Neural systems contributing to acetylcholinesterase histochemical staining in primary visual cortex of the adult rat

Brain Research, 509 (1990) 181-197

181

Elsevier BRES 15196

Research Reports

Neural systems contributing to acetylcholinesterase histochemical staining in primary visual cortex of the adult rat Richard T. Robertson 1, Charles J. Fehrenbach 1 and Jen Y u 2 Departments of 1Anatomy and Neurobiology and 2physical Medicine and Rehabilitation, College of Medicine, University of California, lrvine, CA 92717 (U.S.A.) (Accepted 18 July 1989)

Key words: Acetylcholinesterase; Basal forebrain; Cortex; Lateral geniculate; Visual system

Histochemical studies demonstrate that cortical area 17 (primary visual cortex) of the adult rat displays a characteristic laminar pattern of acetylcholinesterase (ACHE) activity. While AChE-positive axons are found throughout the cortical layers, most intense staining occurs in a band that corresponds to layer V and the deep portion of layer IV. The present studies were directed toward determining the neural systems containing this AChE activity. Unilateral electrolytic or excitatory amino acid induced lesions of the basal forebrain result in reductions of AChE staining in ipsilateral visual cortex, particularly in layers IV and V. Electrolytic or scalpel lesions, placed in white matter underlying dorsal and lateral neocortex to interrupt basal forebrain projections to visual cortex, also reduce AChE staining in visual cortex. Lesions in the cingulate bundle and supracallosal stria reduced AChE staining in retrosplenial cortex but did not affect staining in visual cortex. Placement of electrolytic lesions in the hypothalamus produced no detectable change in the pattern of AChE in visual cortex. Electrolytic lesions in the midbrain tegmentum, placed to interrupt ascending axons from brainslem monoamine neurons, produced no detectable change in the pattern of AChE in visual cortex. Placement of lesions in the dorsal thalamus that include all of the dorsal lateral geniculate nucleus did not alter AChE staining in visual cortex. The results indicate that AChE activity in adult visual cortex is found primarily within afferent axons from the basal forebrain system. These data demonstrate further that the AChE staining characteristic of adult visual cortex is associated with neural systems that are distinctly different from those associated with AChE staining in visual cortex of the infant rat. INTRODUCTION Several recent studies have examined the distribution of acetylcholinesterase (ACHE) activity in primary visual cortex of the rat 1"8'9"31~36"43"48-54'5~'64.These histochemical studies have revealed that laminar patterns of A C h E activity in visual cortex of the adult are different from A C h E patterns in the developing rat. In the adult, A C h E is found throughout the cortical laminae, but primarily in a broad band that includes layer V and the deepest portion of layer IV 36"43'66. Prominent staining also occurs in the superficial portion of layer I. In the developing rat, light A C h E staining is found in the same cortical laminae in which staining occurs in the adult, but remarkably intense A C h E activity also occurs in a band that corresponds to cortical layer IV and the deeper portion of layer Il148'5°'51'54. interestingly, the p r o m i n e n t band of A C h E in layer I I I - I V in the developing animal is transient; it first appears at about postnatal day 6 (PND 6), reaches peak intensity at PND 10-12, and gives way to the adult pattern by about PND 214~'5°'51~54. We sought to determine whether the change in laminar distribution of A C h E from the developing to the mature

brain is due to redistribution of an existing ACHEpositive neural system or due to replacement of one system of AChE-positive afferents by another. We selected the lesion technique to help distinguish these two possibilities. Recent work from this laboratory has demonstrated that placement of lesions within the dorsal lateral geniculate nucleus ( d L G N ) or the geniculocortical radiations results in marked reduction of A C h E staining in visual cortex of the infant rat 5~. Placement of lesions in the basal forebrain did not have this effect. In the present paper, we investigated whether similar lesions in the adult rat would have similar or different effects on A C h E staining in visual cortex. Portions of these data have been presented in abstract form 49. MATERIALS AND METHODS

Surgical procedures Experiments were performed on male and female SpragueDawley rats weighing 250-350 g. Animals were anesthetized with chloral hydrate (300 mg/kg, i.p.) supplemented with sodium pentobarbital (10 mg/kg, i.p.). Animals were prepared for stereotaxic surgery using aseptic precautions. Unilateral lesions of visual cortical afferents from the basal forebrain were produced by several methods. Electrolytic lesions

Correspondence." R.T. Robertson, Department of Anatomy and Neurobiology, College of Medicine, University of California, lrvine, CA 92717, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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Fig. 2. Photomicrographs showing the effects of a basal forebrain electrolytic lesion on A C h E histochemical staining m occipital cortex. A: A C h E - s t a i n e d transverse section through the basal forebrain; arrows indicate the borders of the lesion that includes portions of the nucleus of the diagonal band, substantia innominata and medial globus pallidus. Bar = 1 ram. B: A C h E - s t a i n e d transverse section through occipital cortex showing effects of basal forebrain lesion. Note the decreased A C h E staining, including cortical layers V and deep portion of IV. Bar = 1 m m .

were placed in the basal forebrain in 5 animals. A stainless-steel electrode, insulated except for 250 ~ m at the tip, was placed in the region of the junction between the vertical and horizontal divisions of the diagonal band, i.e. the angular division of the nucleus of the diagonal band. Electrolytic lesions were made by passing a 0.5 m A positive current for 15-30 s. Injections of excitatory amino acids were used in 7 animals in an attempt to produce localized basal forebrain lesions without involvement of passing axons. Injections were m a d e with a microsyringe through a 33 gauge needle or glass

micropipette placed stereotaxically in the region of the angular division of the nucleus of the diagonal band. Injections of 0.2-1.0 ~1 of an a q u e o u s solution containing 1 mg/100 kd of ibotenate, kainate, N-methyl-D-aspartate, or a combination of these excitatory amino acids were m a d e over 3-10 min. A x o n s of cortically projecting cells from the basal forebrain 5~' were severed in 12 animals. Lesions were placed in the cortex and subcortical white matter either by transverse scalpel cuts or by electrolysis (0,4 m A for 10-20 s). Positions of these lesions ranged from the cingulate

Fig. 1. Photomicrographs illustrating the pattern of A C h E histochemical staining in cortical area 17 of the adult rat. A: A C h E - s t a i n e d transverse section through occipital cortex; Tago et al. 56 procedure. Arrows indicate borders of cortical area 17. Calibration bar = 1 m m for A and B. B: Nissl-stained section adjacent to A. C: laminar pattern of A C h E staining in cortical area 17; Tago et al. 5~' m e t h o d . D: Nissl-stained section adjacent to A; cortical layers are indicated. E: laminar pattern of A C h E staining in cortical area 17: Koelle and Friedenwald t7 procedure. Bar in E = 250/~m for C - E .

184 gyrus and supracallosal stria (medially) to lateral parietal cortex (see Fig. 8). Electrolytic lesions were placed in several other subcortical sites. Seven animals received large electrolytic lesions in an attempt to sever ascending axons from brainstem monoamine neurons. Using a vertical or angled stereotaxic approach, a stainless-steel electrode was placed in the central tegmental field of the midbrain. Lesions were produced by passing 1.0 mA current for 30 s. Similar techniques were used in two rats to place unilateral lesions in the hypothalamus. Unilateral electrolytic lesions of the dLGN were produced in 7 rats. Using an angled or a vertical stereotaxic approach, the tip of a stainless steel electrode was placed in the region of dLGN and lesions were produced by passing 0.5 mA of current for 10-20 s. After placement of the lesions, topical antibiotics and local analgesics were administered, wound~ were sutured and the animals allowed to recover from the anesthetic in a temperature and humidity controlled incubator. Some animals receiving lesions of brainstem or basal forebrain were given infusions of glucose/saline solutions during the first few post-operative days. Following post-operative survival periods of 6-21 days, the animals were deeply anesthetized and perfused through the heart with 250-500 ml 10% formalin in 0.1 M sodium phosphate buffer (pH 7.3). The brains were removed and stored in 10% formalin and 20% sucrose overnight at 4 °C.

Histochemistry Transverse sections were cut on a freezing microtome at a thickness of 64 ~m. Every 5th section was processed for AChE histochemistry as described below; adjacent sections were processed for a Nissl stain. Free-floating sections were processed for AChE histochemistry using one of two techniques. Most commonly used was a modified version of the method of Koelle and Friedenwald3']7. The substrate was 1.0 x 10 4 M acetylthiocholine iodide and non-specific cholinesterase was inhibited by 1.14 x 10 4 M iso-OMPA. Sections were incubated for 18-24 h at room temperature• The histochemical reaction product was developed by placing the sections in an aqueous solution of 1% ammonium sulfide for 30 s. Some sections were incubated according to the procedure of Tago et al. 6~. Sections were incubated for 30-90 min in a diluted Karnovsky and Roots ~5 solution containing 1.82 x 10-6 M acetylthiocholine and 5 x 10-5 M /so-OMPA. The reaction product was intensified by placing sections in an aqueous solution of diaminobenzidine and nickel ammonium sulfate for 5 min. Sections were dehydrated, coverslipped and examined under the light microscope• Density of AChE histochemical reaction product was taken as an index of the amount or activity of AChE 3'61.

Data analysis Quantitative assessments of effects of lesions on AChE activity were made using densitometric analyses• AChE reacted sections were viewed with the aid of a video camera and monitor. The video recorded image was digitized and analyzed with deAnza or MCID densitometric systems. The computer system determined a relative grey value for a rectangular area 240/~m × 34/~m. The converted grey value varied from '0' (white) tot '250' (black) and indicated the density of AChE histochemical reaction product• Intensity of illumination, contrast of the image, and sensitivity of the densitometer were adjusted for each brain so that measurements of AChE reaction product density from the internal capsule would produce readings of 70 (arbitrary) units. Densitometric measurements were made starting external to the pial membrane and moving through the cerebral cortex orthogonal to the pial surface. Density measurements from two adjacent readings were averaged; this procedure usually yielded 24 measurements for each pass through cortex. In some cases, measurements of AChE density were taken at 4 different positions across the medial-lateral extent of cortical area 17. Particular attention was paid to comparing the laminar patterns

and density of AChE histochemical reaction product in lesioned and control hemispheres. Most commonly, differences between laminar patterns of AChE activity at homologous positions in the lesioned and the control hemispheres were analyzed with the Kolmogorow Smirnov test sg. In other cases, an attempt was made to calculate the

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Fig. 3. Graphic presentation of results of densitometric measures of AChE through cortical area 17• Cortical depth is represented on the ordinate from outside the pial surface (24) to subcortical white matter (1). Optical density of AChE reaction product is represented on the abscissa• A: laminar distribution of AChE staining in cortical area 17 in the control hemisphere contralateral to the basal forebrain lesion. B: AChE staining in cortical area 17 ipsilaterat to the basal forebrain lesion. C: laminar differences in optical density of AChE staining between the two hemispheres.

185 degree of loss of A C h E staining in visual cortex as a result of particular lesions. Average density measurements in 5-6 laminar positions, corresponding to cortical layer V, were taken for homologous positions between the two hemispheres. Because the densitometric measures were referenced against a baseline of 70 units, the recorded averages were reduced by 70 units and the two hemispheres compared. For example, if measurements in the control hemisphere averaged 110 units and measurements in the lesioned hemisphere averaged 80 units, this would indicate a

reduction in the lesioned hemisphere to 25% of control. RESULTS

Normal pattern of A C h E histochemical staining in cortical area 17 of the adult rat The pattern of AChE

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Fig. 4. Photomicrographs showing the effects of a basal forebrain excitatory amino acid induced lesion on A C h E histochemical staining in occipital cortex. A: AChE-stained transverse section through the basal forebrain. Solid arrows indicate the borders of the lesion; open arrow indicates end of the needle track. Bar = 1 mm. B: AChE-stained transverse section through occipital cortex showing effects of this basal forebrain lesion. Arrows indicate borders of area 17. Note the decreased A C h E staining throughout occipital cortex. Bar = 1 mm.

186 seen in a normal adult rat is illustrated by the photomicrographs in Fig. 1. These photomicrographs demonstrate that while AChE is found throughout occipital cortex, area 17 displays a characteristic density and laminar pattern of staining. In addition to relatively light AChE staining throughout the cortical laminae z~, note the dense AChE staining found as a band that corresponds to the deepest portion of cortical layer IV and layer V. Relatively dense staining also is characteristic of the superficial portion of layer I. This laminar pattern of AChE staining is found throughout the rostral-caudai extent of cortical area 17. These photomicrographs also illustrate the pattern of AChE staining as revealed by two different histochemical methods: a modification of the traditional Koelle and Friedenwald 17 procedure (Fig. 1E) and the recently introduced diaminobenzidene intensification method of Tago et al. 62 (Fig. 1C). Although the two procedures differ in regard to their sensitivity and their ability to reveal AChE-stained individual axons, the laminar and areal distributions of reaction product revealed by the two methods are similar. Although the Tago procedure produced material that was, in the best cases, superior, the technique of Koelle and Friedenwald yielded more consistent results and thus the materials presented in the photomicrographs and densitometric analyses in the present paper are based on the Koelle technique.

adjacent sections. Optical density of the AChE histochemical reaction product in the hemisphere contralateral to the lesion (Fig. 3A) shows a pattern typical for cortical area 17, with dense staining corresponding to layer V and layer I. The laminar profile of AChE staining in cortical area 17 ipsilateral to the lesion (Fig. 3B) clearly is different. Note the modest and relatively even density of

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Pattern of A C h E histochemical staining in cortical area 17 in animals with basal forebrain lesions Fig. 2 presents material from a case in which a large lesion was produced electrolytically in the basal forebrain of an adult rat, and the animal sacrificed 7 days later. The lesion, illustrated by the photomicrograph in Fig. 2A, involved the angular and horizontal divisions of the nucleus of the diagonal band, anterior and middle portions of the substantia innominata, and the most anterior portion of the medial globus pallidus. The photograph in Fig. 2B demonstrates the loss of AChE staining in occipital cortex of the left hemisphere, ipsilateral to the lesion, in this case. Note the decrease in AChE staining, particulary the loss of the dense band of AChE activity in deep layer IV and layer V. AChE staining in the right hemisphere, contralateral to the basal forebrain lesion, does not appear different from staining in non-lesioned control cases. This reduction in AChE staining was found throughout cortical area 17, although lateral and caudal portions were affected less dramatically. Results from densitometric analysis of the case illustrated in Fig. 2 are presented in Fig. 3. Data presented were averaged from densitometric measurements from the tissue section photographed in Fig. 2 and in 3

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Diff, AChE Density Fig. 5. Densitometric data for effects of excitatory amino acidinduced basal forebrain lesion on A C h E staining in cortical area 17. A: laminar distribution of A C h E staining in cortical area 17 in the control hemisphere contralateral to the lesion. B: A C h E density in cortical area 17 ipsilateral to the basal forebrain lesion. C: laminar differences in optical density of AChE staining in cortical area 17 between the two hemispheres.

187 staining throughout the cortical laminae. Fig. 3C presents the difference in laminar profiles between the two hemispheres, and illustrates that the major difference between the lesioned and unlesioned hemispheres is the reduction of A C h E in the infragranular layers. Differences in density profiles of the two hemispheres were examined using the K o l m o g o r o v - S m i r n o v test. A null hypothesis would assume zero differences between density profiles of the two hemispheres; the K o l m o g o r o v Smirnov test allows us to reject the null hypothesis, demonstrating a significant difference from 0 in the cumulative distribution of density scores (D = 0.708; P < 0.01). Fig. 4 presents results from an animal that received an excitatory amino acid induced basal forebrain lesion and was sacrificed after 19 days. The 1/~1 injection contained 0.01 mg each of kainate, ibotenate and N-methylD-aspartate. The extent of the lesion can be seen in the photomicrograph of an AChE-stained section through the basal forebrain, shown in Fig. 4A. Loss of A C h E reaction product, reduced numbers of neurons and gliosis indicated the lesion site. Note that the region involved in this lesion overlaps considerably with the lesioned region shown in Fig. 2, i,e. portions of the nucleus of the diagonal band and the substantia innominata. Fig. 4B

illustrates the reduction in A C h E staining in ipsilateral cortical area 17. Results from densitometric analysis of the case illustrated in Fig. 4 are presented in Fig. 5. A C h E histochemical reaction product in the hemisphere contralateral to the lesion (Fig. 5A) again shows the pattern typical for cortical area 17. The laminar profile of A C h E staining ipsilateral to the lesion (Fig. 5B) shows the even density of staining throughout the cortical laminae, with the absence of the usual peak corresponding to deep layer IV and layer V. Some slight staining appears to remain at the cortical surface. Fig. 5C presents the difference in laminar profiles between the two hemispheres, and ilustrates again the prominent reduction of A C h E in the infragranular layers. Again, the K o l m o g o r o v - S m i r n o v test demonstrates the differences between the two hemispheres to be significant (D = 0.750; P < 0.01).

Pattern of A C h E histochemical staining in cortical area 17 in animals with lesions involving basal forebrain axonal projections to occipital cortex Fig. 6 presents results from two cases that sustained lesions of cortex and subcortical white matter, Fig. 6A illustrates a large lesion, which includes the cingulate gyms, cingulate bundle, and supracallosal stria, along

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with much of dorsal cortex and its underlying white matter. Note in Fig. 6B the general loss of A C h E staining in cortical area 17, as well as in cortical area 18 just medial to area 17 and in the cingulate gyms. A C h E loss in visual cortex is most pronounced in cortical layer I V - V of medial portions of area 17. Interestingly, A C h E staining in the lateral portion of cortical area 17 appears reduced only slightly by the lesion. Results from a case with a smaller cingulate lesion are presented in Fig. 6C and D. This lesion involved the cingulate bundle and supracallosal stria, but avoided much of the dorsal cortex and underlying white matter that were included in the larger lesion in Fig. 6A, In this case, A C h E staining in posterior cingulate cortex was markedly reduced, but staining in cortical area 17 appears unaffected. Densitometric analysis of the case presented in Fig. 6 A and B is presented in Fig. 7. Note that the large lesion resulted in a marked reduction of A C h E staining in the medial portion of cortical area 17 (D = 0.439; P < &01) but no reduction was detected in lateral portions (D -0.161; P > 0.2). Densitometric analysis did not detect differences between hemispheres of the case with the smaller lesion in Fig. 6C. A variety of electrolytic and scalpel cut lesions were

produced in cortical and subcortical tissue of 12 adult rats in an attempt to determine the path(s) of basal forebrain efferent axons projecting to occipital cortex. Fig. 8 summarizes results from 3 of these cases. The drawing of the dorsal surface of the left cerebral hemisphere of a rat in Fig. 8 A represents the positions of these 3 ( B - D ) lesions. Also shown are 4 positions (1-4) within cortical area 17 at which densitometric measures of A C h E staining were taken. Differences between the two hemispheres in A C h E staining intensity in layer V are presented for medial to lateral positions within cortical area 17 for each case. Reduction of A C h E activity for each medial-lateral point was determined for the region corresponding to cortical layer V. It can be seen that a small lesion involving the cingulate bundle and supracallosal stria has virtually no affect on A C h E staining in cortical area 17 (Fig. 8B) while slightly more laterally placed lesions reduce staining in medial regions of cortical area 17 (Fig. 8C). Progressively more lateral lesions affect progressively more lateral regions o f cortical area 17 (Fig. 8D). Only relatively large lesions, involving much of dorsal-lateral cortex and subjacent white matter, reduced A C h E staining throughout cortical area 17.

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Pattern of A ChE histochemical staining in cortical area 17 in animals with hypothalamic lesions The cerebral cortex also receives afferent projections from neurons in the hypothalamus 6"18"39'4°'57 and the lesions of basal forebrain could interfere with ascending projections from AChE-positive hypothalamic neurons. The possible contribution of efferents from hypothalamocortical systems was investigated by placement of lesions. Fig. 9 illustrates a case in which a lesion was produced electrolytically in the hypothalamus of a rat, and the animal sacrificed 6 days later. The lesion included neurons of the ventral, dorsal and much of the lateral hypothalamus, and likely destroyed many ascending axons passing through this region. However, AChE staining in cortical area 17, as shown in Fig. 9B, appears similar in the two hemispheres of this case. Densitometric analysis of this case revealed no difference in AChE staining between the two hemispheres indicating that the hypothalamic lesion did not affect AChE staining in visual cortex.

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Pattern of A C h E histochemical staining in cortical area 17 in animals with brainstem lesions The lesions of basal forebrain or subcortical white matter presented above could interfere with ascending projections from brainstem monoamine systems. Because some of these neurons contain ACHE, this projection could contribute to AChE staining in occipital cortex. The possible contribution of efferents from brainstem systems was investigated by placement of brainstem lesions. Fig. 10 illustrates a case in which a large lesion was produced electrolytically in the midbrain tegmentum of a rat, and the animal sacrificed 8 days later. The large lesion included the central tegmentum, through which most of the monoaminergic axons destined for cerebral cortex travel. The electrode was introduced with an angled approach and hence did not pass through visual cortex. As illustrated in Fig. 10B, AChE staining in cortical area 17 appears similar in the two hemispheres of this case, indicating that the brainstem lesion did not affect AChE staining in visual cortex. Densitometric analysis of this case revealed no difference in AChE staining between the two hemispheres (D = 0.125; P > 0.2). Other cases with similar lesions and with postoperative survival times of up to 3 weeks revealed no change of AChE staining in visual cortex following placement of brainstem lesions. Pattern of A C h E histochemical staining in cortical area 17 in animals with lesions of the lateral geniculate body Previous work from this laboratory has demonstrated that placement of lesions in the dLGN of the infant rat

190

Fig. 9. AChE-stained transverse section showing effects of a unilateral hypothalamic lesion on AChE histochemical staining m occipital cortex. A: lesion of the hypothalamus of the right hemisphere; borders of the lesion are indicated by arrows. Bar = 1 mm. B: section through occipital cortex showing AChE staining in the two hemispheres. Borders of cortical area 17 are indicated by arrows. Note the similarity of AChE staining in the two hemispheres. Bar = 1 mm.

results in a m a r k e d loss of A C h E staining in visual cortex 47. Q u i t e different results were o b t a i n e d from adult animals. Fig. 11 presents results from an e x p e r i m e n t in which an adult rat received a lesion of the lateral geniculate body, and was sacrificed 8 days later. The lesion, shown in Fig. l l A , d e s t r o y e d all of the lateral geniculate body, with minimal involvement of adjacent thalamic or hippoc a m p a l tissue. The p h o t o m i c r o g r a p h in Fig. l i B illus-

trates the similarity of A C h E staining in the two hemispheres of occipital cortex in this case. Densitometric analysis p r e s e n t e d in Fig. 12 further d e m o n s t r a t e s the similarity of A C h E staining in the two hemispheres (D = 0.125; P > 0.2). O t h e r cases with similar lesions and post-operative survival periods ranging up to 3 weeks revealed no evidence of change in A C h E staining in visual cortex following p l a c e m e n t of d L G N lesions.

191

Fig. 10. AChE-stained transverse section showing effects of a unilateral brainstem lesion on AChE histochemical staining in occipital cortex. A: lesion site in left hemisphere of the brainstem tegmentum; borders of the lesion are indicated by arrows. Bar = 1 ram. B: AChE staining in occipital cortex. Borders of cortical area 17 are indicated by arrows. Note the similarity of AChE staining in the two hemispheres. Bar = 1 mm.

DISCUSSION

Overview of results and technical considerations Results presented in this manuscript demonstrate that A C h E histochemical staining in cortical area 17 of the adult rat is derived largely from AChE-positive, cortically projecting n e u r o n s of the basal forebrain. Thus, the origins of A C h E staining in the adult are clearly different from the origins of A C h E staining in the infant 51. The

present data, taken together with those from experiments with developing animalssl, indicate a f u n d a m e n t a l change in AChE-positive elements in primary visual cortex during development. P r o m i n e n t A C h E staining of the geniculocortical projection system declines and gives way to A C h E staining in the system of basal forebrain projections to visual cortex. The present results rely upon use of lesion techniques that result in anterograde degeneration and loss of

192

Fig. 11. Photomicrographs showing the effects of a lesion of the dLGN on AChE histochemical staining in occipital cortex. A: AChE-stained transverse section through the caudal thalamus: note the absence of dLGN in the left hemisphere. Bar = 1 mm B: AChE-stained transverse section through occipital cortex from the case with the lesion shown in A. Note the similarity of AChE staining in the two hemispheres. Bar = 1 mm.

e n z y m e activity. The lesion technique is widely used in e x p e r i m e n t a l neurobiology, but certainly is not without its d r a w b a c k s ~6. A s we have discussed in some detail previously 5~, the lesion technique can suffer from flaws that could yield either false positive and false negative results. T h e possibility of obtaining false positive results warrants particular attention in regard to the fiber of passage p r o b l e m in the basal forebrain. These lesions were relatively large by design because the cholinergic cells of origin of A C h E - p o s i t i v e projections to visual cortex are scattered throughout several cytoarchitecturally defined nuclear groups of the basal forebrain, including the nucleus of the diagonal band, the substantia

innominata and the medial globus p a t l i d u s 1'5'6'9'29'3°' 38-40.55,56,58.64 The electrolytic lesions very likely also interrupted ascending axons of h y p o t h a l a m i c cholinergic and brainstem m o n o a m i n e neurons, which pass through the basal forebrain on their route to cortex, including cortical a r e a 172'7'1°'33'41 . This may be an i m p o r t a n t point because neurons of the hypothalamus, locus coeruleus and the raphe have been d e m o n s t r a t e d to be ACHE° positive 2'4. A l t h o u g h we are aware of no evidence that indicates axons of these neurons t r a n s p o r t A C h E to cerebral cortex, this is a possibility. Thus, the m a r k e d reduction in A C h E histochemical activity in cortical area 17 following p l a c e m e n t of lesions in the basal forebrain could have been a result of d a m a g e to passing axons,

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axons. Cytotoxin-induced lesions had essentially the same affect on patterns of A C h E staining in visual cortex as did electrolytic lesions. Our use of these cytotoxins, however, resulted in lesions quite varied in size and we were not convinced that passing axons were spared in cases with the more evident lesions. Further, evidence is accumulating that cytotoxin-induced lesions may result in slow demyelination and axon damage 65. Thus we also investigated the possibility of false positive results from basal forebrain lesions by placing electrolytic lesions in the hypothalamus or midbrain tegmentum. The hypothalamic lesions would have destroyed many of the cell bodies of origin and likely most of the efferent axons of hypothalamic neurons projecting to cortex. Further, hypothalamic or brainstem lesions likely destroyed the vast majority of ascending projections from the locus coeruleus and raphe groups as their axons pass through these regions 2"7"m'3341. Placement of these lesions was not followed by reduction or change in the pattern of A C h E staining in cortical area 17, indicating that the reduction in A C h E staining in cortical area 17 following placement of lesions in the basal forebrain is a result of destruction of cortically projecting, AChE-positive cholinergic neurons in the basal forebrain. The possibility of false negative results must be addressed in the case of lesions of the d L G N , particularly so because d L G N lesions in the developing rat result in a marked reduction in A C h E staining in cortical area 1751 . However, we detected no loss of ACHE, either visually or by densitometry, in cortical area 17 of animals sacrificed with post-lesion survival times ranging from 6 to 21 days. It could always be argued that use of even longer survival times may have revealed changes in A C h E staining following d L G N lesions. However, changes with longer survival times seem to us to be unlikely and would, in turn, raise other problematic issues of false positive results, in particular the problem of transneuronal degeneration 5~.

2 0

Diff, AChE Density Fig. 12. Densitometric data for effects of dLGN lesion on AChE staining in cortical area 17. A: AChE staining in cortical area 17 in the control hemisphere. B: AChE staining in cortical area 17 ipsilateral to the dLGN lesion. C: laminar differences in optical density of AChE staining between the two hemispheres.

rather than basal forebrain projections, to cortex. We investigated this possibility directly by two approaches. First, lesions were produced in basal forebrain by injection of excitatory amino acids 42"63. The cytotoxic effects of these excitatory amino acids results from receptor mediated uptake and thus these agents are relatively specific for neuronal somata, sparing passing

Basal forebrain projections to visual cortex A number of recent papers have described projections from the basal forebrain to visual cortex ~'5'~''9'11'14' 29-32.36-40,42.45,47,55,56.58,64. B a s a l forebrain neurons projecting to cortex include both cholinergic and noncholinergic neurons, and virtually all cortically projecting cholinergic neurons contain A C h E 3~'~5. The present data demonstrating reduction of A C h E activity in visual cortex following placement of lesions in basal forebrain lend support to results presented previously by Shute and Lewis 5s who, using essentially the same techniques employed here, first demonstrated reduction in cortical A C h E staining following basal forebrain lesions. The present results also are in agreement with those of Kristt

194 et ai. 23 who demonstrated that basal forebrain lesions reduced levels of AChE histochemical staining in somatosensory cortex of the adult rat. The present results are the first to note a reduction of AChE in visual cortex following basal forebrain lesions and support other studies that have demonstrated reduction in levels of choline acetyltransferase in occipital cortex following basal forebrain lesions 14'63. In studies using anterograde transport of 3H-amino acids, Saper 56'57 introduced the idea that corticopetal axons from basal forebrain neurons travel through two distinguishable paths to reach the cortex: a relatively well confined medial pathway through the cingulate bundle and supracallosal stria and a more dispersed lateral pathway. This scheme has received support from studies using anterograde transport of lectins 34 and retrograde transport of peroxidase 6. In general, neurons in the anterior portion of the basal forebrain complex send their axons through a medial path through the septum, perforating the corpus callosum and joining the cingulate bundle before innervating medial regions of cortex, particularly cingulate and parahippocampal cortices 6" 11,29,34,38,40,56,63 Neurons from middle and caudal portions of the basal forebrain complex send their axons more laterally, through the internal capsule, before innervating lateral cortex. Cortical area 17 appears to occupy a region that is on the border of cortices innervated by, respectively, the medial and lateral pathways 6'56. Indeed, Saper 56 concluded that medial parts of area 17 received basal forebrain input via the medial path while lateral area 17 received innervation through the lateral path. Carey and Rieck 6 reported that injection of H R P in medial area 17 resulted in retrogradely labeled neurons in anterior portions of the basal forebrain complex, while injections into lateral area 17 resulted in labeled cells in more caudal portions of the basal forebrain complex. Curiously, lesions restricted to the cingulate bundle and supracallosal stria resulted in no detectable change in AChE staining in cortical area 17, although AChE staining in cingulate and retrosplenial cortical regions was drastically reduced. Apparently, axons of AChE-positive basal forebrain neurons bound for visual cortex travel in a position lateral to those in the cingulate bundle. Our lesion data indicate that axons of basal forebrain projections to occipital cortex are not confined to a particular bundle, but appear to be quite dispersed in the mediallateral dimension as they travel caudally. Results from experiments using anterograde 29'3°'34'56 and retrograde 47 transport techniques have indicated that basal forebrain projections terminate primarily in the infragranular layers of cortical area 17 of the rat. These data provide experimental support to histochemical and

immunocytochemical studies demonstrating relatively more activity of cholinergic enzymes in deeper layers than in superficial layers 36'43'66. As noted above, many of the cortically projecting neurons of the basal forebrain complex are cholinergic ~'31"3~'4°'55 and virtually all of these cholinergic neurons stain positively for AChE 1'~55 Thus the presently used AChE stain appears to be a reliable measure of cholinergic basal forebrain projections to visual cortex. We must note, however, that basal forebrain neurons that are not cholinergic and do not stain for AChE may also project to visual cortex 55 and our techniques would not be sensitive to these neurons. It is of particular relevance to note that the results of basal forebrain lesions in the adult, as reported in this communication, are distinctly different from the results of similar lesions placed in the developing animal 51. This will be discussed in more detail below. Hypothalamic projections to visual cortex Several studies using retrograde or anterograde transport techniques have demonstrated hypothalamic projections to cerebral cortex 18'39'4°'57. These cells can be found primarily in the lateral hypothalamus, but appear to extend into para-mammillary regions as well as the fields of Forel and zone incerta 57. Many of these hypothalmic neurons stain positively for AChE TM but not for choline acetyltransferase 39 and hence appear not to be cholinergic. Whether these neurons simply represent the caudal extention of the population of cortically projecting neurons found in the basal forebrain or whether they should be viewed as a distinct group remains unsettled. In either case, there is little evidence suggesting a strong projection from the hypothalamus to visual cortex and the present results from lesion experiments indicate that these neurons do not contribute appreciably to AChE staining in visual cortex. Brainstem projections to visual cortex Studies using a variety of techniques have provided convincing evidence that brainstem monoamine neurons send axonal projections to visual cortex 4"7'1°'33'41. Some of these monoaminergic neurons contain AChE 2'4 and the axon terminals of these neurons must be considered as possibly contributing to AChE staining in visual cortex. AChE-positive dopaminergic projections from the ventral midbrain seem least likely, as their axons terminate only very sparsely in cortical area 177. Noradrenergic neurons of the locus coeruleus are ACHEpositive and their axons reach occipital cortex, but the laminar pattern of termination appears quite different from the pattern of AChE staining 41. Serotonergic projections from the raphe group appear to terminate more densely in area 17 than do other monoaminergic

195 systems, but again the laminar pattern of labeled fibers does not correspond to the laminar pattern of A C h E staining 33. Thus, although brainstem monoaminergic projections could be considered as possible, albeit not probable, candidates, the present results from lesion experiments present convincing evidence that these neurons do not contribute appreciably to A C h E staining in visual cortex.

17, particularly in the A C h E bands in layer I and in III-IV. Thus, A C h E staining in cortical area 17 in adult animals is clearly different from A C h E staining in developing animals. First, adult and developing animals have different laminar patterns of A C h E staining. In adults, A C h E is found primarily in layer l and in a band that corresponds to cortical layer V and the deep part of layer IV 36"43"66. In developing animals 48"5°'51"s3'54, partic-

Dorsal lateral geniculate projections to visual cortex A wealth of evidence demonstrates that the d L G N projects to cortical area 1712"35"44"46"66. It is interesting that the d L G N of the adult rat displays A C h E activity 43' 52, but does not contribute to A C h E staining in visual cortex. Inspection of the adult d L G N does not reveal AChE-positive neuronal somata52; indeed A C h E staining appears characteristic of axons, probably from cholinergic neurons of the dorsal pons. The absence of effects of d L G N lesions on A C h E staining in visual cortex of adult animals is in stark contrast to results of d L G N lesions in the developing animal 5~.

ularly during the second postnatal week, A C h E is found primarily in cortical layer I and in a band that corresponds to layer IV and the deep part of layer III. If one only studied normal material, the differences in the laminar pattern of A C h E staining could be dismissed as insignificant or perhaps due to slight shifts in terminal fields during development. However, the results from lesion studies in the adult and the developing animals clearly demonstrate that the neuronal systems contributing to A C h E staining in the developing visual cortex are clearly different from those contributing to A C h E staining in visual cortex of mature animals.

Differences between mature and developing visual cortex O u r primary interest centers around the differences in results obtained in the present study of adult animals and our previously reported results from studies of developing infant rats. The differences are striking. In the adult, placement of lesions in the basal forebrain is followed by marked reduction of A C h E staining in occipital cortex of the hemisphere ipsilateral to the lesion. The reduction in staining is most obvious in the band that corresponds to cortical layers V and deep IV. In contrast, placement of lesions in the d L G N result in no detectable change in A C h E staining in visual cortex. On the other hand, results from developing animals are quite different. In infant rats 5~, basal forebrain lesions result in a slight loss of A C h E staining throughout the laminae in cortical area 17, and in other posterior cortical regions, but the distinct band of A C h E characteristic of cortical layer IV and deep III remains unaffected. Lesions involving the d L G N , in contrast, result in a dramatic reduction of A C h E staining in cortical area

Transiently expressed A C h E activity in development Geniculocortical neurons express A C h E activity transiently during development but not in the mature state. A C h E activity in the developing animal is found associated with protein synthetic machinery in d L G N neurons and with axon terminals in cortical area 1753. Transient expression of A C h E is not limited to the rat geniculocortical system, but appears to be a c o m m o n characteristic of sensory thalamocortical projections in rodents TM 22 26 and of some thalamocortical projections in humans and sub-human primates 19"2°. In each of these instances, it appears that A C h E is expressed transiently by developing thalamocortical neurons during the time when axons of these neurons are growing into thalamorecipient layers of cortex and forming synapses with cortical neurons. The function of the transiently expressed A C h E remains elusive.

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Acknowledgements. Supported in part by NSF Grant 87-08515. We thank Kathy Gallardo for technical assistance and Drs, J.H. Fallon, S.H.C. Hendry and E.G. Jones for stimulating discussions.

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