐, 100‐μm radius circles (Figure 5) have been observed in CC of 15‐day‐old chick, G. domesticus (Table 1). Average axonal length has been observed up to the visible point of axon, and the direction of axon and axon collaterals is noted towards which axons are projecting (Table 3).

TABLE 1.

The total number and percentage of neurons, mean values of dendritic field and soma diameter, and average number of dendritic branches at 25‐, 50‐, and 100‐µm radius circle from soma center in different neuronal classes observed in the intermediate corticoid area (CI) and dorsolateral corticoid area (CDL) of corticoid complex (CC) of 15‐day‐old nonstress (NS) and acute stress (AS) chick, Gallus domesticus

Field	Type of neuron	Number of neurons		%age of neurons		Soma diameter (µm) (Mean ± SEM)		Dendritic field (µm) (Mean ± SEM)		Average dendritic branches at 25‐µm radius from soma		Average dendritic branches at 50‐µm radius from soma		Average dendritic branches at 100‐µm radius from soma
		NS	AS	NS	AS	NS	AS	NS	AS	NS	AS	NS	AS	NS	AS
CI	Multipolar	153	345	37.50	51.11	20.20 ± 0.62	18.29 ± 0.67	283.00 ± 2.73	253.60 ± 7.52	11.17	12.17	13.67	12.67	5.83	4.17
	Pyramidal	135	120	33.09	17.78	20.03 ± 0.93	20.38 ± 0.84	254.70 ± 8.35	257.20 ± 5.66	13.00	10.50	18.00	11.5	7.00	4.50
	Stellate	120	210	29.41	31.11	19.21 ± 0.74	20.86 ± 0.67	140.30 ± 6.74	166.10 ± 7.14	9.50	5.50	4.00	4.00	0.00	0.00
CDL	Multipolar	255	405	51.52	60.00	18.80 ± 0.92	16.92 ± 1.15	244.20 ± 5.10	239.40 ± 6.75	11.17	9.83	13.00	16.67	5.83	5.33
	Pyramidal	105	165	21.21	24.44	17.79 ± 0.94	17.03 ± 0.65	242.90 ± 7.55	265.90 ± 6.09	9.75	11.00	10.25	11.00	3.75	3.00
	Stellate	135	105	27.27	15.56	18.54 ± 0.73	16.61 ± 0.73	145.10 ± 5.62	157.30 ± 4.49	9.00	6.00	4.50	3.50	0.00	0.00

TABLE 2.

Showing the mean dendritic diameter of different neuronal cell types along with various dendritic spine characteristics observed in the intermediate corticoid area (CI) and dorsolateral corticoid area (CDL) of corticoid complex (CC) of 15‐day‐old nonstress (NS) and acute stress (AS) chick, Gallus domesticus

Field	Type of neuron	Dendritic diameter (µm) (mean ± SEM)		Spine length (µm) (mean ± SEM)		Spine head diameter (µm) (mean ± SEM)		Spine density (n) per 25 µm (mean ± SEM)		Corrected spine density (N) per 25 µm (mean ± SEM)
		NS	AS	NS	AS	NS	AS	NS	AS	NS	AS
CI	Multipolar	1.41 ± 0.06	1.41 ± 0.06	1.66 ± 0.07	1.89 ± 0.08	1.22 ± 0.03	1.28 ± 0.04	18.10 ± 0.72	21.60 ± 0.86	69.05 ± 5.83	73.34 ± 6.53
	Pyramidal	1.34 ± 0.06	1.43 ± 0.05	1.57 ± 0.05	1.73 ± 0.07	1.22 ± 0.02	1.39 ± 0.04	18.70 ± 0.73	19.90 ± 0.60	80.22 ± 7.20	89.19 ± 5.26
	Stellate	1.55 ± 0.08	1.53 ± 0.09	1.72 ± 0.07	1.80 ± 0.02	1.42 ± 0.06	1.28 ± 0.04	18.20 ± 0.55	19.60 ± 0.50	87.56 ± 4.37	67.77 ± 3.07
CDL	Multipolar	1.38 ± 0.06	1.31 ± 0.04	1.91 ± 0.07	2.06 ± 0.08	1.20 ± 0.03	1.26 ± 0.04	18.50 ± 0.79	23.80 ± 1.40	54.61 ± 1.50	67.01 ± 5.28
	Pyramidal	1.45 ± 0.07	1.28 ± 0.04	1.99 ± 0.10	2.07 ± 0.07	1.24 ± 0.05	1.21 ± 0.02	16.90 ± 0.38	22.30 ± 1.12	50.37 ± 2.65	59.39 ± 3.95
	Stellate	1.34 ± 0.08	1.38 ± 0.10	1.89 ± 0.05	1.84 ± 0.12	1.17 ± 0.02	1.27 ± 0.05	17.10 ± 1.22	20.20 ± 0.71	49.30 ± 4.02	68.33 ± 5.37

TABLE 3.

The average axonal length and axonal projections of the different neurons observed in both the intermediate (CI) and dorsolateral corticoid (CDL) subfields of the corticoid complex (CC) of nonstress (NS) and under acute stress (AS) of 15‐day‐old chick, Gallus domesticus

Field	Type of neuron	Average axonal length (µm)		Axonal projection
		NS	AS	NS	AS
CI	Multipolar	46.67	42.33	APH; CDL; local; dorsal and ventral	APH; CDL; dorsal and ventral
	Pyramidal	49.5	40.5	APH; CDL; dorsal and ventral	APH; CDL; dorsal and ventral
	Stellate	17.5	19.5	Local; dorsal and ventral	Local; dorsal and ventral
CDL	Multipolar	35.67	36.33	CI; local; dorsal and ventral	CI; local; dorsal and ventral
	Pyramidal	61.25	49.25	CI; dorsal and ventral	CI; local; dorsal and ventral
	Stellate	22	25	CI; local; dorsal and ventral	Local; dorsal and ventral

FIGURE 3.

Microphotographs representing the Golgi impregnated multipolar projection neurons with spinous dendrites, axon, and axon collaterals of CI subfield of CC in nonstressed (a, c, e) and stressed (b, d, f) 15‐day‐old chick, Gallus domesticus. d—dendrites, ax—axon, c—axon collaterals, and arrow—spines. Scale bars = 50 μm

FIGURE 4.

Microphotographs representing the Golgi impregnated (a–d) pyramidal projection neurons and (e–f) stellate neurons with spinous dendrites, axon, and axon collaterals of CI subfield of CC in (a, c, e) nonstressed and (b, d, f) stressed 15‐day‐old chick, Gallus domesticus. d—dendrites, ax—axon, c—axon collaterals, and arrow—spines. Scale bars = 50 μm

FIGURE 5.

Camera lucida drawings of the neurons with spinous dendrites, axon, and axon collaterals, observed in the intermediate corticoid area of corticoid complex in nonstressed 15‐day‐old chick, Gallus domesticus. Cell 1—multipolar projection neurons; Cell 2—pyramidal projection neurons. Camera lucida drawing of one pyramidal projection neuron showing how branches were counted at the circles of radius 25, 50, and 100 μm from soma center. The branches crossing the circle were counted. The location of these neuronal classes corresponds to the serial section E in Figure 1. Inset shows the respective position of these cells. d—dendrites, ax—axon, c—axon collaterals, and arrow—dendritic spines. Scale bars = 50 μm

FIGURE 6.

Camera lucida drawings of the neurons with spinous dendrites, axon, and axon collaterals, observed in the intermediate corticoid area of corticoid complex in nonstressed 15‐day‐old chick, Gallus domesticus. Cell 1—multipolar projection neurons; Cell 3—stellate neuron showing d—dendrites, ax—axon, c—axon collaterals, and arrow—dendritic spines. The location of these neuronal classes corresponds to the serial section D in Figure 1. Inset shows the respective position of these cells. Scale bars = 50 μm

FIGURE 7.

Camera lucida drawings of the neurons with spinous dendrites, axon, and axon collaterals, observed in the intermediate corticoid area of corticoid complex in stressed 15‐day‐old chick, Gallus domesticus. Cell 1—multipolar projection neurons; Cell 2—pyramidal projection neurons; Cell 3—stellate neuron showing d—dendrites, ax—axon, c—axon collaterals, and arrow—dendritic spines. Insets show the respective position of these cells. Scale bars = 50 μm

FIGURE 9.

Microphotographs representing the Golgi impregnated multipolar projection neurons with spinous dendrites, axon, and axon collaterals of CDL subfield of CC in nonstressed (a, c, e) and stressed (b, d, f) 15‐day‐old chick, Gallus domesticus. d—dendrites, ax—axon, c—axon collaterals, and arrow—dendritic spines. Scale bars = 50 μm

FIGURE 10.

Microphotographs representing the Golgi impregnated (a–d) pyramidal projection neurons and (e,f) stellate neurons with spinous dendrites, axon, and axon collaterals of CDL subfield of CC in (a, c, e) nonstressed and (b, d, f) stressed 15‐day‐old chick, Gallus domesticus. d—dendrites, ax—axon, c—axon collaterals, and arrow—dendritic spines. Scale bars = 50 μm

FIGURE 11.

Camera lucida drawings of the neurons with spinous dendrites, axon, and axon collaterals observed in the dorsolateral corticoid area of corticoid complex in nonstressed 15‐day‐old chick, Gallus domesticus. Cell 1—multipolar projection neurons; Cell 2—pyramidal projection neurons showing d—dendrites, ax—axon, c—axon collaterals, arrow—dendritic spines. Inset shows the respective position of these cells. Scale bars = 50 μm

FIGURE 12.

Camera lucida drawings of the neurons with spinous dendrites, axon, and axon collaterals, observed in the dorsolateral corticoid area of corticoid complex in nonstressed 15‐day‐old chick, Gallus domesticus. Cell 1—multipolar projection neurons; Cell 2—pyramidal projection neurons; Cell 3—stellate neuron showing d—dendrites, ax—axon, c—axon collaterals, and arrow—dendritic spines. Insets show the respective position of these cells. Scale bars = 50 μm

FIGURE 13.

Camera lucida drawings of the neurons with spinous dendrites, axon, and axon collaterals, observed in the dorsolateral corticoid area of corticoid complex in stressed 15‐day‐old chick, Gallus domesticus. Cell 1—multipolar projection neurons; Cell 2—pyramidal projection neurons; Cell 3—stellate neuron showing d—dendrites, ax—axon, c—axon collaterals, and arrow—dendritic spines. Inset shows the respective position of these cells. Scale bars = 50 μm

FIGURE 14.

Camera lucida drawings of the neurons with spinous dendrites, axon, and axon collaterals, observed in the dorsolateral corticoid area of corticoid complex in stressed 15‐day‐old chick, Gallus domesticus. Cell 1—multipolar projection neurons; Cell 2—pyramidal projection neurons showing d—dendrites, ax—axon, c—axon collaterals, and arrow—dendritic spines. Inset shows the respective position of these cells Scale bars = 50 μm

Both Golgi techniques show that the spinous neurons have the spine covering over their dendritic branches. The dendritic spines are the tiny morphological specializations of variable shapes and size that protrude from the main shaft of neuronal cylindrical dendrites into the visible flanking zones at some distance away from the dendritic base point. A spine consists of two parts: a bulbous head and a narrow neck. The spine head is connected to the dendritic shaft with the help of its neck. The morphology of the spines is highly variable: some have a short neck with a small head and others have long neck with large oval heads while some are stubby or sessile. Some spines are perpendicular to the shaft and others are oblique. Thus, morphologically four types of dendritic spines, namely, filopodia, stubby, thin, and mushroom, were observed in the CI subfield of both nonstressed (Figure 8a–f) and stressed (Figure 8g–l) chick as well as in the CDL subfield of both nonstressed (Figure 15a–f) and stressed (Figure 15g–l) chick. The dendritic spine characteristics observed in nonstress (NS) and under AS chick of the neurons of CI and CDL region have been shown in Table 2. The 25‐μm‐long segments of the spinous cylindrical dendritic shaft that did not leave the plane of focus and also lying in a plane parallel to the section have been selected for the study. The mean dendritic diameter, mean spine length (perpendicular linear distance from the surface of the dendritic shaft to the distal tip of the spine head), mean spine head diameter and visible spine density observed, and the corrected spine density have been calculated for 25‐μm dendritic segment (Table 2).

FIGURE 8.

Microphotographs and camera lucida drawings show the Golgi impregnated dendritic segments with different types of spines observed in (CI) intermediate corticoid area of nonstressed (a–f) and stressed (g–l) 15‐day‐old chick, Gallus domesticus. Panels (a, d, g, j) represent the spinous dendritic segments of multipolar, (b, e, h, k) pyramidal, and (c, f, i, l) stellate neurons. Arrows‐1, 2, 3 and 4 indicate the filopodia, stubby, thin and mushroom‐shaped dendritic spines respectively. Scale bars = 20 μm

FIGURE 15.

Microphotographs and camera lucida drawings show the Golgi impregnated dendritic segments with different types of spines observed in (CDL) dorsolateral corticoidal neurons of nonstressed (a–f) and stressed (g–l) 15‐day‐old chick, Gallus domesticus. Panels (a, d, g, j) represent the spinous dendritic segments of multipolar, (b, e, h, k) pyramidal, and (c, f, i, l) stellate neurons. Arrows 1, 2, 3, and 4 indicate the filopodia, stubby, thin, and mushroom‐shaped dendritic spines, respectively. Scale bars = 20 μm

3.3. Effects of AS on the CI area

3.3.1. Multipolar projection neurons

Multipolar spiny neurons are the most dominant neurons and distributed uniformly (37.5%, x = 153, nonstress; 51.11%, x = 345, AS) in the CI. They possess medium‐ to large‐sized somata with a diameter of 20.20 ± 0.62 µm in nonstress and 18.29 ± 0.67 µm in AS. The oval, spherical, rectangular, multiangular to irregularly shaped somata of these neurons extend five to eight (five to seven in nonstress; six to eight in AS) long spinous dendrites (Figure 3 and Table 1). The extension of dendrites is in all direction with a mean dendritic field of 283.00 ± 2.73 µm in nonstress and 253.60 ± 7.52 µm in AS (Table 1). The primary dendrites after a short course further give off oblique secondary and tertiary dendritic side branches that also extend in all possible directions and fashioned as tree‐shaped structure in nonstressed (Figures 3a,c,e; 5.1; and 6.1) as well as in stressed chick (Figures 3b,d,f and 7.1). These neurons have average dendritic branches 11.17 (NS) and 12.17 (AS) at 25‐μm radius from the soma, 13.67 (NS) and 12.67 (AS) at 50‐μm radius from the soma, and 5.83 (NS) and 4.17 (AS) at 100‐μm radius from the soma (Table 1).

The dendritic spine characteristics observed in nonstress (NS) and under AS chick of multipolar neurons (Figure 8a,d,g,j) have been shown in Table 2. The mean dendritic diameter is 1.41 ± 0.06 μm (both in NS and AS), mean spine length observed is 1.66 ± 0.07 μm (NS) and 1.89 ± 0.08 μm (AS), and mean spine head diameter is 1.22 ± 0.03 μm (NS) and 1.28 ± 0.04 μm (AS) (Table 2). The multipolar projection neurons of CI region in both nonstressed and stressed chick show the mean values of 18.10 ± 0.72 (NS) and 21.60 ± 0.86 (AS) for visible spine density and 69.05 ± 5.83 (NS) and 73.34 ± 6.53 (AS) for corrected spine density within 25 μm, long spinous dendritic segment (Table 2).

The axon originates from the soma of the multipolar neurons with an average length of 46.67 μm (NS) and 42.33 μm (AS) from the soma center, and sometimes after a short course, it bifurcates into two side branches and form finer axon collaterals (c) which run‐up to the dendrites of the same neuron or the dendrites of neighboring neurons. The axon and axon collaterals run in all possible directions local, towards APH region, CDL area, dorsal surface, and ventral side in CI of nonstressed chick (Figure 3a,c,e; 5.1; and 6.1) and towards APH region, CDL area, dorsal surface, and ventral side in stressed chick (Figures 3b,d,f and 7.1 and Table 3).

3.3.2. Pyramidal projection neurons

The uniformly distributed pyramidal neurons are the second most dominant type of projection neurons found in CI of chick, accounting for 33.09% (x = 135) in the nonstress condition in comparison to 17.78% (x = 120) under AS (Table 1). The pyramidal projection neurons possessed medium‐ to large‐sized triangular‐, pyramidal‐, or cone‐shaped somata having a mean soma diameter of 20.03 ± 0.93 µm in nonstress and 20.38 ± 0.84 µm in AS (Table 1). The soma gives rise to the thick apical dendrites that extend towards the dorsal wall of the telencephalon, whereas the basal dendrites go towards the ventricle. The apical and basal dendrites are not clearly visible, and sometimes additional dendrites from other positions are also identified in presently studied bird. Thus, pyramidal neurons are identified and classified here mostly on the basis of somata shape. The dendritic extension is mostly in apical (towards the dorsal pial surface) and basal (towards ventricle) direction with a mean of 254.70 ± 8.35 µm in nonstress and 257.20 ± 5.66 µm in AS (Table 1). The primary dendrites after a short course further give off oblique secondary and tertiary dendritic side branches that also extend in all possible directions and fashioned as tree‐shaped structure in both nonstressed (Figures 4a,c and 5.2) as well as in stressed chick (Figures 4b,d and 7.2). These neurons have average dendritic branches of 13.00, 18.00, and 7.00 in nonstress and 10.50, 11.50, and 4.50 in AS at 25‐, 50‐, and 100‐μm radius circles from soma (Table 1).

The dendritic spine characteristics of pyramidal neurons (Figure 8b,e,h,k and Table 2) show the mean dendritic diameter of 1.34 ± 0.06 μm (NS) and 1.43 ± 0.05 μm (AS), mean spine length 1.57 ± 0.05 μm (NS) and 1.73 ± 0.07 μm (AS), and mean spine head diameter 1.22 ± 0.02 μm (NS) and 1.39 ± 0.04 μm (AS) (Table 2). The pyramidal projection neurons of CI region in both nonstressed and stressed chick show the mean values of 18.70 ± 0.73 (NS) and 19.90 ± 0.60 (AS) for visible spine density and 80.22 ± 7.20 (NS) and 89.19 ± 5.26 (AS) for corrected spine density within 25 μm, long spinous dendritic segment (Table 2).

The axon originates from the basal pole of the pyramidal neurons having an average length of 49.5 μm (NS) and 40.5 μm (AS) from the soma center, and sometimes after a short course, it bifurcates into two side branches and form finer axon collaterals (c), which run‐up to the dendrites of the same neuron or the dendrites of neighboring neurons in all possible directions, namely, in local, towards APH region, the CDL area, the dorsal surface and ventral side in CI of nonstressed (Figures 4a,c and 5.2) and towards APH region, the CDL area, the dorsal surface, and ventral side in CI of in stressed chick (Figures 4b,d and 7.2 and Table 3).

3.3.3. Stellate neurons

The uniformly distributed stellate neurons were the least abundant type of neurons found in CI of nonstressed chick, accounting for 29.41% (x = 120), whereas in CI of stressed chick they are the second most abundant cell type and accounting for 31.11% (x = 210) (Table 1). The stellate neurons possessed small‐ to medium‐sized oval, spherical, or circular perikaryon with a mean soma diameter of 19.21 ± 0.74 µm in nonstress and 20.86 ± 0.67 µm in AS and show 5–10 (nonstress), 3–6 (acute stress) thin wavy dendrites heading to all directions (Figures 6.3 and 7.3 and Table 1). Dendritic extensions were seen 140.30 ± 6.74 μm during nonstress, while somewhat increased 166.10 ± 7.14 µm during AS (Table 1). The primary dendrites after a short course further give off oblique secondary and tertiary dendritic side branches that also extend in all possible directions and fashioned as tree‐shaped structure in both nonstressed (Figures 4e and 6.3) as well as in stressed chick (Figures 4f and 7.3). These neurons show 9.50, 4.00, and 0.00 average dendritic branches in CI of nonstressed chick and 5.50, 4.00, and 0.00 average dendritic branches in CI of stressed chick at 25‐, 50‐, and 100‐μm radius circle, respectively, from the center of the neuronal soma (Table 1).

The dendritic spine characteristics of stellate neurons (Figure 8c,f,i,l and Table 2) show the mean dendritic diameter of 1.55 ± 0.08 μm (NS) and 1.53 ± 0.09 μm (AS), mean spine length 1.72 ± 0.07 μm (NS) and 1.80 ± 0.02 μm (AS), and mean spine head diameter 1.42 ± 0.06 μm (NS) and 1.28 ± 0.04 μm (AS) (Table 2). The stellate neurons of CI region in both nonstressed and stressed chick show the mean values of 18.20 ± 0.55 (NS) and 19.60 ± 0.50 (AS) for visible spine density and 87.56 ± 4.37 (NS) and 67.77 ± 3.07 (AS) for corrected spine density within 25 μm, long spinous dendritic segment (Table 2).

The axon comes out either from the somata or from the primary dendrite of the stellate neurons having an average length of 17.5 μm (NS) and 19.5 μm (AS) from the soma center. Sometimes after a short course axon bifurcates into two side branches and form finer axon collaterals (c), which run‐up to the dendrites of the same neuron or the dendrites of neighboring neurons in all possible directions. Axon of stellate neurons shows their extension in local, towards dorsal surface and ventral side in nonstressed (Figures 4e and 6.3) towards CDL area, dorsal surface, and ventral side (Figures 4f and 7.3) in stressed chick (Table 3).

3.4. Effects of AS on the CDL area

3.4.1. Multipolar projection neurons

The multipolar projection neurons are the most dominant type of projection neurons found, accounting for 51.52% (x = 255) and 60.00% (x = 405), and were uniformly distributed throughout the CDL subfield of CC of nonstressed and stressed 15‐day‐old chick, respectively (Table 1). These neurons possessed medium‐ to large‐sized perikaryon with a mean soma diameter of 18.80 ± 0.92 μm (NS) and 16.92 ± 1.15 μm (AS) having oval, spherical, multi‐angular, and irregular soma. The multipolar neurons have 4–9 (NS) and 5–10 (AS) spinous dendritic branches, originating from their cyton towards all possible directions with mean dendritic field 244.20 ± 5.10 μm (NS) and 239.40 ± 6.75 μm (AS) in CDL region of chick (Table 1). The primary dendrites, after a short course, further give off oblique secondary and tertiary dendritic side branches that also extend in all possible directions as tree‐shaped structure both in nonstressed (Figures 9a,c,e, 11.1 and 12.1) and acute stressed (Figures 9b,d,f 13.1, and 14.1) chick. These neurons have average dendritic branches 11.17, 13.00, and 5.83 (in NS chick) and 9.83, 16.67, and 5.33 (in AS chick) at 25‐μm radius from soma, 50‐μm radius from soma, and 100‐μm radius from soma (Table 1).

The dorsolateral corticoidal multipolar neurons of nonstressed chick and stressed chick (Figure 15a,d,g,j) show the mean dendritic diameter of 1.38 ± 0.06 μm (NS), 1.31 ± 0.04 μm (AS) (Table 2). The spines of the cylindrical dendritic shaft of multipolar neurons selected for study show 1.91 ± 0.07 μm (NS) and 2.06 ± 0.08 μm (AS) mean spine length and 1.20 ± 0.03 μm (NS) and 1.26 ± 0.04 μm (AS) mean spine head diameter (Table 2). The multipolar projection neurons of CI region of both nonstressed and stressed chick show the mean values of 18.50 ± 0.79 (NS) and 23.80 ± 1.40 (AS) for visible spine density and 54.61 ± 1.50 (NS) and 67.01 ± 5.28 (AS) for corrected spine density within 25‐μm‐long spinous dendritic segment (Table 2).

The axon originates from the soma of the multipolar neurons with average length of 35.67 μm (NS) and 36.33 μm (AS), from the soma center. Sometimes after a short course, it bifurcates into two side branches and form finer axon collaterals (c), which run‐up to the dendrites of the same neuron or the dendrites of neighboring neurons in all possible directions namely such as in local, towards APH region, CDL area, dorsal surface, and ventral side in nonstressed chick (Figures 9a,c,e, 11.1, and 12.1) and towards APH region, CDL area, dorsal surface, and ventral side in CI (Figures 9b,d,f, 13.1, and 14.1) of stressed chick (Table 3).

3.4.2. Pyramidal projection neurons

The pyramidal neurons are uniformly distributed, least abundant type of projection neurons present in CDL of nonstressed chick, accounting for 21.21% (x = 105), whereas in CDL of stressed chick, they are the second most abundant cell type and accounting for 24.44% (x = 165) (Table 1). The pyramidal projection neurons possessed medium‐ to large‐sized triangular‐, pyramidal‐, or cone‐shaped perikaryon with a mean soma diameter of 17.79 ± 0.94 μm (NS) and 17.03 ± 0.65 μm (AS) (Table 1). The pyramidal neurons have five to nine spinous dendritic branches, originating from their cyton towards different directions having mean dendritic field 242.90 ± 7.55 μm in nonstressed chick, whereas six to seven spinous dendritic branches, originating from their cyton and extending in all possible directions with mean dendritic field of 265.90 ± 6.09 μm in CDL region of stressed chick (Table 1). The primary dendrites after a short course further give off oblique secondary and tertiary dendritic side branches that also extend in all possible directions and fashioned as tree‐shaped structure in nonstressed (Figures 10a,c, 11.2; and 12.2) as well as in stressed chick (Figures 10b,d, 13.2, and 14.2). These neurons have average dendritic branches of 9.75, 10.25, and 3.75 in nonstress and 11, 11, 3 in AS at 25‐, 50‐, and 100‐μm radius from the soma (Table 1).

The dendritic spine characteristics of pyramidal neurons (Figure 15b,e,h,k and Table 2) show the mean dendritic diameter of 1.45 ± 0.07 μm (NS) and 1.28 ± 0.04 μm (AS), mean spine length 1.99 ± 0.10 μm (NS) and 2.07 ± 0.07 μm (AS), and mean spine head diameter 1.24 ± 0.05 μm (NS) and 1.21 ± 0.02 μm (AS) (Table 2). The pyramidal projection neurons of CDL region in both nonstressed and stressed chick show the mean values of 16.90 ± 0.38 (NS) and 22.30 ± 1.12 (AS) for visible spine density and 50.37 ± 2.65 (NS) and 59.39 ± 3.95 (AS) for corrected spine density within 25 μm, long spinous dendritic segment (Table 2).

The axon originates from the soma of the pyramidal neurons having an average length of 61.25 μm (NS) and 49.25 μm (AS) from the soma center, and sometimes after a short course, it bifurcates into two side branches to form finer axon collaterals (c). These axon collaterals may run‐up to the dendrites of the same neuron or the dendrites of neighboring neurons in all possible directions (Table 3), namely, in local, towards APH region, CDL area, dorsal surface, and ventral side in nonstressed (Figures 10a,c, 11.2, and 12.2) chick and towards APH region, CDL area, dorsal surface, and ventral side in stressed chick (Figures 10b,d, 13.2, and 14.2).

3.4.3. Stellate neurons

The unevenly distributed stellate neurons are the second most abundant type of projection neurons found in CDL region of nonstressed chick, accounting for 27.27% (x = 135), whereas in CDL of stressed chick, they are the least abundant cell type and accounting for 15.56% (x = 105) (Table 1). The stellate neurons possessed medium‐ to large‐sized perikaryon with a mean soma diameter of 18.54 ± 0.73 μm (NS) and 16.61 ± 0.73 μm (AS), having variable shapes, namely, oval, spherical, and circular. The stellate neurons have five to eight (in NS) and five to nine (in AS) spinous dendritic branches, originating from their cyton towards all possible directions with mean dendritic field 145.10 ± 5.62 μm (NS) and 157.30 ± 4.49 μm (AS) in CDL region of 15‐day‐old chick (Table 1). The primary dendrites after a short course further give off oblique secondary and tertiary dendritic side branches extending to all directions in nonstressed (Figures 10e and 12.3) and in stressed (Figures 10f and 13.3) chick. These neurons show an average dendritic branch of 9.00, 4.50, and 0.00 in nonstress and 6.00, 3.50, and 0.00 in AS at 25‐, 50‐, and 100‐μm radius circles from soma center (Table 1).

The stellate neurons of CDL show the mean dendritic diameter of 1.34 ± 0.08 μm (NS) and 1.38 ± 0.10 μm (AS) within 25‐μm‐long segment of the spinous cylindrical dendritic shaft (Figure 15c,f,i,l and Table 2). The dendritic shaft of stellate neurons observed shows mean spine length of 1.89 ± 0.05 μm (NS), 1.84 ± 0.12 μm (AS), and mean spine head diameter of 1.17 ± 0.02 μm (NS) and 1.27 ± 0.05 μm (AS) (Table 2). The stellate neurons of CDL region of both nonstressed and stressed chick show the mean values of 17.10 ± 1.22 and 20.20 ± 0.71 for visible spine density and 49.30 ± 4.02 and 68.33 ± 5.37 for corrected spine density within 25‐μm long spinous dendritic segment (Table 2).

The axon originates from the soma of the stellate neurons having an average length of 22 and 25 μm from the soma center in both nonstressed and stressed chick, and sometimes after a short course, it bifurcates into two side branches to forms finer axon collaterals (c). These axon collaterals run‐up to the dendrites of the same neuron or the dendrites of neighboring neurons locally, towards dorsal surface, and ventral side in nonstressed chick (Figures 10e and 12.3) and towards the CDL area, dorsal surface, and ventral side in stressed (Figures 10f and 13.3) chick (Table 3).

3.5. Statistical data analysis

Statistical analysis of the data given in the results of the present study revealed that the single AS of 24‐h food and water deprivation in the 15‐day‐old chick, G. domesticus, induces a remarkable fluctuations in neuronal plasticity in terms of spine density in CI and CDL subfields of CC.

3.5.1. Spine density in CI subfield

The results of the unpaired t‐test with Welch's correction for corrected spine number of the different neurons observed in nonstress (NS) and under AS (Table 4) show that the stellate neurons of CI region show a significant decrease (p < 0.05) in their spine density within 25‐μm dendritic segment due to AS (Table 4; Graph), whereas the multipolar and pyramidal projection neurons show an insignificant increase (p < 0.05) in their spine density within the 25‐μm dendritic segment due to AS (Table 4 and Figure 16).

TABLE 4.

Showing both the significant and insignificant results of unpaired t‐test with Welch's correction for corrected spine number per 25‐µm‐long spinous dendritic segment of the different neuronal cells observed in the corticoid complex (CI and CDL) of 15‐day‐old nonstress (NS) and acute stress (AS) chick, Gallus domesticus

Region	Type of neurons	Corrected spine number / 25 µm (mean ± SEM)		t test (with Welch's correction) at p < 0.05
		Non stress	Acute stress	Degree of freedom	ttable values	Tcalculated values	Significant/insignificant
CI	Multipolar	69.05 ± 5.827	73.34 ± 6.526	17	2.11	0.4911	Insignificant
	Pyramidal	80.22 ± 7.201	89.19 ± 5.263	16	2.12	1.006	Insignificant
	Stellate	87.56 ± 4.367	67.77 ± 3.068	16	2.12	3.707	*Significant
CDL	Multipolar	54.61 ± 1.498	67.01 ± 5.282	10	2.23	2.258	*Significant
	Pyramidal	50.37 ± 2.648	59.39 ± 3.947	15	2.13	1.899	Insignificant
	Stellate	49.30 ± 4.015	68.33 ± 5.372	16	2.12	2.838	*Significant

Stellate neurons of both CI and CDL and multipolar neurons of CDL neurons show *data with variance significantly different at level p < 0.05.

FIGURE 16.

Graph showing the results of corrected spine number of the different neurons observed in nonstress (NS) and under acute stress (AS) of 24‐h food and water deprivation in the corticoid complex (CC) of 15‐day‐old chick, Gallus domesticus. Stellate neurons of both CI and CDL and multipolar neurons of CDL neurons show *data with significant differences at p < 0.05

3.5.2. Spine density in CDL subfield

The results of the unpaired t‐test with Welch's correction for corrected spine number of the different neurons observed in nonstress (NS) and under AS (Table 4) shows that the multipolar and stellate neurons of the CDL region show a significant increase (p < 0.05) in their spine density within 25‐μm dendritic segment due to AS (Table 4 and Figure 16), whereas the pyramidal projection neurons show an insignificant increase (p < 0.05) in their spine density within the 25‐μm dendritic segment due to AS (Table 4 and Figure 16).

4. DISCUSSION

The present study investigates the variations and fluctuations occurred in neuronal morphology most especially in the dendritic spine density and dendritic branching pattern in CC (CI & CDL) of 15‐day‐old chick as a result of single AS of 24‐h food and water deprivation. The Cresyl Violet study reveals that the CC of chick is present towards the dorsolateral side of the cerebral cortex after the replacement of HA and includes two subfields CDL complex and CI complex, which is also shown by different investigators in their study in the birds (Chand et al., 2013; Montagnese et al., 1996; Srivastava, Chand, et al., 2007, 2009; Srivastava & Gaur, 2013; Srivastava et al., 2014; Srivastava, Singh, et al., 2012; Tömböl et al., 2000). The CI region is found to be further composed of three layers: Layer‐I (present towards the dorsal surface), Layer‐II (present in the center of CI region), and Layer‐III (present beneath the Layer‐II, towards the ventricle) which is in line with the observations in Zebra finch (Montagnese et al., 1996), Chick and pigeon (Tömböl et al., 2000), Strawberry finch (Srivastava, Chand, et al., 2009), Indian house crow (Srivastava et al., 2014). At the caudal most level, only the parahippocampal area is observed limited laterally by the CDL, as the CI and the various subfields of the HCC progressively disappeared. In CDL layering pattern is absent and moderately packed, small‐ to medium‐sized purple blue colored neuronal somas are uniformly distributed throughout the region. CDL was observed to be narrower in most of the birds by different workers (Matochik et al., 1991; Srivastava, Chand, et al., 2009; Székely & Krebs, 1996), same as observed in the present study.

The Golgi study of the CC of chick revealed two main types of spinous neurons, namely, projection neurons (comprising multipolar and pyramidal neurons) and stellate neurons based on soma shape, soma diameter, dendritic field, axonal length, and axonal projection. The diverse neuronal classes of CI and CDL regions of presently studied 15‐day‐old chick share some homologies with neuronal types in other birds such as Zebra finch (Montagnese et al., 1996), Strawberry Finch (Srivastava, Chand, et al., 2009), and Indian house crow (Srivastava et al., 2014). The projection neurons of the CC of the chick are comparable with projection neurons of the Chick ventral telencephalon (Tömböl et al., 1988), CC of Zebra finch (Montagnese et al., 1996), CC of Strawberry finch (Srivastava, Chand, et al., 2009), Corvus splendens, (Srivastava et al., 2014), Psittacula krameri (Srivastava, Singh, et al., 2012), and entorhinal cortex of mammals (Canto et al., 2008; Hamam et al., 2002, 2000). In the present study the multipolar neurons are found to be dominant‐type neurons which are also reported in other birds (Srivastava, Chand, et al., 2009; Srivastava et al., 2014; Tömböl et al., 2000) and their morphology may play a major role in their survivals due to their adaptations to varied ecological niches as CC has been proposed to participate in spatial memory (Atoji & Wild, 2005; Colombo et al., 2001). The multipolar neurons were also reported in the lower vertebrate group such as in lizard but as a nondominant type of neurons because in lizards mostly bitufted neurons were found to be the dominant type (Maurya & Srivastava, 2006; Sakal et al., 2010; Srivastava & Maurya, 2009; Srivastava, Maurya, et al., 2009; Srivastava, Sakal, et al., 2012; Srivastava et al., 2007).

The pyramidal neurons of the chick CC show only the shape of their soma which is comparable to mammalian pyramidal neurons. The presence of pyramidal projection neurons in CDL may be related to memory and cognitive ability as in Indian house crow (Srivastava et al., 2014). Pyramidal neurons of CDL in chick are comparable with pyramidal neurons of Chick ventral telencephalon (Tömböl et al., 1988), various parts of the entorhinal cortex of mammalian hippocampus (Alonso & Klink, 1993; Canto et al., 2008; Gloveli et al., 1997, 2001; Hamam et al., 2002, 2000), and CC of C. splendens (Srivastava et al., 2014). The main afferent source of stellate neurons is the collaterals of the projection neurons; it may have some role in local circuitry (Srivastava et al., 2014; Tömböl, 1988; Tömböl et al., 2000). The stellate neurons of CC of chick show local arborization of their axon collaterals with spinous wavy dendrites and are comparable with type‐2 neurons of field L complex in the male Zebra Finch (Fortune & Margoliash, 1992), stellate neurons of CC in the Zebra Finch (Montagnese et al., 1996), neurons of Island Canary (Nixdorf et al., 1989), and CC of C. splendens (Srivastava et al., 2014). It is worth mentioning that dendritic field extents of the stellate neuron of chick are larger than those of house crow but morphologically they are similar.

The dendritic spine density of different neuronal types was significantly variable between two subfields of the corticoid region. In addition, dendritic diameter, spine length, and diameter of spine head showed an almost uniform pattern in these regions. Previous studies (Chand et al., 2013; Montagnese et al., 1996; Srivastava, Chand, et al., 2007, 2009; Srivastava & Gaur, 2013; Srivastava, Singh, et al., 2012; Srivastava et al., 2014; Tömböl et al., 2000) are in agreement with our finding that spine density more or less depends on neuronal activity, supporting the presence of more spine density on the neuronal dendrites in case of stressed chick and comparatively less spine density on the neuronal dendrites in nonstressed chick. This study may provide valuable insights about the neuronal plasticity in terms of spine density produced as a result of AS exposure. The basic characteristic feature of neurons is the presence of spines which are present in abundance on the dendrites and function as primary postsynaptic structures (Ballesteros et al., 2006; Feldman & Peters, 1979). Spines are commonly characterized by the presence of a head and a neck, whose morphologies have been described in birds (Srivastava, Chand, et al., 2009; Srivastava et al., 2014; Tömböl et al., 2000).

Studies have reported that due to AS, under extreme conditions, if the stress response is insufficient, an individual may develop numerous pathologies, such as anxiety and depressive disorder (Otte et al., 2016). The learning and memory depend upon the timing, intensity, and extent of stress exposure (Joëls et al., 2006). The impact of AS on memory formation and its retrieval may be negative or positive (Joëls et al., 2006). Various evidence have indicated that small, thin, and motile spines form weak synapses, while the more stable mushroom spines are associated with the formation of strong synapses of prominent postsynaptic density (Kasai et al., 2003, 2010). Dendritic spines are highly dynamic structures and provide a substrate to be reshaped under stimulus (Aguayo et al., 2018). The influences of AS on dendritic morphology have been studied by few investigators in the hippocampus. The change in spine number is found to be dependent on variations in globular and filamentous actin (Lei et al., 2016).

Interestingly, in the present study, we detected significant decrease in the spine density of stellate neurons in the CI region due to AS, while multipolar and pyramidal neurons of this region show insignificant increase. The number of average dendritic branches at 25‐, 50‐, and 100‐µm radius circle from soma is observed to be decreased in each neuron of CI due to AS. A significant increase in the spine density of multipolar and stellate neurons in CDL region had been observed due to AS but pyramidal neurons of this region show an insignificant increase. The number of average dendritic branches at 25‐, 50‐, and 100‐µm radius circle from the soma depicted no significant modification in CDL region due to AS. Thus, the present study indicates that the dendritic spine changes due to AS, and the projection neurons of both CI and CDL region show an increase in spine density which will provide enhanced mental ability to cope with the effects that occurred due to single AS. The morphological changes in the form of decreased dendritic branches due to stress in the CI region could be linked to more effect of stress in this region in comparison to CDL region. There is evidence that even during the stress hyporesponsive period; the stress response may be modulated by environmental factors. For example, food restriction in Black‐legged kittiwake Rissa tridactyla and red‐legged kittiwake R. brevirostris chicks led to enhanced stress response (Brewer et al., 2008; Kitaysky et al., 1999, 2001).

Thus, present finding provide valuable data about the effects of AS on three‐dimensional neuronal morphology especially the spine density in the CC of stressed and nonstressed chick. In stressed chick changes provoked by stress result in characteristic fluctuations in neuronal classes, in terms of increased dendritic thickness, the density of spines, spine length, and spine head diameter. The variations produced as a result of stress mediated neuronal plasticity that could have important outcomes for CC functions. The results of the present study will open new dimensions to understand the effect of many environmental stresses in the neurons of these regions. Further studies will be required to determine the effect of unpredictable mild stresses in the CC and other regions of the avian telencephalon. Finally, these results will contribute to the search and discovery of new targets for treating many neuro‐disorders of CC derived from maladaptive responses to stress.

5. CONCLUSION

The present study shows that in CI only the stellate neurons show a significant decrease whereas in CDL, multipolar and stellate neurons both show a significant increase in their spine density as compared to other neurons. The presence of more dendritic spine density in the case of stressed chick and comparatively less spine density in nonstressed chick supports that spine density is more or less dependent on neuronal activity. A single AS exposure of 24 h duration is differentially produced variations in the expression of spine number. AS may influence particular brain circuits by modulating synaptic plasticity, which finally influences the animal's behavior. Neural plasticity is absolutely necessary for the adequate functioning of an individual in the continuously changing environment. Furthermore, our findings open new avenues of research to understand how a single stress exposure may trigger a fast‐adaptive response, thereby predisposing an individual to neuropsychiatric diseases.

AUTHOR CONTRIBUTIONS

Dr. Ram Chandra Maurya and Mr. Adarsh Kumar have designed this research work plan. Mr. Adarsh Kumar has performed the laboratory investigations in which Miss. Hemlata Arya and Miss. Kavita Tamta have helped. All the authors have contributed to data acquisition and interpretation, drafting and critical revision of the manuscript, and finally the approval of the article.

Kumar, A., Arya, H., Tamta, K. & Maurya, R.C. (2021) Acute stress‐induced neuronal plasticity in the corticoid complex of 15‐day‐old chick, Gallus domesticus . Journal of Anatomy, 239, 869–891. 10.1111/joa.13483

DATA AVAILABILITY STATEMENT

We have shared new data in this research article. The data that support the findings of this study are available from the corresponding author upon reasonable request.