Studies on the hippocampal formation: From basic development to clinical applications: Studies on schizophrenia

Abstract

The hippocampal formation plays a critical role in cognitive function. The developmental events that shape the hippocampal formation are continuing to be elucidated and their implications for brain function are emerging as well as applying those advances to interventions that have important possibilities for the treatment of brain dysfunction. The story told in this chapter is about the use of the in oculo transplant method to illuminate intrinsic and extrinsic features that underlie the development of the dentate gyrus and adjacent hippocampus and the role of one molecule in the hippocampus and schizophrenia. Schizophrenia, originally conceptualized as a dysfunction in dopaminergic neurotransmission, is now known to involve multiple neuronal systems. Dysfunction of hippocampal neurons is emerging as one of its signature pathological features. Basic insights into the development and function of hippocampal interneurons form the basis of a new treatment initiative for this illness. Evidence for the role of the alpha 7-nicotinic acetylcholine receptor in the development and function of these neurons in rodents has led to human trials of nicotinic agonists for cognitive dysfunction in schizophrenia and the possibility of improving hippocampal development in children at risk for schizophrenia by perinatal supplementation with choline, which can act as an alpha 7-nicotinic acetylcholine receptor agonist.

Figure 1

a). Hippocampal formation from a 30-mm crown-to-rump length (CRL) embryo stained with cresyl violet. Boxed-in area approximates the region dissected to be used as the area dentata transplant.. b). An in oculo area dentata transplant from a 30-mm CRL embryo after 7 months in oculo. × 12. c). Cresyl violet stained frozen section of an area dentata transplant from a 28–30 mm CRL embryo.. d). Cresyl-violet stained paraffin section of the area dentata in situ from a 15-day-old rat. . Scale bar in A and B=500μm, C and D=250μm. From Goldowitz et al., 1982.

Figure 2

The weekly growth (in mm2) of area dentata transplants from 30–35 mm CRL embryos. Areae dentatae were transplanted to anterior eye chambers that had sham operated (Sham + AD), or had received a septal transplant (Septum + AD), or an entorhinal cortical transplant (ERC + AD). The increase in area dentata transplant size was somewhat less in the double transplant situation than in the single area dentata group. The inset compares the size of the area dentata transplants during the first 5 days after transplantation in a group of single area dentate transplants (solid line) and in a group of double area dentate/entrorhinal cortex transplants (broken lines). From Goldowitz et al., 1984a.

Figure 3

Falck-Hillarp histofluorescence of locus coeruleus/area dentata double transplants. a–c). Ten weeks joint survival. Fluorescent locus coeruleus neurons are shown in a and c with enlarged nuclei. There were approximately 91 noradrenergic neurons in this locus coeruleus transplant. The border between locus coeruleus and area dentata transplants is highlighted in a by arrows. Noradrenergic fibers are sparse in the locus coeruleus transplant and more prevalent in the area dentata transplant. For the most part, these noradrenergic fibers in the area dentata have prominent varicosities with small intervaricose segments (see b). d,e). Four-month joint survival. The locus coeruleus transplant contained approximately 200 noradrenergic neurons. In (d), the crack in the tissue marks the locus coeruleus/area dentata boundary. The noradrenergic innervation of area dentata is very dense. At higher magnification, in (e), the dense noradrenergic innervation of the granule cell region of the area dentata transplant is shown. f). Six months joint survival. This locus coeruleus transplant contained approximately 390 noradrenergic neurons. Noradrenergic hyperinnervation was characteristic of area dentata transplants, with an adjoining locus coeruleus transplant containing about 100 or more noradrenergic neurons with a joint survival of 6 months. Scale bar for a,d,f =50μm; b,c,e=25μm. From Goldowitz et al., 1984a.

Figure 4

Double, sequential transplants of septum and area dentata. a). In oculo appearance of the small septal transplant (left) joined with the larger area dentata transplant (right). Scale bar=500μm. b). Septal/area dentata transplants processed for AChE-histochemistry 7 months after joint survival. The septal transplant is on the left. Arrows point to the AChE-rich region occupied by cholinergic cells. The adjacent area dentata transplant has both a zone rich in AChE-positive fibers (arrowheads) and a zone poor in AChE-positive fibers. c,d). Septal/area dentata transplants taken for AChE and Timm’s histochemistry 8 months after joint survival. Dense AChE-positive material is present in the septal transplant in (c), and AChE-postive fibers are prevalent in two regions of the area dentate transplant (arrowheads). These two regions in an adjacent section stained for Timm’s method (d) lack the typical mossy fiber staining that is found elsewhere in the area dentata transplant (arrowheads, Timm’s positive mossy-fiber-like staining). e,f). Similar comparison as in (c) and (d) from another double transplant taken for AChE and Timm’s histochemistry after 8 months joint survival. The area dentata region most proximal to the AChE-positive rich septal transplant (arrows) in (e) receives a modest amount of AChE-positive fibers (arrowheads), while the distal region receives very few AChE-positive fibers. It is this AChE-poor zone in the area dentata transplant that has extensive mossy fiber-like staining with the Timm’s procedure in (f). Area dentata proximal to the septal transplant has very little mossy fiber-like staining although there are many pyramidal cell targets. Scale bar for b–f=50μm. From Goldowitz et al., 1984a.

Figure 5

Schematic representation of the four types of intraocular triple transplantation combinations are shown in b–e (Goldowitz et al., 1984b) compared to the double transplant shown earlier (Goldowitz et al., 1984a). AD = area dentata, ERC = entorhinal cortex, S = septum, LC = locus coeruleus. The density of LC-derived noradrenergic innervation of AD is schematically depicted by the density of cross-hatching (i.e., the denser the hatching the denser the noradrenergic innervation of the area dentata transplant). Note that the noradrenergic innervation in other transplants than AD is not shown. The order of grafting in (a) is AD, then LC, and in (b–e) ERC or S, then AD, then LC. From Goldowitz et al., 1984b.

Figure 6

Triple transplant with septal nuclei (S) intervening between area dentata (AD) and locus coeruleus (LC) transplants. a) The granule cell layer (arrowheads) and scattered pyramidal and polymorphic cells in the area dentata transplant are evident in this section stained with cresyl violet. The crack in the section passes through the septal transplant. At either end of the transplants is iris tissue. b) The fluorescent noradrenergic fibers are shown in this photomontage taken from a section adjacent to (a). In the center of the septal neuropil are the “clubs” or “swellings” (arrowheads) of histofluorescent axons that are characteristic of this triple transplant configuration, but are unusual in the other triple transplant configurations that were examined. The area dentata also contained such swellings (arrows). An unusual feature of this transplant configuration was the greater density of histofluorescent axons in the locus coeruleus compared to the area dentata. The histofluorescent axons at the edge of the septal transplant appeared to be destined for the area dentata neuropil. Other noradrenergic fibers reached the area dentata via the iris. c) This photomicrograph shows some of the approximately 288 fluorescent neurons present in the locus coeruleus transplant. The septal bridge is relatively fiber-free and the moderately innervated area dentate is shown to the left. d)The density of fluorescent noradrenergic fibers in the granule cell region of area dentata is shown at higher magnification in this photomicrograph. Calibraion bars: a) 0.5 mm; b–d) 100 μm. From Goldowitz et al., 1984b.

Figure 7. The role of α7nAchRs and CHRNA7 in auditory sensory gating and schizophrenia

A pedigree with two schizophrenic siblings (black symbols). Sibling A has a P50 auditory evoked response (positive polarity down) to the first of paired stimuli and normal inhibition of response to the second stimulus. Sibling B, who has schizophrenia, lacks this inhibitory sensory gating function and has a large response to the second sound, compared to Sibling A. Sibling C, who is unaffected, shares Sibling B’s electrophysiological trait. Abnormal sensory gating (A) segregates with the 2 allele of D15S1360, which is in intron 2 of CHRNA7. Normal gating (N) segregates with the 1 allele. From Freedman et al., 1997. The P50 was selected and its amplitude measured by a computer algorithm that identified the largest positive wave between 40 and 70 ms following the initial stimulus. The wave following the second stimulus was selected to be within ± 10 ms of the latency of the first wave. This criterion was derived from hippocampal single neuron recordings in laboratory animals, where the timing of neuronal discharge to each stimulus could be rigorously determined. From Freedman et al., 1996). Hence, the earlier wave in the response to the second stimulus in Sibling A is not a P50 response to this stimulus. From Freedman et al., 1997

Figure 8

Co-localization of ¹²⁵I-α-bungarotoxin labeling of α7-nicotinic acetylcholine receptors (right panels) and GABA-immunocytochemical reactivity as detected by fluorescence histochemistry (left panels) on rat hippocampal interneurons. For all panels but b, the fluorescence histochemistry was performed on perfused and fixed brain sections, followed by ¹²⁵I-α-bungarotoxin binding. For b, the ¹²⁵I-α-bungarotoxin was injected intraventricularly, and then the brain was perfused, fixed, and sectioned. Panels a and b show large neurons (arrowheads) that contain GABA and also bind ¹²⁵I-α-bungarotoxin, located in the stratum oriens of CA3. Similar CA3 neurons are shown in stratum oriens and stratum lucidum in d. c shows a series of smaller neurons on the inner layer of the distal portion of the dorsal blade of the dentate gyrus. e shows several small neurons in CAl, in striatum radiatum, and in stratum lacunosum moleculare. Scale bar: 50 μm for a–d, 100 μm for e. From Freedman et al., 1993.

Figure 9

Co-localization of α7-nicotinic acetylcholine receptors and nitric oxide synthetase in rat hippocampal interneurons. Photomicrographs of interneurons in stratum oriens of area CA1 which were first found to be immunoreactive for nitric oxide synthetase (indicated with white arrowheads in A, B, C) and then subsequently found to also bind α-bungarotoxin (indicated with black arrowheads in the alternate A, B and C frames). The small black or white arrows indicate blood vessels visible in associated pairs of photomicrographs (e.g. A and A, B and B, C and C). Calibration bar=48 μm. From Adams et al., 1997.

Figure 10

Blockade of α7-nicotinic acetylcholine receptors activates nerve growth factor gene transcription. In situ hybridization using a probe for Nerve Growth Factor mRNA in rat hippocampus, 3 hr after intraventricular administration of α-bungarotoxin (1 pg in 10 μL; panel a) or and equal volume of Ringer’s solution (b). The animal treated with α-bungarotoxin shows prominent labeling in the dentate. The animal treated with Ringer’s shows no labeling in the dentate, with scattered cells labeled in the hilus. Scale bar for A and B=100μm. From Freedman et al., 1993.

Figure 11

DBA/2 mice have abnormal inhibitory sensory gating and expression of hippocampal α7-nicotinic acetylcholine receptors, linked to the Chrna7 locus. Left panel. C3H mice have hippocampal evoked responses to repeated auditory stimuli that resemble those of normal humans, whereas DBA-2 mice have responses similar to persons with schizophrenia. Right panel. Photomicrographs of ¹²⁵I-α-bungarotoxin labeling of transverse sections of hippocampus. Panel (A) illustrates the pattern of ¹²⁵I-α-bungarotoxin labeling characteristic of parental C3H mice, showing that C3H mice have prominent labeling at the CA3/CA2 border (arrowheads). The photomicrograph in (B) is from a backcrossed strain which has a C3H Chrna7 marker introgressed into a C3H genetic background; the characteristic C3H α- bungarotoxin binding phenotype is preserved. At higher power (C, D), the α-bungarotoxin-positive neurons embedded in the dense, diffuse binding can be more easily discerned (arrows). DBA/2 mice (E) lack this diffuse labeling, but instead display numerous α-bungarotoxin-positive neurons in CA1 lacunosum-moleculare (arrows). Introgression of the DBA/2 Chrna7 marker into a C3H background to produce a second backcrossed strain results in a pattern of α-bungarotoxin binding characteristic of the DBA/2 phenotype (F). Panels G and H illustrate the area CA1 lacunosum-moleculare α-bungarotoxin-positive neurons (arrows) at a higher power. These mice also have abnormal inhibitory sensory gating like the DBA/2 parental strain. Scale bars (A, B, E, F)=125 μm; (C, D, G, H)=50 μm. Modified from Adams et al., 2001.

Figure 12

Alpha7-nicotinic acetylcholine receptors are maximally expressed in the perinatal period. Graph shows the levels of ¹²⁵I-α-bungarotoxin binding in the hippocampus of the developing rat from just prior to birth to adulthood (postnatal day 60). From Adams et al., 2002.

Figure 13

Effects of perinatal choline supplementation on the development of sensory gating. DBA/2 mice have diminished expression of α7-nicotinic cholinergic receptors, due to Chrna7 polymorphisms, and also have hippocampal sensory gating deficits that resemble the P50 gating deficits in schizophrenia. Choline was supplemented at twice the normal dietary level in the maternal diet from conception through weaning. Thereafter, both groups of mice received diets with normal choline levels. Each point is the ratio of amplitude of the hippocampal auditory evoked responses to paired stimuli, amplitude of the second response divided by the first. In adulthood, the offspring had decreased amplitude ratios, indicative of improved sensory gating. Data reported in Stevens et al., 2008.