Histochemical Distribution of Acetylcholinesterase in the Forebrain Nuclei of an Indian Non Catfish Channa punctatus

Though the teleostean brain represents a typical vertebrate pattern, it shows markable differences over tetrapod pattern (Burmeister et al., 2009). The telencephalon of teleosts which consists of dorsal pallium and ventral subpallium, folds outward during development, in contrast to tetrapod telencephlon which folds inward during development (Tripathi and Prakash, 2020). These initial developmental differences are coupled with subsequent cellular rearrangements (Yamamoto et al., 2007), that together set the stage for numerous differences among homologous pallial structures. Thus a thorough investigation of fore brain structures and functions of teleosts and how it differs from those of tetrapods may possibly reveal some of the fundamental principles of brain evolution.
       
Acetylcholinesterase (AChE) which is an effective marker enzyme of cholinergic and cholinoceptive neurons, clearly demarcates cell populations and regions of the brain thus giving an anatomical distinction between different forebrain centres which is particularly difficult in teleosts due to poor cytological differentiation.
       
In addition, the cholinergic (Soreq and seidman, 2001) and non cholinergic (Chub et al., 1980; Chub et al., 1982; Downes and Granto, 2004; Silman and Sussman, 2005; Tripathi and Srivastava, 2008) roles of AChE, which have been elucidated in last two decades, provide adequate base to functionally correlate its variable distribution in the different telencephalic regions of the presently studied fish.
       
The distribution of cholinesterases has been carried out in the brain of several mammalian (Krnjevik and Silver,1964; Bennet et al.,1966; Ishii and Friede, 1967; Bhatt and Tewari,1978; Giris,1980), avian (Cavanagh and Lolland,1961; Zuschratter and Scheich,1990; Cookson et al., 1996; Sadananda, 2004) reptilian (Sethi and Tewari, 1976; Sethi and Tewari, 1977; Subhedar and Rama Krishna, 1990; Tripathi and Srivastava, 2007) species. Data available on enzyme localization in the brain of fishes (Contestebile and Zannoni, 1975; Northcut and Butler, 1993; Clement et al., 2004; Tripathi et al., 2013; Tripathi and Rahman, 2014) particularly in the forebrain of Indian teleosts is inadequate and scattered.
       
Hence present study was conductedto study histochemical nature of AChE distribution in the telencephalic and diencephalic centres of Channa punctatus. In the present study the neuro anatomical nomenclature follows recent cytoarchitectonic studies (Northcut and Butler, 1993; Butler, 2000; Nieuwenhuys, 2009; Nieuwenhuys et al., 1998).

Seven adult male Channa punctatus weighing 35-40 gm and ranging in length between 16-20 cm. were used in the  present study. The animals were acclimatized according to  laboratory conditions before sacrificing them. Experimental procedures were performed according to the guidelines of the Institutional Animal Ethics Committee (IAEC). The fish were anesthetized with MS-222 (Sigma, St. Lovis, MO) and decapitated. Brains were fixed in the solution of 0.5% paraformaldehyde and 1.5% gluteraldehyde in 0.1 M phosphate buffer (pH 7.4) for 6 hours at 4°C. The tissue was then given 2-3 changes in 15% sucrose solution in 0.1M phosphate buffer and stored in the same solution for 2-3 days. 30 micron thick frozen sections were cut by A O Histostat at -22°C and stored serially in 0.1 M phosphate buffer. AChE histochemistry was carried out by using a modified histochemical technique ( Hedreen et al., 1985). The dark brown coloured patches appeared in sections which designated AChE activity. Omission of the substrate acetylthiocholine iodide was carried out as control for the AChE histochemistry and no residual activity was observed.

The forebrain in the presently studied animal comprises telencephalic lobes and diencephalic centres which are composed of many subcentres and nuclear groups. Telencephalon consists of dorsal pallium and the ventral subpallium. Sub divisions of these telencephalic areas are currently denoted by descriptive topographical terms, dorsal, medial, central and lateral nuclei (Tripathi and Rahman, 2014).
 
Telencephalon
 
The pallium of C.punctatus is characterized by extensive lateral (Dl), medial (Dm) and central (Dc) divisions with many distinct cell groups and uniform dorsal (Dd) and posterior (Dp) sub divisions. In the present study the AChE intensity in various telencephalic centres has been described in Table1.
 

       
In the rostral sections the medial nucleus (Dm) is characterized by tightly packed small cells at a position medial to dorsal nucleus (Dd). These cells showed strong activity for AChE (Fig 1, 2). However in middle and caudal sections as the subpallium becomes more prominent Dm takes on a more dorsal position and its AChE activity is gradually reduced (Fig 3-5).
 

 

       
The dorsal part of the dorsal division (Dd) of the pallium appears dorsolaterally at about all the levels as a triangular bunch of small round cells. In the rostral parts it showed mild activity (Fig 1, 2) but in middle and caudal parts it showed intense activity in continuance with central nucleus (Dc) (Fig 3, 4). The dorsolateral nucleus (Dl) is differentiated in to dorsal (Dld) and ventral (Dlv) parts in rostral parts (Fig 1, 2). This demarcation is less prominent in middle and caudal parts due to appearance of posterior nucleus (Dp) (Fig 3, 4). Dorsal part of dorsolateral nucleus demonstrated intense activity for AChE in rostral and middle parts (Fig 1, 2) while it demonstrated moderate intensity in caudal sections (Fig 4, 5). The ventral part (Dlv) which is a poor zone of dense cells, showed moderate to mild activity almost at all levels (Fig 1-5). The central nucleus of pallium (Dc) which appears from rostral to caudal section is highly prominent zone which is placed lateral to Dm and ventral to Dd and Dl (Fig 1). In the present histochemical analysis, Dc can be subdivided in to three zones (Dc₁, Dc₂, Dc₃) on the basis of size of soma, group and intensity for AChE (Fig 1). Most anterior is DC1 which is a group of loosely arranged cells that appear in rostral sections. This zone showed mild activity for AChE (Fig 1, 2). Dc₂ which is the most prominent zone among three, consists of largest cells which occur in clusters and appear at all levels of pallium. Dc₂ showed very high intensity for AChE (Fig 1, 6A). Dc₃ which is located between Dld and Dc₂ is also composed of very large sized rounded cells. It also showed very high intensity at all levels (Fig 1, 2, 6A).
 

       
The posterior part of pallium (Dp) appears in middle and caudal sections and is post commissurally placed in the ventrolateral pole of the pallium (Fig 3, 4). The cells of Dp are small to medium sized and are loosely arranged. These cells show strong activity for AChE (Fig 4). The ventral telencephalon consists of four main cell groups, dorsal (Vd), ventral (Vv), supracommissural (Vs) and entopeduncular (E) nuclei like in all other teleosts.
       
The cells of Vd represent clustered arrangement and demonstrated very high activity at all levels (Fig 1-4). In the rostral sections Vv appears ventral to Vd and shows a distinct cluster of medium sized cells with intense AChE activity (Fig 1, 2), in middle and caudal sections after the appearance of Vs, both Vv and Vd showed diffused arrangement of cells with very high intensity for AChE (Fig 3-4, 6C). Vc and ventrolateral subdivision (Vl) demonstrated moderate activity (Fig 1, 6D). Vs which is dorsally placed to anterior commissure showed very high intensity (Fig 6C). Entopeduncular nucleus (E) which appears in middle and caudal sections post commissurally showed intense activity (Fig 3, 5). Commissures and fibre tracts showed negativity for AChE (Fig 3, 4).
       
However in caudal sections all the dorsal and ventral telencephalic nuclei after the appearance of diencephalon showed recession and the AChE activity also diminished (Fig 5).
 
Diencephalon
 
Various nuclear groups identified in the diencephalon of presently studied animal are rostrocaudally extended starting from anterior diencephalic region up to tegmental region of mesencephalon. Acetylcholinesterase activity was detected in a number of fibre bundles and nuclei of the diencephalon.
       
In C. Punctatus, preoptic area (POA) surrounds the preoptic recess of the third ventricle. It is bounded rostrally by anterior commissure and dorsally by the telencephalon (Fig 2, 6B). The most rostral cell group in the PoA is nucleus preopticus parvocellularis anterioris (PPa). It consists of small sized, oval, scattered cells. This nucleus showed intense activity for AChE staining (Fig 2, 6B).
       
Further caudally, nucleus preopticus parvocellularis posterioris (PPp) is present and is characterized rostrally by four to five, mostly single celled lamina. This nucleus demonstrated intense reaction (Fig 3-4, 7A, 7B). Dorsal to the rostral part of PPp, there is a small area of more scattered, slightly larger cells which are believed to be nucleus preopticus magnocellularis pars magnocellularis (PMm) based on previous cytoarchitectonic studies. This nucleus also demonstrated intense activity (Fig 3, 7A).
 
@figure7
       
Rostral most nucleus of the ventral thalamus is nucleus ventromedialis rostrales (vmr). It lies dorsal to PMm and is composed of a group of non-laminated scattered cells. This cell group exhibited intense activity (Fig 3, 7A). Slightly farther caudally, the distinct tightly packed cells of ventromedialis caudalis (VMc) are present which showed very intense reaction (Fig 4, 7B). Nucleus intermedius (I) lies immediately dorsolateral to VMc and comprises very small cell group. This nucleus demonstrated moderate activity (Fig 4, 7B), ventrolateral to this nucleus, nucleus ventrolaterales (Vl) is present which also showed moderate activity (Fig 7B).
Nucleus Anterior (ATN) of the dorsal thalamus lies dorsal to nucleus ventromedialis caudalis and ventral to anterior commissure. It is several celled thick that are separated by a region of neuropil. (Table2) In the anterior level it showed moderate while in caudal levels it showed mild to negative activity (Fig 4-5, 7B). In the caudal sections, posterior thalamic nucleus (PTN) appears ventral to ATN and exhibited moderate activity (Fig 5). Habenular ganglia (Hb) demonstrated moderate activity (Fig 5, 7D).
 

       
Tuberal area (TA) in the C. punctatus consists of dorsal (Hd), ventral (Hv) and lateral (Hl) hypothalamic nuclei (Fig 5, 7C). Dorsal and ventral hypothalamic nuclei comprise small sized, densely packed cells which demonstrated high intensity (Fig 5, 7C). The lateral hypothalamic nucleus showed moderate activity (Fig  7C).
       
In the present study the Dm of pallial area in the rostral and middle sections demonstrated strong activity for AChE. The earlier connectional findings in gold fish (Northcut, 2006) reveal that Dm receives many ascending projections of preglomerular nuclei which additionally receive auditory and chemosensory inputs. The central posterior thalamic nucleus sends second auditory input to Dm. In addition, Dm also appears to receive gustatory information. Dm is also reported earlier, to be connected to Vd and Vv which are reported to be homologues of the tetrapod striatum and septum respectively (Northcut, 2006).
       
Thus Dm is highly involved in the processing of these inputs which needs many cholinergic and non cholinergic metabolic activities as suggested in the present findings, due to intense activity of AChE in this zone.
       
The Dc, which is subdivided into three distinct cell groups in our study from Dc₁ to Dc₃, comprise variable number of zones among teleosts such as from Dc₁ to Dc₅ in Astatotilapia and from Dc₁ to Dc₄ in Clarias (Clement et al., 2004), a single group in Heteropneustes (Tripathi Rahman, 2014). The central zone Dc, represents a collection of the centrally lying parts of each superficial pallial zone (Braford, 1995; Northcut, 2006). Dc demonstrated moderate activity for AChE except Dc₂, suggesting that most of superficial pallial zones are basically non-cholinergic in nature, since AChE is the marker of cholinergic neurons. Moreover the cholinergic innervations in these pallial zones may have been contributed by the neuronal inputs and outputs from other brain centres.
       
The Dl in the presently studied fish resembles in many respects to that of other teleosts (Clement et al., 2004).  The preglomerular nuclei that primarily project to Dl receive auditory, lateral line and visual inputs. In addition it receives afferents from most of the pallial and subpallial centres (Northcut, 2006). Thus like Dm, Dl is also involved in high level processing of these inputs controlling many physiological and behavioral activities which further needs cholinergic and non cholinergic synaptic transmission as evident in the present study due to presence of AChE intense cells.
       
The absence of cholinergic cells in the dorsal area of the telencephalon has been considered as a primitive ch ergic neurons are absent almost entirely from cortical regions in birds (Medina and Reiner, 1994), though they are reported to be present in the cortex of the rat (Blaker et al., 1988). Thus the presence of cholinergic neurons seems to be a secondary feature acquired relatively late during the evolution of vertebrates.
       
Dp which appears in the caudal sections and may be considered as the part of Dl showed intense activity for AChE. This nucleus is supposed to be homologous to hippocampal pallium of amniotes (Butler, 2000). This zone has been reported to be intense for AChE in amniotes (Wullimann and Rink, 2002) as in present study. This fact corroborates the homology of Dp and amniotic hippocampal pallium.
       
In the subpallium of C. Punctatus, dorsal, ventral, lateral and supra commissural nuclei showed high intense activity for AChE. On the basis of earlier anatomical studies (Northcut, 2006) Vd and Vv are supposed to be the homologous of the striatum and septum respectively and Vs is believed to be homologous to the amygdala in other vertebrates (Mathisen and Blackstad,1964). In the previous studies (Giris, 1980; Tripathi and Srivastava, 2007) these nuclei in reptiles and mammals are reported to be highly intense for AChE like Vd, Vv and Vs in the present study. This observation further supports the homology of Vd, Vv and Vs to striatum, septum and amygdala of tetrapods respectively.
       
Among the diencephalic nuclei in the presently studied animal, in the preoptic area, PPa, PMm and PPp showed intense AChE staining in their cell bodies as well as in neuropil areas. These findings are in agreement with previous studies (Northcut and Butler, 1993; Clement et al., 2004). The AChE positive cells were observed in the ventral most region of anterior part of the parvocellular preoptic nucleus (PPa) of zebrafish. These AChE positive cells were located in the same region as the cholinergic cells within preoptic area of dogfish and trout (Perez et al., 2000; Anadon et al., 2000). However in the zebrafish, these cells were ChAT immunonegative; moreover, they received a strong cholinergic input, which suggests a possible cholinoceptive nature of PPa. The magnocellular preoptic nucleus projects massively to the neurohypophysis in cyprinids (Anglade et al., 1993). Moreover a cholinergic projection from this nucleus to the hypophysis has been described in the Siberian sturgeon and in the trout (Adrio et al., 2000). It is suggested therefore that this nucleus might be involved in neurosecretory roles. Furthermore, PPa projects to the telencephalon in teleosts (Striedter, 1990) and could be the origin of the cholinergic innervations of the telencephalon in the presently studied fishes. However this needs more investigation. The epithalamus in the presently studied animals consists of habenular ganglia which exhibited moderate intensity for AChE. Areas within habenula are strongly positive for AChE in the long nose gar (Northcut and Butler,1993) as in the case of Pantodon (Butler and Saidal,1991) and euteleosts (Wullimann and Meyer,1990) also. However cholinergic cells were absent from this diencephalic region in cyprinid (Ekstrom, 1987), batrachoid (Brantley and Bass,1988). But ChAT intense immunoreactive cells were observed in amphibians (Marin et al.,1997), birds (Sorenson et al.,1989) and mammals (Contestebile et al., 1987). With these findings it was suggested that the existance of cholinergic cells within habenular complex is exclusive to tetrapods and appeared initially in early amphibians which could be supported by present results. However ChAT immunoreactive cells were described among some primitive fishes (Adrio et al., 2000). It may be concluded therefore that the presence of cholinergic cells within habenular ganglia may be a common feature of the cholinergic system in vertebrates. Moreover this could be a primitive feature of fishes that has been modified in some teleosts and has been conserved in tetrapods.
       
Among the thalamic nuclei identified in present study, the rostral; caudal and lateral subdivisions of nucleus ventromedialis demonstrated intense activity for AChE. These subdivisions are similar to that of long nose gar (Northcut and Butler, 1993). These nuclei are also AChE positive in Pantodon (Butler and Saidal, 1991).
       
In addition, anterior and posterior thalamic nuclei showed moderate to mild AChE staining along with nucleus intermedius. In the thalamus of all the fish species hitherto studied, cholinergic cells are absent or scarce and are exclusively located in the dorsal thalamus (Brantley and Bass, 1988; Anadon et al., 2000; Adrio et al., 2000). In the ventromedial and ventrolateral thalamic nuclei of zebrafish, AChE positive neurons were present but ChAT immunoreactive cells were not observed in any thalamic nuclei (Clement et al., 2004). Moreover the presence of cholinergic neurons in the thalamus has been described in birds (Cookson et al., 1996) and in mammals (Rico and Cavade,1998). This data is suggestive of the fact that the cholinergic neurons appeared in the thalamus several times during evolution so they are homoplastic (parallel evolution).
       
Among the hypothalamic nuclei, dorsal and ventral subdivisions of hypothalamic nucleus showed intense reaction for AChE in our study. Among the other fish species studied earlier, regional differences in AChE occur within the periventricular part of the hypothalamus in longnose gar (Northcut and Butler, 1993). Further, no AChE positive or ChAT immunoreactive cell somata was observed in zebra fish hypothalamus (Clement et al., 2004). The presence of cholinergic or cholinoceptive cell groups is poorly represented in most teleosts studied to date. Thus in the cyprinid Phoxinus phoxinus, ChAT positive cells were detected only in the caudal zone of the periventricular hypothalamus (Ekstrom,1987). In the trout, a salmonid, two groups of putative cholinergic cells were described exclusively in the anterior tuberal nucleus and a zone lateral to the Para ventricular organ (Perez et al., 2000). In non teleostean fishes such as dog fish some cholinergic cells appeared in the boundary between the posterior recess organ with the hypophysis and in the lateral tuberal nucleus (Anadon et al., 2000). In contrast, cholinergic cells in the hypothalamus of the rest of the vertebrates are more widely distributed, cholinergic cells have been described in the periventricular hypothalamus of amphibians (Marin et al., 1997) reptiles (Medina and Reiner,1994), birds (Giris, 1980) and in different hypothalamic regions of mammals (Tago et al., 1987). Therefore this poor representation or absence of cholinergic/cholinoceptive system in the teleostean hypothalamus may be a derived characteristic acquired during the evolution radiation of teleosts.
       
The presence of a dense background staining of AChE is often suggested to be an accurate enough marker for cholinergic centres though the simultaneous presence of AChE and ChAT (Choline-Acetyltransferase), enzyme responsible for acetylcholine synthesis, serves as a good indicator, although indirect of both cholinergic and cholinoceptive brain regions (Ryan and Arnold, 1981). But there are also indications of non parallel distribution of AChE and ChAT as discussed earlier among different vertebrates, which indicate that only a co-occurence of AChE and ChAT denote cholinergic and cholinoceptive nature of neurons, while AChE staining alone denotes cholinoceptive neurons.
       
Other findings have shown that AChE hydrolyses substance P, met and leu-eukephelin and could degrade other neuropeptides as well (Chub et al., 1980; Chub et al.,1982; Downes and Granto, 2004; Silman and Sussman, 2005; Tripathi and Srivastava, 2008). Moreover AChE can facilitate neurite growth during embryogenesis (Silman and Sussman, 2005). It also acts as neuronal adhesion protein (Tripathi and Srivastava, 2008). Hence the cholinergic and noncholinergic roles of the enzyme are directly correlated with its widespread differential staining in the different cholinergic or cholinoceptive centres and nuclei of forebrain thereby giving a clear neuro-anatomical distinction. On the basis of these findings various homologous brain centres of vertebrate groups can be compared which give an insight about the evolution of vertebrate brain.

This work is financially supported by the UGC MRP No. FPSJ-07/10-11 granted to corresponding author.