Acta Neuropathot. (Berl.) 44, 197
Acta Neuropathologica
202 (1978)
Springer-Verlag 1978
Redirected Perforant and Commissural Connections of Eutopic* and Ectopic Neurons in the Hippocampus of M[ethylazoxymethanol-Acetate Treated Rats S. C. Singh 1 Department of Physiology, Monash University, Clayton, Victoria 3168, Australia
Summary. In an earlier study, it was reported that clusters of ectopic neurons developed postnatally in the hippocampus of rats which were exposed to Methylazoxymethanol acetate (MAMac) during fetal development (Singh, 1977b). This paper describes the perforant tract and commissural connections of hippocampal eutopic and ectopic neurons. These connections were traced with a reduced-silver method (Eager, 1970). Two observations of significance were made: (i) Ectopic neurons misplaced in stratum radiatum received terminals from axons in the perforant tract. The upper boundary for these redirected fibers was stratum pyramidale - approximately 350~t outside the normal boundary which is situated near the hippocampal fissure. (ii) Ectopic neurons received a dramatically reduced commissural projection, compared with eutopic pyramidal neurons in Ammon's horn. Eutopic neurons in the hippocampus were found to receive afferent perforant tract and cornmissural fibers in the same way - i.e., density and distribution, as in control rats. Key words" Ectopic neurons -- Hippocampus Connections - Methylazoxymethanol acetate Treated rats.
Methylazoxymethanol acetate (MAMac) has been shown to preferentially methylate guanine nucleotides of nucleic acids (Nagata and Matsumoto, 1969). MAMac acts principally on the developing nervous * Eutopic refers to normally sited neurons 1 Aided by the N.H. & M. R. C. of Australia
system - adults usually show no neurological effects unless huge doses are administered. Fetuses which have been exposed to MAMac develop anomalies of hippocampal cytoarchitecture (Singh, 1977b). Woodward et al. (1975) showed that neonatal rat pups develop anomalies in cerebellar cytoarchitecture after M A M a c intoxication. In the latter case there was no report of hippocampal anomalies. The behavior and fate of the ectopic neurons is not known. The appearance of such neurons in the hippocampus was easily detected because this structure is, under normal conditions, composed of a single layer of tightly packed pyramidal cells. Therefore, the MAMac model of ectopic neurons provides a suitable system for a comprehensive analysis of the life history of these misplaced cells, e.g., proliferation, migratory patterns, survival etc. In addition, the hippocampus of the normal animal receives afferent connections which terminate in precisely laminated dendritic fields; thus, unusual patterns of connections to these misplaced neurons could also be investigated. A detailed study of ectopic neurons is of interest because of repeated observations of such cells in human diseases, e.g., Zellweger's syndrome (Volpe and Adams, 1972; Evrard et al., 1978) and Trisomy 17/18 (Michaelson and Gilles, 1972). Of other human diseases, Larroche (1977, p.498) provides a useful summary of heterotopias in the nervous system under various conditions. In addition, ectopic neurons also occur in non-primates, e.g., the 'reeler' mutant mouse (Caviness and Sidman, 1973; Bliss and Chung, 1974) and in the developing chick (Clarke and Cowan, 1976). This paper asks the question: Are afferent connections of eutopic and ectopic neurons in the hippocampus of MAMac-treated rats altered? An answer to this question should shed light on the overall understanding of the production, stability and behavior ofectopic neurons in the central nervous system.
0001-6322/78/0044/0197/$1.20
198
Materials and Methods Pregnant Hooded rats were injected with either 0.9 % physiological saline or 20 mg/kg MAMac (Schwarz/Mann, New York) intraperitoneally. MAMac was dissolved in 0.9 % physiological saline. The gestation period (mode 21 days), parturition and litter size were very similar in the treated and untreated dams.
Lesions and Post-Operative Survial Scalpel blades were ground-down to a fine tip and neck (less than or equal to 0.5 mm). Antiseptic conditions were adopted; instruments, skin and wounds were cleaned with 0.5 % Chlorhexidine (Hibitane, ICI). The perforant 1 and commissurai tracts were lesioned in salineand MAMac-treated rats (Table 1). The commissural axons were severed by transection of the ventral hippocampal commissure close to the midline. The rats were allowed to survive for 2 - 3 days before the brains were perfused with 4 % paraformaldehyde (in 0.1 M phosphate buffer at pH 7.4) via the left cardiac ventricle. Staining for terminal degeneration 2 5 - 3 5 gm sections were cut on a CO2 freezing microtome. These sections were stained for degeneration debris using Eager's silver method (1970, also see Singh, 1977a). The site and extent of the lesion was determined on sections stained with cresyl-violet. These sections were also used to determine the size, location and frequency of ectopic cell clusters.
Results
The commissural and perforant tract connections of the hippocampus in the normal rat have already been described in detail by previous workers, e.g., Gottlieb and Cowan (1973) and Hjorth-Simonsen and Jeune (1972), respectively. The distribution of these fibers in the brains of MAMac-treated animals was compared with the above reports, and with control material from this study (see Table 1).
Acta Neuropathol. (Berl.) 44 (1978) Table 1
Intraperitoneal injections of M A M a c or saline on day 15 of gestation
Type of lesion
Number of offspring
MAMac 0.9% saline MAMac 0.9 % saline
perforant tract commissural tract
9" 3b 4 3b
(treated) (control) (treated) (control)
a Three of these animals were exposed to three maternal doses of 20 mg/kg on days 13,15, and 17 of gestation b Some of the observation on control material were obtained from tissue used in a previous report (Singh, 1977a)
In the dentate gyrus, the black dots were confined to a discrete lamina which occupied the outer two-thirds of the dendritic field in the molecular layer (ML in Fig. 1A and 2). This terminal-degeneration pattern was entirely consistent with that seen in the normal rat (Hjorth-Simonsen and Jeune, 1972). Stratum lacunosum-moleculare of Ammon's horn (SLM in Figs. 1 A and 2), contained axon terminals between subfields CA1 to CA3 in animals with perforant tract lesions (Figs. 3 and 4). The distribution of these axon terminals was similar to that seen in the control material. From the above observations it was concluded that rats which were exposed to MAMac during prenatal development, had a normal projection of perforant tract fibers to eutopic neurons in the hippocampus and the dentate gyrus. b) Ectopic Neurons
A. Perforant Tract Connections 1
Twenty-one-day-old rats were used because by this age the projection of the entorhinal/prepyriform areas to the hippocampus (via the perforant tract) resembles that found in adult rat (Singh, 1977a). A 2 - 3 day survival period produced optimal staining of axon terminals after lesioning the perforant tract. a) Eutopic Neurons Silver-impregnated axon terminals of lesioned axons (seen as 'black dots') were present in stratum moleculare of the dentate gyrus and stratum lacunosunmoleculare of the Ammon's horn. 1 Hjorth-Simonsen (1972) suggested that terminal degeneration in stratum lacunosum-moleculare of hippocampal subfield CA I may be due to lesions of axons en passage through the prepyriform cortex. Therefore, I have not excluded the possibility that fibers projecting to field CA I may originate from the entorhinal/pyriform cortices and have termed these fibers collectively as the perforant tract throughout this paper
The distribution of the perforant tract projection to ectopic neurons was studied in the same sections as described above. In Fig. 1 A, ectopic neurons can be seen around Ammon's horn. The main cluster of these somata is located in stratum radiatum (SR in Fig. 1 A), and a few in stratum oriens (SO) in the plane of the section illustrated. The distribution, density, and frequency of similar clusters of ectopic cells has already been described (Singh, 1977b). A heavy concentration of argyrophilic axon terminals was seen among ectopic neurons in Ammon's horn in animals with perforant tract lesions (Fig. 1 A). The density and distribution of these terminals is better illustrated at the higher magnification of Fig. 1 B. The perforant tract axons terminated diffusely around these ectopic neurons. There was no obvious organization of these terminals into destinct laminae, in contrast with the normal perforant tract projection. The density of axon terminals decreased towards stratum pyramidale and stratum oriens. The occur-
S. C. Singh: Redirected Connections of Ectopic Neurons
199
Fig. 1
Pattern of terminal argyrophilia in the hippocampus of a 21-day-old, MAMac-treated rat. Entorhinal lesion, 2 days survival, Eager's silver stain. The dendritic patterns of a pyramidal and a dentate granule cell are superimposed in A. The insert in A is shown at a higher magnification in B. A x 108, B x 263. Abbreviations apply to all figures (except Fig. 5). AL alveus; SO stratumoriens; SP stratum pyramidale; SR stratum radiatum; SLM stratum lacunosummoleculare; HF hippocampal fissure; ML molecular layer; DG dentate gyrus
fence of these terminals in stratum radiatum and stratum oriens is most striking because these afferent axons had moved away for more than about 350 jam from the boundary of the perforant tract. B. Cornmissural Connections
a) Eutopic Neurons
pyramidal somata (Fig. 5A and C). It should be noted that there were some commissural axon terminals among ectopic cell bodies (Fig. 5B), but when this contribution is compared with the normal projection, it is extremely reduced, but nevertheless present. In conclusion, the results obtained indicate that the ectopic neurons in the hippocampus receive a redirected or adaptive perforant tract projection and a dramatically reduced commissural projection.
Commissural fibers terminated on apical and basal dendrites of eutopic pyramidal neurons of A m m o n ' s horn (Fig. 5A), as they do in normal untreated or control saline-treated rats. In the dentate gyrus, commissural fibers terminate mainly in the inner molecular layer (IML) as illustrated in Fig. 3. These figures were obtained from MAMacand saline-treated rats with combined perforant tract and commissural lesions. The contribution of terminals from both these afferent sources to the dentate gyrus of the treated and control rats is very similar (compare Figs. 3 and 4).
In the results presented above, it was shown that ectopic neurons in the hippocampus, a) received a redirected afferent projection from the perforant tract, and b) received a drastically reduced commissural projection. Two explanations are provided which may account for the changes mentioned above.
b) Ectopic Neurons
1. Redirection of Perforant Tract Axons
Figure 5 illustrates a section of the hippocampus which has ectopic cells among eutopic cell bodies in the A m m o n ' s horn. Figure 5B shows ectopic cells in subfield CA 1. After commissural lesions, a drastically reduced projection of terminals could be stained by Eager's method among these neurons. This was in marked contrast with the high density of terminal argyrophilia a m o n g the dendrites of adjacent eutopic
In the hippocampus of the normal rat, eutopic pyramidal cells receive axon terminals specifically on dendrites which project into stratum lacunosummoleculare (the preferred dendritic segment, Fig. 6). It is reasonable to assume that there is considerable specificity for this pattern of connectivity because, under normal conditions, the afferent fibers are restricted to this belt of dendritic tree. When a cell soma is
Discussion Changes in Afferent Connections
200
Acta Neuropathol. (Berl.)44 (1978) 2. Reduction of Commissural Afferent Terminals
Fig.2. Terminal argyrophilia in the dentate gyrus of a MAMactreated rat which had receiveda lesion of the perforant tract, x 254
displaced, there follows a certain amount of spatial disorganization as far as dendritic development is concerned (personal observations on material stained by the Golgi-Cox method). The dendritic segments normally destined for stratum lacunosum-moleculare are now located in stratum radiatum2: Under these conditions, the afferent axons which would specifically terminate on the preferred dendritic segment would now seek these surfaces which are at the ectopic sites so as to make the appropriate synaptic contacts. The foregoing explanation would be one way of explaining the remarkable redirection of perforant tract axons to ectopic sites. 2 Devoret al. (1975)have also shown that in the prepiriformcortex of the reeler mutant mouse, pyramidal cells which normally send dendrites into the molecular layer, fail to do so when they are spatially displaced (e.g., see their "double asterisked" pyramidal cell in Fig.2B, p. 473)
The drastic reduction of commissural fiber input may be caused by the following conditions. First, ectopic cells may have a reduced dendritic surface which is available for commissural fibers as depicted in cell '2' in Fig. 6 B. Secondly, there may be no reduction in the dendritic surface available for commissural fibers, but for unknown reasons, the commissural fibers largely fail to establish contacts with dendrites of ectopic neurons, but in contrast, proceed to make great numbers of topographically exact terminations upon nearby eutopic pyramidal cells. The second condition is more likely than the first because preliminary studies on dendritic patterns have revealed ectopic neurons with sturdy, spinous apical dendrites of relatively normal lengths. Bliss and Chung (1974) and Devor et al. (1975) also illustrate misplaced pyramidal cells (in the hippocampus and prepiriform cortex, respectively) which have normal dendritic morphology. Devor et al. (1975) came to the conclusion that the olfactory afferents to the prepyriform cortex remain normal, in spite of abnormal location of cells, caused by the "reeler' mutation in mice. However, it is puzzling, in the findings of Devor and his colleagues, why the afferent olfactory fibers are not redirected to those misplaced or ectopic pyramidal cells that fail to send their dendrites into stratum moleculare where they are normally present (also see footnote No. 2). It is possible that they failed to stain these redirected axon terminals or is it possible that olfactory axon terminals fail to establish contacts with these neurons because another afferent system had already done so ? If there is no clear demonstration of afferent terminals upon these ectopic pyramidal cells, then one would be required to conclude that these cells lack the usual input from the olfactory bulb. Bliss and Chung (1974), on the other hand, present convincing electrophysiological data that indicate an intact afferent input, from the perforant tract, upon misplaced pyramidal cells in the hippocampus of the reeler mutant mouse. The foregoing discussion is pertinent in relation to the question of adaptability of ectopic neurons in forming appropriate connections with afferent axons. The present results support the notion that although connectivity of ectopic neurons may be altered in certain ways, there still develops a strong connection between the ectopic neurons and afferent axons in the perforant tract. Altman (1973) found that ectopic granule cells in the cerebellum of x-irradiated rats received connections with mossy fibers. He proposed that these afferent axons grew past their usual terminal site towards the ingrowing granule cells and "whenever granule cells form synapses with mossy fibers they became immobi-
S. C. Singh: Redirected Connections of Ectopic Neurons
201
Figs. 3 and 4
Dark- and light-field photomicrographs of terminal argyrophilia in the hippocampi from MAMac-treated (Fig. 3) and saline-treated (Fig. 4) rats. Perforant tracts were lesioned at 21 days in both animals. Two days survival Eager's silver stain, x 263
Fig. 5
Silver-stained, horizontal section illustrating the hippocampus from a MAMac-treated rat which had received a lesion of the commissural tract on postnatal day 21. Two days survival. Enclosures are shown at higher magnifications in A - C. 5 x 44, 5 A--C x 167
lized". Ebels (1972) also suggested a similar hypothesis. In view o f A l t m a n ' s a n d Ebels' findings it w o u l d be very interesting to d e t e r m i n e w h e t h e r or n o t the ectopic n e u r o n s t h a t develop in the h i p p o c a m p u s o f M A M a c treated rats stop because these cell s o m a t a meet axons f r o m the p e r f o r a n t t r a c t d u r i n g their u n u s u a l m i g r a t i o n
t h r o u g h s t r a t u m r a d i a t u m . It w o u l d be possible to deafferentate the h i p p o c a m p u s by lesioning the e n t o r h i n a l / p r e p y r i f o r m region in n e w b o r n animals. I f these afferent do p l a y a role in s t o p p i n g the m i g r a t o r y neurons, then in the absence, a greater m o b i l i t y o f ceils should be noticed instead.
202
Acta Neuropathol. (Bed.) 44 (1978)
Con~n
p
t
1
-
) ~
~
6A CONTROL
SO
References
SP
-- SLiM
6B MAMac - treated
Fig. 6 A and B. A diagram depicting how afferent connections to ectopic neurons are altered. Dendritic segments indicated by arrows, refer to dendritic areas preferred by perforant tract axons. Cell I is from a control animal (A). Cells 2 - 4 are from a MAMac-treated animal (B) with cells 2 and 3 at an ectopic site and ceil 4 at the eutopic site. CommissuraI terminals are shown in white while the perforant tract terminals are black
Intrinsic and Efferent Connections. In the normal rat, pyramidal neurons in subfield CA 1 - 2 receive connections from schaffer collaterals (Hjorth-Simonsen, 1973). Any alterations in this projection would be difficult to elucidate because these connections are extremely short and are therefore difficult to label by conventional methods. Since a large portion of Ammon's horn remains intact (see Fig. 13, Singh, 1977b), it would be out of the present tracing methodology to selectively map out the trajectories of this small sample of cells. It might perhaps be possible to determine whether or not ectopic pyramidal cells project to the contralateral hippocampus by injecting horseradish peroxidase on the ipsilateral side and subsequently examining the ectopic cells for evidence of retrograde uptake of this marker. This project remains to be carried out. However, the ability of ectopic neurons to develop and project axons to. terminal structures was demonstrated in another system. Using the procedure outlined above, Clarke and Cowan (1976) showed that ectopic neurons in the isthmo-optic nucleus became labeled with horseradish peroxidase after intravitreal injections. This clearly demonstrates the ability of ectopic neurons to generate axons. The ability of ectopic neurons to form adaptive connections has been demonstrated. The functional validation of these connections remains to be determined. Detailed studies of "birthdays", migratory patterns, dendritic growth, and survival of ectopic neurons are in progress. These extended investigations would allow a better understanding of the 'why' and 'how' of ectopic neurons.
Altman, J.: Experimental reorganization of the cerebellar cortex. III. Regeneration of the external germinal layer and granule cell ectopia. J. Comp. Neurol 149, t53-180 (t973) Bliss, T. V. P., Chung, S. H. : An electrophysiological study of the hippocampus of the "reeler" mutant mouse. Nature 252, 153155 (1974) Caviness, V. S., Sidman, R. L. : Retrohippocampal, hippocampal and related structures of the forebrain in the reeler mutant mouse. J. ComP. Neurol. 147, 235-253 (1973) Clarke, P. G. H., Cowan, W. M. : The development of the isthmooptic tract in the chick, with special reference to the occurrence and correction of developmental errors in the location and connections of isthmo-optic neurons. J. Comp. Neurol. 167, 143164 (1976) Devor, M., Caviness, V. S., Jr., Deter, P.: A normally laminated afferent projection to an abnormally laminated cortex: some olfactory connections in the reeler mouse. J. Comp. Neuroi. 164, 471-482 (1975) Eager, R. P. : Selective staining of degenerating axons in the central nervous system by a simplified silver method: spinal cord projections to the external cuneate and inferior olivary nuclei in the cat. Brain Res. 22, 137-14l (1970) Ebels, E. J. : Studies on ectopic granule cells in the cerebellar cortex with a hypothesis as to their aetiology and pathogenesis. Acta Neuropathol. (Bed.) 21, 117-127 (1972) Evrard, P., Caviness, V. S., Jr,, Prats-Vinas, J., Lyon, G.: The mechanism of arrest of neuronal migration in the Zellweger malformation: A hypothesis based upon cytoarchitectonic analysis. Acta Neuropathol. (Berl.) 41, 109-117 (1978) Gottlieb, D. I., Cowan, W. M.: Autoradiographic studies of the commissural and ipsilateral association connections of the hippocampus and dentate gyrus of the rat I. The commissural" connections. J. Comp. Neurol. 149, 393-422 (1973) Hjorth-Simonsen, A. : Projection of the lateral part of the entorhinal area to the hippocampus and fascia dentate. J. Comp. Neurol. 146, 219-232 (1972) Hjorth-Simonsen, A.: Some intrinsic connections of the hippocampus in the rat: An experimental analysis. J. Comp. Neurol. 147, 145-162 (1973) Hiorth-Simonsen, A., Jeune, B.: Origin and termination of the hippocampal perforant path in the rat studied by silver impregnation. J. Comp. Neurol. 144, 215-232 (1972) Larroche, J.-C. : Developmental Pathology of the Neonate. p. 525. Amsterdam: Excerpta Medica 1977 Michaelson, P. S., Gilles, F. H. : Central nervous system abnormalities in Trisomy E (17-18) syndrome. J. Neurol. Sci. 15, 193208 (1972) Nagata, Y., Matsumoto, H.: Studies on methylazoxymethanoh Methylation of nucleic acids in the fetal rat brain. Proc. Soc. Exp. Biol. Med. 132, 383-385 (1969) Singh, S. C.: The development of olfactory and hippocampal pathways in the brain of the rat. Anat. Embryol. (Berl.) 15, 183 199 (1977a) Singh, S. C. : Ectopic neurones in the hippocampus of the postnatal rat exposed to methylazoxymethanol during foetal development. Acta Neuropathol. (Berl.) 40, 111 - 116 (1977 b) Volpe, J. J., Adams, R. D.: Cerebro-hepato renal syndrome of Zellweger: An inherited disorder of neuronal migration. Acta Neuropathol. (Berl.) 20, 175-198 (1972) Woodward, D. J., Bickett, D., Chanda, D.: Purkinje cell dendritic alterations after transient developmental injury of the external granular layer. Brain Res. 97, 195-214 (1975)
Acknowledgement. I am grateful to Drs. Brian Cragg and Sandra Rees for criticism on the manuscript.
Received May 16, 1978/Accepted June 14, I978
Acta Neuropathologica
202 (1978)
Springer-Verlag 1978
Redirected Perforant and Commissural Connections of Eutopic* and Ectopic Neurons in the Hippocampus of M[ethylazoxymethanol-Acetate Treated Rats S. C. Singh 1 Department of Physiology, Monash University, Clayton, Victoria 3168, Australia
Summary. In an earlier study, it was reported that clusters of ectopic neurons developed postnatally in the hippocampus of rats which were exposed to Methylazoxymethanol acetate (MAMac) during fetal development (Singh, 1977b). This paper describes the perforant tract and commissural connections of hippocampal eutopic and ectopic neurons. These connections were traced with a reduced-silver method (Eager, 1970). Two observations of significance were made: (i) Ectopic neurons misplaced in stratum radiatum received terminals from axons in the perforant tract. The upper boundary for these redirected fibers was stratum pyramidale - approximately 350~t outside the normal boundary which is situated near the hippocampal fissure. (ii) Ectopic neurons received a dramatically reduced commissural projection, compared with eutopic pyramidal neurons in Ammon's horn. Eutopic neurons in the hippocampus were found to receive afferent perforant tract and cornmissural fibers in the same way - i.e., density and distribution, as in control rats. Key words" Ectopic neurons -- Hippocampus Connections - Methylazoxymethanol acetate Treated rats.
Methylazoxymethanol acetate (MAMac) has been shown to preferentially methylate guanine nucleotides of nucleic acids (Nagata and Matsumoto, 1969). MAMac acts principally on the developing nervous * Eutopic refers to normally sited neurons 1 Aided by the N.H. & M. R. C. of Australia
system - adults usually show no neurological effects unless huge doses are administered. Fetuses which have been exposed to MAMac develop anomalies of hippocampal cytoarchitecture (Singh, 1977b). Woodward et al. (1975) showed that neonatal rat pups develop anomalies in cerebellar cytoarchitecture after M A M a c intoxication. In the latter case there was no report of hippocampal anomalies. The behavior and fate of the ectopic neurons is not known. The appearance of such neurons in the hippocampus was easily detected because this structure is, under normal conditions, composed of a single layer of tightly packed pyramidal cells. Therefore, the MAMac model of ectopic neurons provides a suitable system for a comprehensive analysis of the life history of these misplaced cells, e.g., proliferation, migratory patterns, survival etc. In addition, the hippocampus of the normal animal receives afferent connections which terminate in precisely laminated dendritic fields; thus, unusual patterns of connections to these misplaced neurons could also be investigated. A detailed study of ectopic neurons is of interest because of repeated observations of such cells in human diseases, e.g., Zellweger's syndrome (Volpe and Adams, 1972; Evrard et al., 1978) and Trisomy 17/18 (Michaelson and Gilles, 1972). Of other human diseases, Larroche (1977, p.498) provides a useful summary of heterotopias in the nervous system under various conditions. In addition, ectopic neurons also occur in non-primates, e.g., the 'reeler' mutant mouse (Caviness and Sidman, 1973; Bliss and Chung, 1974) and in the developing chick (Clarke and Cowan, 1976). This paper asks the question: Are afferent connections of eutopic and ectopic neurons in the hippocampus of MAMac-treated rats altered? An answer to this question should shed light on the overall understanding of the production, stability and behavior ofectopic neurons in the central nervous system.
0001-6322/78/0044/0197/$1.20
198
Materials and Methods Pregnant Hooded rats were injected with either 0.9 % physiological saline or 20 mg/kg MAMac (Schwarz/Mann, New York) intraperitoneally. MAMac was dissolved in 0.9 % physiological saline. The gestation period (mode 21 days), parturition and litter size were very similar in the treated and untreated dams.
Lesions and Post-Operative Survial Scalpel blades were ground-down to a fine tip and neck (less than or equal to 0.5 mm). Antiseptic conditions were adopted; instruments, skin and wounds were cleaned with 0.5 % Chlorhexidine (Hibitane, ICI). The perforant 1 and commissurai tracts were lesioned in salineand MAMac-treated rats (Table 1). The commissural axons were severed by transection of the ventral hippocampal commissure close to the midline. The rats were allowed to survive for 2 - 3 days before the brains were perfused with 4 % paraformaldehyde (in 0.1 M phosphate buffer at pH 7.4) via the left cardiac ventricle. Staining for terminal degeneration 2 5 - 3 5 gm sections were cut on a CO2 freezing microtome. These sections were stained for degeneration debris using Eager's silver method (1970, also see Singh, 1977a). The site and extent of the lesion was determined on sections stained with cresyl-violet. These sections were also used to determine the size, location and frequency of ectopic cell clusters.
Results
The commissural and perforant tract connections of the hippocampus in the normal rat have already been described in detail by previous workers, e.g., Gottlieb and Cowan (1973) and Hjorth-Simonsen and Jeune (1972), respectively. The distribution of these fibers in the brains of MAMac-treated animals was compared with the above reports, and with control material from this study (see Table 1).
Acta Neuropathol. (Berl.) 44 (1978) Table 1
Intraperitoneal injections of M A M a c or saline on day 15 of gestation
Type of lesion
Number of offspring
MAMac 0.9% saline MAMac 0.9 % saline
perforant tract commissural tract
9" 3b 4 3b
(treated) (control) (treated) (control)
a Three of these animals were exposed to three maternal doses of 20 mg/kg on days 13,15, and 17 of gestation b Some of the observation on control material were obtained from tissue used in a previous report (Singh, 1977a)
In the dentate gyrus, the black dots were confined to a discrete lamina which occupied the outer two-thirds of the dendritic field in the molecular layer (ML in Fig. 1A and 2). This terminal-degeneration pattern was entirely consistent with that seen in the normal rat (Hjorth-Simonsen and Jeune, 1972). Stratum lacunosum-moleculare of Ammon's horn (SLM in Figs. 1 A and 2), contained axon terminals between subfields CA1 to CA3 in animals with perforant tract lesions (Figs. 3 and 4). The distribution of these axon terminals was similar to that seen in the control material. From the above observations it was concluded that rats which were exposed to MAMac during prenatal development, had a normal projection of perforant tract fibers to eutopic neurons in the hippocampus and the dentate gyrus. b) Ectopic Neurons
A. Perforant Tract Connections 1
Twenty-one-day-old rats were used because by this age the projection of the entorhinal/prepyriform areas to the hippocampus (via the perforant tract) resembles that found in adult rat (Singh, 1977a). A 2 - 3 day survival period produced optimal staining of axon terminals after lesioning the perforant tract. a) Eutopic Neurons Silver-impregnated axon terminals of lesioned axons (seen as 'black dots') were present in stratum moleculare of the dentate gyrus and stratum lacunosunmoleculare of the Ammon's horn. 1 Hjorth-Simonsen (1972) suggested that terminal degeneration in stratum lacunosum-moleculare of hippocampal subfield CA I may be due to lesions of axons en passage through the prepyriform cortex. Therefore, I have not excluded the possibility that fibers projecting to field CA I may originate from the entorhinal/pyriform cortices and have termed these fibers collectively as the perforant tract throughout this paper
The distribution of the perforant tract projection to ectopic neurons was studied in the same sections as described above. In Fig. 1 A, ectopic neurons can be seen around Ammon's horn. The main cluster of these somata is located in stratum radiatum (SR in Fig. 1 A), and a few in stratum oriens (SO) in the plane of the section illustrated. The distribution, density, and frequency of similar clusters of ectopic cells has already been described (Singh, 1977b). A heavy concentration of argyrophilic axon terminals was seen among ectopic neurons in Ammon's horn in animals with perforant tract lesions (Fig. 1 A). The density and distribution of these terminals is better illustrated at the higher magnification of Fig. 1 B. The perforant tract axons terminated diffusely around these ectopic neurons. There was no obvious organization of these terminals into destinct laminae, in contrast with the normal perforant tract projection. The density of axon terminals decreased towards stratum pyramidale and stratum oriens. The occur-
S. C. Singh: Redirected Connections of Ectopic Neurons
199
Fig. 1
Pattern of terminal argyrophilia in the hippocampus of a 21-day-old, MAMac-treated rat. Entorhinal lesion, 2 days survival, Eager's silver stain. The dendritic patterns of a pyramidal and a dentate granule cell are superimposed in A. The insert in A is shown at a higher magnification in B. A x 108, B x 263. Abbreviations apply to all figures (except Fig. 5). AL alveus; SO stratumoriens; SP stratum pyramidale; SR stratum radiatum; SLM stratum lacunosummoleculare; HF hippocampal fissure; ML molecular layer; DG dentate gyrus
fence of these terminals in stratum radiatum and stratum oriens is most striking because these afferent axons had moved away for more than about 350 jam from the boundary of the perforant tract. B. Cornmissural Connections
a) Eutopic Neurons
pyramidal somata (Fig. 5A and C). It should be noted that there were some commissural axon terminals among ectopic cell bodies (Fig. 5B), but when this contribution is compared with the normal projection, it is extremely reduced, but nevertheless present. In conclusion, the results obtained indicate that the ectopic neurons in the hippocampus receive a redirected or adaptive perforant tract projection and a dramatically reduced commissural projection.
Commissural fibers terminated on apical and basal dendrites of eutopic pyramidal neurons of A m m o n ' s horn (Fig. 5A), as they do in normal untreated or control saline-treated rats. In the dentate gyrus, commissural fibers terminate mainly in the inner molecular layer (IML) as illustrated in Fig. 3. These figures were obtained from MAMacand saline-treated rats with combined perforant tract and commissural lesions. The contribution of terminals from both these afferent sources to the dentate gyrus of the treated and control rats is very similar (compare Figs. 3 and 4).
In the results presented above, it was shown that ectopic neurons in the hippocampus, a) received a redirected afferent projection from the perforant tract, and b) received a drastically reduced commissural projection. Two explanations are provided which may account for the changes mentioned above.
b) Ectopic Neurons
1. Redirection of Perforant Tract Axons
Figure 5 illustrates a section of the hippocampus which has ectopic cells among eutopic cell bodies in the A m m o n ' s horn. Figure 5B shows ectopic cells in subfield CA 1. After commissural lesions, a drastically reduced projection of terminals could be stained by Eager's method among these neurons. This was in marked contrast with the high density of terminal argyrophilia a m o n g the dendrites of adjacent eutopic
In the hippocampus of the normal rat, eutopic pyramidal cells receive axon terminals specifically on dendrites which project into stratum lacunosummoleculare (the preferred dendritic segment, Fig. 6). It is reasonable to assume that there is considerable specificity for this pattern of connectivity because, under normal conditions, the afferent fibers are restricted to this belt of dendritic tree. When a cell soma is
Discussion Changes in Afferent Connections
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Acta Neuropathol. (Berl.)44 (1978) 2. Reduction of Commissural Afferent Terminals
Fig.2. Terminal argyrophilia in the dentate gyrus of a MAMactreated rat which had receiveda lesion of the perforant tract, x 254
displaced, there follows a certain amount of spatial disorganization as far as dendritic development is concerned (personal observations on material stained by the Golgi-Cox method). The dendritic segments normally destined for stratum lacunosum-moleculare are now located in stratum radiatum2: Under these conditions, the afferent axons which would specifically terminate on the preferred dendritic segment would now seek these surfaces which are at the ectopic sites so as to make the appropriate synaptic contacts. The foregoing explanation would be one way of explaining the remarkable redirection of perforant tract axons to ectopic sites. 2 Devoret al. (1975)have also shown that in the prepiriformcortex of the reeler mutant mouse, pyramidal cells which normally send dendrites into the molecular layer, fail to do so when they are spatially displaced (e.g., see their "double asterisked" pyramidal cell in Fig.2B, p. 473)
The drastic reduction of commissural fiber input may be caused by the following conditions. First, ectopic cells may have a reduced dendritic surface which is available for commissural fibers as depicted in cell '2' in Fig. 6 B. Secondly, there may be no reduction in the dendritic surface available for commissural fibers, but for unknown reasons, the commissural fibers largely fail to establish contacts with dendrites of ectopic neurons, but in contrast, proceed to make great numbers of topographically exact terminations upon nearby eutopic pyramidal cells. The second condition is more likely than the first because preliminary studies on dendritic patterns have revealed ectopic neurons with sturdy, spinous apical dendrites of relatively normal lengths. Bliss and Chung (1974) and Devor et al. (1975) also illustrate misplaced pyramidal cells (in the hippocampus and prepiriform cortex, respectively) which have normal dendritic morphology. Devor et al. (1975) came to the conclusion that the olfactory afferents to the prepyriform cortex remain normal, in spite of abnormal location of cells, caused by the "reeler' mutation in mice. However, it is puzzling, in the findings of Devor and his colleagues, why the afferent olfactory fibers are not redirected to those misplaced or ectopic pyramidal cells that fail to send their dendrites into stratum moleculare where they are normally present (also see footnote No. 2). It is possible that they failed to stain these redirected axon terminals or is it possible that olfactory axon terminals fail to establish contacts with these neurons because another afferent system had already done so ? If there is no clear demonstration of afferent terminals upon these ectopic pyramidal cells, then one would be required to conclude that these cells lack the usual input from the olfactory bulb. Bliss and Chung (1974), on the other hand, present convincing electrophysiological data that indicate an intact afferent input, from the perforant tract, upon misplaced pyramidal cells in the hippocampus of the reeler mutant mouse. The foregoing discussion is pertinent in relation to the question of adaptability of ectopic neurons in forming appropriate connections with afferent axons. The present results support the notion that although connectivity of ectopic neurons may be altered in certain ways, there still develops a strong connection between the ectopic neurons and afferent axons in the perforant tract. Altman (1973) found that ectopic granule cells in the cerebellum of x-irradiated rats received connections with mossy fibers. He proposed that these afferent axons grew past their usual terminal site towards the ingrowing granule cells and "whenever granule cells form synapses with mossy fibers they became immobi-
S. C. Singh: Redirected Connections of Ectopic Neurons
201
Figs. 3 and 4
Dark- and light-field photomicrographs of terminal argyrophilia in the hippocampi from MAMac-treated (Fig. 3) and saline-treated (Fig. 4) rats. Perforant tracts were lesioned at 21 days in both animals. Two days survival Eager's silver stain, x 263
Fig. 5
Silver-stained, horizontal section illustrating the hippocampus from a MAMac-treated rat which had received a lesion of the commissural tract on postnatal day 21. Two days survival. Enclosures are shown at higher magnifications in A - C. 5 x 44, 5 A--C x 167
lized". Ebels (1972) also suggested a similar hypothesis. In view o f A l t m a n ' s a n d Ebels' findings it w o u l d be very interesting to d e t e r m i n e w h e t h e r or n o t the ectopic n e u r o n s t h a t develop in the h i p p o c a m p u s o f M A M a c treated rats stop because these cell s o m a t a meet axons f r o m the p e r f o r a n t t r a c t d u r i n g their u n u s u a l m i g r a t i o n
t h r o u g h s t r a t u m r a d i a t u m . It w o u l d be possible to deafferentate the h i p p o c a m p u s by lesioning the e n t o r h i n a l / p r e p y r i f o r m region in n e w b o r n animals. I f these afferent do p l a y a role in s t o p p i n g the m i g r a t o r y neurons, then in the absence, a greater m o b i l i t y o f ceils should be noticed instead.
202
Acta Neuropathol. (Bed.) 44 (1978)
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Fig. 6 A and B. A diagram depicting how afferent connections to ectopic neurons are altered. Dendritic segments indicated by arrows, refer to dendritic areas preferred by perforant tract axons. Cell I is from a control animal (A). Cells 2 - 4 are from a MAMac-treated animal (B) with cells 2 and 3 at an ectopic site and ceil 4 at the eutopic site. CommissuraI terminals are shown in white while the perforant tract terminals are black
Intrinsic and Efferent Connections. In the normal rat, pyramidal neurons in subfield CA 1 - 2 receive connections from schaffer collaterals (Hjorth-Simonsen, 1973). Any alterations in this projection would be difficult to elucidate because these connections are extremely short and are therefore difficult to label by conventional methods. Since a large portion of Ammon's horn remains intact (see Fig. 13, Singh, 1977b), it would be out of the present tracing methodology to selectively map out the trajectories of this small sample of cells. It might perhaps be possible to determine whether or not ectopic pyramidal cells project to the contralateral hippocampus by injecting horseradish peroxidase on the ipsilateral side and subsequently examining the ectopic cells for evidence of retrograde uptake of this marker. This project remains to be carried out. However, the ability of ectopic neurons to develop and project axons to. terminal structures was demonstrated in another system. Using the procedure outlined above, Clarke and Cowan (1976) showed that ectopic neurons in the isthmo-optic nucleus became labeled with horseradish peroxidase after intravitreal injections. This clearly demonstrates the ability of ectopic neurons to generate axons. The ability of ectopic neurons to form adaptive connections has been demonstrated. The functional validation of these connections remains to be determined. Detailed studies of "birthdays", migratory patterns, dendritic growth, and survival of ectopic neurons are in progress. These extended investigations would allow a better understanding of the 'why' and 'how' of ectopic neurons.
Altman, J.: Experimental reorganization of the cerebellar cortex. III. Regeneration of the external germinal layer and granule cell ectopia. J. Comp. Neurol 149, t53-180 (t973) Bliss, T. V. P., Chung, S. H. : An electrophysiological study of the hippocampus of the "reeler" mutant mouse. Nature 252, 153155 (1974) Caviness, V. S., Sidman, R. L. : Retrohippocampal, hippocampal and related structures of the forebrain in the reeler mutant mouse. J. ComP. Neurol. 147, 235-253 (1973) Clarke, P. G. H., Cowan, W. M. : The development of the isthmooptic tract in the chick, with special reference to the occurrence and correction of developmental errors in the location and connections of isthmo-optic neurons. J. Comp. Neurol. 167, 143164 (1976) Devor, M., Caviness, V. S., Jr., Deter, P.: A normally laminated afferent projection to an abnormally laminated cortex: some olfactory connections in the reeler mouse. J. Comp. Neuroi. 164, 471-482 (1975) Eager, R. P. : Selective staining of degenerating axons in the central nervous system by a simplified silver method: spinal cord projections to the external cuneate and inferior olivary nuclei in the cat. Brain Res. 22, 137-14l (1970) Ebels, E. J. : Studies on ectopic granule cells in the cerebellar cortex with a hypothesis as to their aetiology and pathogenesis. Acta Neuropathol. (Bed.) 21, 117-127 (1972) Evrard, P., Caviness, V. S., Jr,, Prats-Vinas, J., Lyon, G.: The mechanism of arrest of neuronal migration in the Zellweger malformation: A hypothesis based upon cytoarchitectonic analysis. Acta Neuropathol. (Berl.) 41, 109-117 (1978) Gottlieb, D. I., Cowan, W. M.: Autoradiographic studies of the commissural and ipsilateral association connections of the hippocampus and dentate gyrus of the rat I. The commissural" connections. J. Comp. Neurol. 149, 393-422 (1973) Hjorth-Simonsen, A. : Projection of the lateral part of the entorhinal area to the hippocampus and fascia dentate. J. Comp. Neurol. 146, 219-232 (1972) Hjorth-Simonsen, A.: Some intrinsic connections of the hippocampus in the rat: An experimental analysis. J. Comp. Neurol. 147, 145-162 (1973) Hiorth-Simonsen, A., Jeune, B.: Origin and termination of the hippocampal perforant path in the rat studied by silver impregnation. J. Comp. Neurol. 144, 215-232 (1972) Larroche, J.-C. : Developmental Pathology of the Neonate. p. 525. Amsterdam: Excerpta Medica 1977 Michaelson, P. S., Gilles, F. H. : Central nervous system abnormalities in Trisomy E (17-18) syndrome. J. Neurol. Sci. 15, 193208 (1972) Nagata, Y., Matsumoto, H.: Studies on methylazoxymethanoh Methylation of nucleic acids in the fetal rat brain. Proc. Soc. Exp. Biol. Med. 132, 383-385 (1969) Singh, S. C.: The development of olfactory and hippocampal pathways in the brain of the rat. Anat. Embryol. (Berl.) 15, 183 199 (1977a) Singh, S. C. : Ectopic neurones in the hippocampus of the postnatal rat exposed to methylazoxymethanol during foetal development. Acta Neuropathol. (Berl.) 40, 111 - 116 (1977 b) Volpe, J. J., Adams, R. D.: Cerebro-hepato renal syndrome of Zellweger: An inherited disorder of neuronal migration. Acta Neuropathol. (Berl.) 20, 175-198 (1972) Woodward, D. J., Bickett, D., Chanda, D.: Purkinje cell dendritic alterations after transient developmental injury of the external granular layer. Brain Res. 97, 195-214 (1975)
Acknowledgement. I am grateful to Drs. Brian Cragg and Sandra Rees for criticism on the manuscript.
Received May 16, 1978/Accepted June 14, I978
