Neuroscience Letters, 116 (1990) 75-80 75 Elsevier Scientific Publishers Ireland Ltd.

NSL 07045

Tyrosine hydroxylase-like (TH) immunoreactivity in human mesolimbic system

Richard M. Torack I and John C. Morris 1,2

Departments of IPathology (Neuropathology) and eNeurology and Neurological Surgery (Neurology), Washington University School of Medicine, St. Louis, MO 63110 (U.S.A.)

(Received 12 March 1990; Revised version received 15 April 1990; Accepted 19 April 1990)

Key words." Bouin's fixative; TH-like immunoreactive fiber; TH-like immunoreactive terminal; Hippo- campal complex; Perforant pathway; Alzheimer's disease

A practical methodology has been described for the use of human postmortem brain tissue in both tyro- sine hydroxylase and dopamine fl-hydroxylase immunohistochemistry in which in situ perfusion with para- formaldehyde is followed by immersion fixation in Bouin's fixative. These studies indicate that TH-like immunoreactive fibers and terminals are not uniformly distributed in the human hippocampal complex. A distinctive lesser innervation is noted for the structures that compose the perforant pathway and may be important for the predilection of these areas for pathological change particularly as occurs in Alz- heimer's disease.

The involvement of the ventral tegmental area (VTA) and the mesolimbic system in human brain disease has been underappreciated until recent reports of histological changes in this network in some cases of Parkinson's disease (PD) that are complicat- ed by dementia [16, 20, 21]. This omission is due largely to the lack of neuronal dis- tinction between ventromedial pigmented neurons that project to the limbic system and dorsolateral neurons that project to the striatum. Moreover, the neuron loss, free pigment, reactive gliosis and Lewy bodies that occur in the VTA are identical to changes that are present in the substantia nigra in uncomplicated PD. The occurrence of neuron loss, gliosis and neurofibrillary tangles in the projection areas of the meso- limbocortical system is indirect evidence that the dopaminergic cells of origin in the VTA are pathologically involved [20]. A more definite indication of change would be a reduction of the dopaminergic terminal field in the hippocampus. This pathway was unrecognized until H6kfelt [10] suggested that some of the fluorescence of cate- cholamines could be due to dopamine. Since that time dopaminergic fibers originat- ing in the VTA have been demonstrated in laboratory animals by pharmacological assay [3], by tracer studies in both directions [18, 19] and most recently by electron

Correspondence." R.M. Torack, Department of Pathology (Neuropathology), Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 631 I0, U.S.A.

0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

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microscopic localization of peroxidase-labeled antibody against tyrosine hydroxylase [13]. The topical application of dopamine to the hippocampus depresses both sponta- neous and glutamate induced firing [9].

Although the mesolimbic system has been thoroughly studied in laboratory ani- mals, relatively little is known about the biochemical anatomy of this system in man. In pigmented neurons of the substantia nigra, tyrosine hydroxylase-like immunoreac- tivity (TH) has been shown to localize equally to the ventromedial and the dorsola- teral area [15]. Biochemical studies of the hippocampus have revealed a low concen- tration ofdopamine [7] but a relatively high concentration of DI receptor particularly in CAI [5]. However the demonstration of dopaminergic fibers by TH immunoreacti- vity in putative terminal fields such as the hippocampus has been unsuccessful in postmortem material. Recently the use of perfusion fixation to localize dopamine fl- hydroxylase-like immunoreactivity (DBH) in human autopsy material [2] suggests that a similar procedure might be effective for TH immunoreactivity as well. We now report the identification of TH fibers in the normal human hippocampal formation including the entire perforant pathway.

The brain tissue for this study was obtained at the time of postmortem examina- tion in our medical center from 4 patients aged 40-80 years who had no history of neurologic disease and who had a cause of death unrelated to the central nervous system. The postmortem interval was 4~18h. Following removal from the cranial cavity the flesh brain was perfused first with 60 ml phosphate-buffered saline (PBS) through each carotid artery and the basilar artery, followed by 60 ml of 4 % parafor- maldehyde in 0.1 M phosphate buffer pH 7.4 at 4.0~'C. The hippocampus was isolated by clamping both the middle cerebral artery just distal to the origin of the anterior choroidal arteries and the posterior cerebral artery just distal to the origin of the pos- terior choroidal arteries. Each cerebral artery was re-perfused with 500 ml of the cold paraformaldehyde solution under 40 lbs. of pressure over a period of 30 min. The hippocampus then was removed as a block of tissue from the level of the mammiUary bodies to a point 4 cm caudally. The block included the hippocampus proper and the entorhinal cortex, and was sectioned into 0.5 cm thick slices so that usually 10 slices were available for study. Blocks 5 and 6 usually represented the middle of the hippocampal formation and were fixed by immersion for another 48 h either in 4% paraformaldehyde alone or in paraformaldehyde containing 1% Bouin's solution. The latter was found to be superior for the demonstration of TH immunoreactivity.

The blocks of tissue were dehydrated in graded alcohol and embedded in paraffin. Tissue sections were cut at 7 pm, mounted on slides, placed in a 56°C oven for 1 h and in a 37°C oven overnight. Following removal from the oven, the tissues were brought to room temperature~ washed for 5 min in 2 changes of xylene and for 3 rain in 3 changes of 100% ethyl alcohol. The tissue sections were rehydrated using decreasing percentages of ethyl alcohol down to distilled water. The sections were treated with 0.3% hydrogen peroxide in 0.05 M Tris buffer, pH 7.2 for 30 min. Fol- lowing a Tris buffer rinse, the tissues were reacted in a solution containing 0.1 M Tris buffer, pH 7.6, 0.1% Triton and 1% normal goat serum. Rabbit antibodies to TH (Eugene Tech) and DBH (Eugene Tech) were used as the primary antibody

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diluted to 1:400, 1:800, 1:1600, and 1:3200. TH was diluted in 0.05 M, pH 7.2 Tris buffer and reacted for 2 h at room temperature. Following a Tris buffer rinse, the sections were reacted with biotinylated goat anti-rabbit IgG diluted 1:25 in a Tris buffer at pH 7.2. Following the Tris rinse, the tissue sections were reacted with avi- din-biotin-peroxidase complex (ABC) (Vector Labs) for 1 h at room temperature. DBH was diluted in 0.1 M, pH 7.6 Tris-buffered saline (TBS) containing 0.1% Triton and 1% normal goat serum, and incubated overnight at 4°C. Following a TBS rinse, the sections were reacted with biotinylated, goat anti-rabbit IgG 1:25 dilution in 0.I M, pH 7.6 TBS containing 0.1% Triton and 1% normal goat serum, for 2 h at room temperature. Following a Tris buffer rinse, the tissue sections were reacted with ABC for 2 h at room temperature. Both sets of tissue (TH and DBH) were developed in the following mixture. Fifteen mg diaminobenzidine (DAB) in 19 ml of 0.5 M, pH 7.4 Tris buffer, 1% nickel sulfate in 0.05 M, pH 7.2 Tris buffer, 25 ~tl hydrogen perox- ide in 9.0 ml 0.05 M, pH 7.2 Tris buffer. Equal amounts of these solutions were mixed and filtered through a 5.0/lm acrodisk onto the slides.

The TH-immunoreactivity of the hippocampal formation was localized to small fibers containing numerous varicosities and small dots that have been identified as terminals in the rat hippocampus (Fig. 1). When the tissues were reacted in normal rabbit serum no beaded fibers or dots were found (Fig. 2). In these studies, TH immu- noreactivity always revealed a greater number of fibers and dots than those that were DBH immunoreactive, however, this difference was obvious only when Bouin's fixa- tive was used for the post-perfusion immersion fixation. No neuronal cell bodies in the hippocampus showed any TH immunoreactivity. A comparision of TH immu- noreactivity in the various components of the hippocampal formation revealed a striking topographical specificity that involved both beaded fibers and dot-like termi- nals. The densest fiber population appeared to be in the CA4, CA3 and in the presu- biculum (Fig. 3). Distinctly fewer number of fibers were present in CAI, subiculum and entorhinal cortex (Fig. 4). TH immunoreactivity also was found in cell bodies of pigmented neurons in both the substantia nigra and the VTA.

The TH immunoreactivity of the human hippocampal formation is localized to beaded fibers and dot-like terminals that are identical to that described in rat brain [13]. Beaded fibers have been described in noradrenergic fibers and dopaminergic fibers in laboratory animals and constitute varicosities where numerous dense core vesicles are localized [8]. The majority of the dots having TH immunoreactivity repre- sent terminals that synapse with either distral dendrites or dendritic spines [13]. The prominence of TH-immunoreactive fibers in the CA4 and CA3 of the hippocampus corresponds to the distribution of TH immunoreactivity in the rat hippocampus [13] but the similar density of fibers and dots in the presubiculum is a new observation. The relative paucity of TH immunoreactivity in CA1 also has been noted in the rat hippocampus but not in the subiculum or the entorhinal cortex. The differing densit- ies of fibers and dots in these parahippocampal structures may represent a unique situation in the human brain but more likely these areas have not been included in the animal studies. The specific localization of TH reactivity in the human perforant pathway assumes importance because of the frequency with which the senile plaques

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Fig. I. A photomicrograph of TH immunoreactivity which is localized to beaded fibers and dot-like termi- nals. TH 1:400. × 800.

Fig. 2. A photomicrograph of CA4 which shows no immunoreactivity when normal rabbit serum is substi- tuted for TH antibody. NRS 1:400. × 450.

Fig. 3. TH immunoreactivity in human CA4 is localized to numerous beaded fibers and dot-like terminals. TH 1:400. × 45.

Fig. 4. TH immunoreactivity in human CA 1 reveals few stained beaded fibers and dots. TH 1:400. × 450.

and neurofibrillary tangles of Alzheimer's disease occur in these areas in contrast to

the presubiculum and CA3 which are relatively spared [1, 11]. The perforant pathway, originally described in detail by Ramon y Cajal [4], has

involved direct and indirect fibers from the entorhinal cortex that comprise one of three main fiber inputs into the hippocampus. This hippocampal innervation has been amplified by Lorente de No [12] who had described a distinctive nerve supply to different parts of the hippocampus rather than the random arrangement proposed by Cajal. The distinctive dopaminergic innervation reported here would be consistent

with this concept. The perforant pathway, particularly CAI, has been recognized to have greater vulnerability to injury than the remainder of these tissues. In an animal

epilepsy model this susceptibility has been demonstrated by neuron loss in CA l and has been related to hyperactive neurons with an increase in the level of their transmit- ter glutamate [17]. Glutamate is excitotoxic and has been shown to cause paired heli- cal filaments in tissue culture [6] and tau fibril formation in vitro [14]. In AD a de- crease in D1 receptors has been reported [5] so the low level of inhibitory fibers in

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areas like CA1 probably is further diminished and could result in hyperactive neu- rons similar to those of the epilepsy model.

The use of human autopsy material to identify different fiber systems by means of immunohistochemistry has not been possible using routinely immersion-fixed tis- sue. Studies of this type have been unsuccessful, probably because of technical diffi- culties arising from an uncontrolled postmortem interval in which the brain is auto- lyzing and forming clots that impair perfusion effectiveness. In contrast, experimental animals used in the demonstration of dopaminergic fibers usually are killed by perfu- sion fixation through the ascending aorta. The human situation has been improved greatly by the introduction of perfusion fixation [2]. In our studies, the second perfu- sion through the isolated choroidal arteries and particularly the use of Bouin's sol- ution in the immersion fixative appear to have greatly enhanced the demonstration of dopaminergic nerve fibers by labeled antibody. The picric acid in this solution seems to result in an enzyme that is more efficiently blocked in situ. The use of this fixation procedure results in the increased TH immunoreactivity that permits the rec- ognition of the distinctive innervation of the perforant pathway.

The authors wish to thank Janet Chavez for her technical assistance and Sandy Brickey for her help in the preparation of this manuscript.

This work was supported by Grant 11RG-87-119 from the Alzheimer Disease and Related Disorders Association.

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