Reduction of transiently expressed acetylcholinesterase activity in developing thalamocortical projections does not affect the mature pattern of basal forebrain projections to visual cortex

DEVELOPMENTAL BRAIN RESEARCH

ELSEVIER

Developmental Brain Research 85 (1995) 283-287

Short communication

Reduction of transiently expressed acetylcholinesterase activity in developing thalamocortical projections does not affect the mature pattern of basal forebrain projections to visual cortex Edward J. Kimm a, Celia E. Perez a, Christopher

C. Yu a, Jen Yu b, Richard T. Robertson

FL*

a Department ofAnatomy and Neurobiology. College of Medicine. University of California. Irvine, CA 92717, USA ’ Department of Physical Medicine and Rehabilitation, College of Medicine, University of California, IrL:ine. CA 92717, USA Accepted

20 December

1994

Abstract Experiments tested the hypothesis that acetylcholinesterase (AChE) activity, expressed transiently in developing thalamocortical projections, serves to limit the growth of basal forebrain cholinergic projections into thalamocortical recipient zones. Newborn rats were subjected to enucleation, a procedure that eliminates transient AChE activity in developing visual cortex.

After 3-8 weekssurvival, AChE histochemicaltechniquesrevealedno alteration in the pattern of AChE positive basalforebrain axonsin visual cortex. Thesedata indicate that transient AChE activity in developingsensorycortex doesnot limit ingrowth of basalforebrain cholinergic axons. Keywords:

Acetylcholinesterase;Basal forebrain; Cholinergic; Deafferentation; Thalamocortical; Visual cortex

Several recent studies have demonstrated the transient presence of acetylcholinesterase (AChE) activity in developing cerebral cortex of mammals [12,13,15,18, 19,21-231. In rats, the transient AChE activity is found in primary sensory cortical regions, particularly in a band corresponding to layer IV and the deep part of layer III [15,18,19,21,22]. This transient AChE activity is first detectable shortly after birth, reaches peak intensity during the second postnatal week, and then subsidesto adult levels during the third postnatal week [lS]. Experimental studies indicate that the transiently expressed AChE is produced by thalamocortical neurons and transported to their terminals in primary sensory cortical regions [21,22]. This transient pattern of AChE activity in thalamocortical projections can be distinguished from the AChE activity associated with basal forebrain cholinergic axons in cerebral cortex by morphological differences in staining patterns and by differences in response to thalamic and basal forebrain lesions [6,21-231.

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While the normal function of AChE is generally agreed to be the cleavage of acetylcholine in cholinergic transmission, the function of transiently expressed AChE in developing thalamocortical projections remains enigmatic [23]. A suggestion that the transiently expressed AChE may have a morphogenic role in neuronal development has attracted interest for several years [12,13,15,18,19,21,23,25]. One possibility is that the AChE activity expressed transiently in developing thalamocortical projections may serve to aid the growing thalamocortical axons in establishing their terminal fields in cortex. This idea that transiently expressed AChE serves to establish synaptic terminal fields for thalamocortical axons can be viewed in the context of a Hebbian [7] model of synapse formation. Available evidence indicates that the noncholinergic, but AChE positive, axons from the thalamus [6,15,21,22] grow into cortex during the late fetal and early postnatal period. At approximately the same time, the cholincrgic, and AChE positive, axons from basal forebrain [3,5,6,21,22] grow into the same cortical areas. Thus, the two populations of afferents could be in competition for synaptic space in cortex. In the mature brain, these two popula-

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tions of afferents show laminar distributions in cortex that are roughly complementary; thalamocortical axons terminate mainly in layer IV and deep layer III [4,6,9,15,18,21,251 while basal forebrain afferents largely avoid these layers [3,6,8,14,17,20,22,24]. The mature complementary pattern of thalamocortical and basal forebrain projections to cortex suggests that these two populations of afferents may be involved in competition for synaptic space. Perhaps the transiently expressed AChE is playing a role in this putative competitive interaction. In a Hebbian [7] model, the presence of AChE secreted by thalamocortical axon terminals could serve to reduce cholinergic transmission between basal forebrain afferents and cortical neurons, thereby

Fig. 1. Low power photomicrographs of age of a sham operated control visual cortex (area 17). Calibration

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reducing the probability of cholinergic synapse formation within the domain of the thalamocortical axon terminals. In conjunction, recent evidence from Appleyard and Jahnsen [l] has shown that AChE released onto neurons can render those neurons more responsive to the excitatory amino acid transmitters glutamate and aspartate, which are likely to be the transmitters of thalamocortical projection neurons. In this way, the transiently expressed AChE released from thalamocortical axons could both increase the probability of thalamocortical (excitatory amino acid) synapse formation and decrease the probability of basal forebrain (cholinergic) synapse formation within layer IV of cerebral cortex.

illustrating AChE histochemical staining (Tag0 et al. [26] technique) in the right occipital cortex animal (A) and a littermate subjected to bilateral enucleation at birth (B). Arrowheads indicate bar = 500 pm.

at 4 weeks borders of

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The present investigation tested the hypothesis that transiently expressed AChE serves to limit the formation of synapses of basal forebrain projections onto cortical neurons in layer IV. Comparisons are made between control brains and brains subjected to neonatal enucleation, a procedure that has been demonstrated to markedly reduce transient expression of AChE in developing thalamocortical projections [ 191. If transiently expressed AChE serves to reduce basal forebrain axonal ingrowth into layer IV, then these manipulations should result in a greater than normal density of termination of basal forebrain projections in layer IV of visual cortex. Portions of these data have been presented previously [ 101. Experiments used 15 Sprague-Dawley albino rats. On the day of birth (PO), pups were anesthetized by hypothermia and subjected to unilateral or bilateral enucleation [19] or sham surgery. One or both eyes were surgically removed and the enucleated orbits were treated with topical procaine and filled with gelfoam.

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Following post-operative survival periods of 3-8 weeks, pups were deeply anesthetized with sodium pentobarbital and sacrificed by perfusion with 4% paraformaldehyde in 0.1 M sodium phosphate buffer at pH 7.4. Frozen transverse sections cut at 50 pm were processed for AChE histochemistry using modified versions of the methods of Tago et al. [261 or of Koelle and Friedenwald [ll]. Sections of cortex from experimental and control littermates were processed together and compared for patterns of AChE histochemical labeling. As previously described in detail [6,21], an MCID image analysis system was used to take series of measurements through each hemisphere from the pial surface to the white matter. The measures taken were the optical density of AChE histochemical reaction product per unit area, and total length of AChE positive elements (presumed axons) per unit area. Density of AChE histochemical reaction product and total length of AChE stained elements were taken as indices of the quantity of basal forebrain cholinergic innerva-

Fig. 2. Medium power photomicrographs illustrating AChE histochemical staining [26] in the right operated control animal (A) and a littermate subjected to bilateral enucleation at birth (B). Layers bar = 200 pm.

visual cortex at 4 weeks of age of a sham of visual cortex are indicated. Calibration

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AChE Density Fig. 3. Results of analyses of optical density of AChE histochemical reaction product across cortical layers of visual cortex from a sham operated control animal and a littermate bilaterally enucleated at birth. Positions of the measurement in cortex are noted on the ordinate; density of AChE is indicated on the abscissa. Positions of cortical layers are indicated.

tion of cortex. Two to four measurements at each cortical depth were averaged, and equivalent regions in experimental and control hemispheres were compared. Figs. 1 and 2 present examples of AChE histochemical staining in occipital cortices of normal and neonatally enucleated rats sacrificed at 4 weeks of age. Note in Figs. 1A and 2A that AChE histochemically stained axons in visual cortex of sham operated control animals are found throughout the cortical layers, but appear densest in layers V and VI, and least dense in layer IV. This pattern in sham operates is identical to the pattern in unoperated controls. Figs. 1B and 2B present material from a littermate that received bilateral enucleation at PO. Comparison of these two sets of photomicrographs, as well as material from all other pairs of experimental and control animals, reveals no difference in the patterns of AChE in visual cortices of enucleated and control cases. The qualitative impression of absence of differences between patterns of AChE stained axons in sham operated control visual cortices and visual cortices of enucleated casesis corroborated by results of quantitative assessments.Fig. 3 presents results of an analysis of optical density of AChE staining per unit area across the laminae of visual cortex of sham operated and neonatally enucleated animals. This quantitative analysis reveals differences in density of AChE staining acrosslaminae of visual cortex similar to the qualitative results reported above, with highest measures occurring in cortical layers V and VI. Further, the laminar pattern in the neonatally enucleated animal is virtually identical to the laminar pattern in the control animal. The Student’s t-test detects no difference between

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laminar patterns in these two cortices, either in measures of laminar profiles of optical density (t = 0.31; P > 0.20) or in measures of total length of AChE positive elements in visual cortex (t = 1.56; P > 0.15). The research hypothesis predicted differences restricted to layer IV, because layer IV exhibits the transient expression of AChE that was markedly reduced by neonatal enucleation. Measurements of AChE density or lengths of AChE stained fibers specifically in layer IV also did not reveal differences between experimental and control animals. Length of survival time appeared not to be a factor, as analysis of data from animals sacrificed at 3, 6, or 8 weeks of age detected no differences between experimental animals and controls. Further, the choice of AChE histochemical staining technique [11,26] did not affect the outcome of the analysis. Although some variation in intensity of histochemical staining can occur between individual cases, the overall intensity of AChE staining, as well as laminar profiles of staining, did not show consistent differences between experimental and control cortices. The lack of consistent differences indicates that enucleation did not produce a general decrement in AChE staining. We also measured AChE density in somatosensory cortex as a general control and found that relative levels of AChE staining between visual and somatosensory cortices were similar, irrespective of whether the visual cortical staining was in control or enucleated cases. The Tago et al. [26] technique for demonstrating AChE positive axons provides a sensitive measure of basal forebrain cholinergic axons in cortex. That is, virtually all basal forebrain cholinergic neurons stain positively for AChE [16], and most of the AChE activity in cerebral cortex of mature animals is associated with basal forebrain cholinergic afferents [14,20]. Two techniques were undertaken to determine quantitatively the laminar pattern of AChE positive axons in visual cortex of mature brains, including the optical density of AChE reaction product and total length of AChE positive elements. Neither of these measures detected differences between neonatally enucleated animals and sham operated controls, in regard to laminar patterns of AChE histochemical activity in visual cortex at maturity. These data demonstrate that the pattern of basal forebrain cholinergic innervation of visual cortex in neonatally enucleated animals does not appear to be different from the pattern of innervation in normal animals. Although the relationship between number, length, or optical density of AChE stained axons in cortex and the number of basal forebrain cholinergic synapses in cortex is unknown, it seems likely that the measures used in this study would be predictive of cholinergic innervation. That is, if neonatal enucleation would have resulted in substantial increase in basal forebrain derived cholinergic synapses

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in cortical layer IV, then that increase in synapses would likely have been associated with an increase in branching of AChE positive axons in layer IV. It is possible that differences could have been detected had a longer survival time been used; however, the research hypothesis would predict specific changes within a limited time, certainly within 8 weeks of age. Results of this study demonstrate that the marked reduction of transiently expressed AChE resulting from neonatal enucleation does not result in an hyperinnervation of layer IV by basal forebrain cholinergic afferents. This result indicates that the intense AChE activity that is expressed transiently in thalamocortical terminal fields during cortical development apparently is not involved in mechanisms of competition between thalamocortical axons and basal forebrain axons for synaptic space within layer IV [23]. This leaves the function(s) of the transiently expressed AChE still unresolved, but the results of the present experiment continue to narrow the range of possibilities.

Acknowledgments Supported by NIH Grant NS 30109 and the Roosevelt Warm Springs foundation. We thank Kimberly Claytor for technical assistance.

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