Ultrastructural evidence for hippocampal target cell-mediated trophic effects on septal cholinergic neurons in reaggregating cell cultures

We have previously demonstrated at the light microscopic level that when embryonic day-15 septal neurons are co-cultured for 21 days with their target cells from the hippocampus, increased numbers of septal cholinergic neurons are present as compared with co-cultures employing cells from the non-target cerebellum. In addition, fine varicose axon-like cholinergic fibers are found to be associated with the hippocampal cells but not with cerebellar cells. We now provide ultrastructural evidence for hippocampal target cell-enhanced cholinergic neuronal survival, axonal proliferation, and synapse formation in this culture system. Dissociated cell suspensions from septal, hippocampal, and cerebellar areas were obtained from 15-day mouse embryos; and hippocampal and cerebellar cells were internally labeled with rhodamine-conjugated wheat germ agglutinin. Combinations of septal and hippocampal cells, and septal and cerebellar cells were allowed to reaggregate in rotation mediated culture for either 15 or 21 days. The reaggregates were then fixed, embedded, sectioned, and processed for acetylcholinesterase-positive acetylcholinesterase-positive cells and fibers, and under fluorescence to locate rhodamine-labeled cell populations. Representative reaggregate profiles were then re-embedded for electron microscopic examination. In both types of reaggregates, either labeled hippocampal target or cerebellar non-target cells segregated from the septal cells so that areas containing each of the respective cell populations could be studied. In sections of septal-hippocampal reaggregates from 15-day cultures, 571 out of 665 (85%) cholinergic neurons examined were intact, whereas 15% of the cells showed some ultrastructural features of degeneration. Similarly, at day 21, 297 out of 335 (88%) of the cholinergic neurons were intact. In sections of septal-cerebellar reaggregates from 15-day cultures, 473 out of 572 (83%) cholinergic neurons were intact. By day 21 of culture, however, only 15 out of 110 (14%) cholinergic neurons examined were intact from the septal-cerebellar reaggregates. In areas of septal-hippocampal reaggregates occupied by rhodamine-labeled hippocampal cells, profiles of acetylcholinesterase-labeled axons were identified, and synaptic specializations were observed between cholinergic terminals and dendrites as well as somata of hippocampal target cells. In contrast, areas of septal-cerebellar reaggregates occupied by rhodamine-labeled cerebellar cells were devoid of cholinergic fibers.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effects of nerve growth factor on the development of septal cholinergic neurons in reaggregate cell cultures

Recent studies suggest that nerve growth factor is present within the central nervous system where it may exert selective trophic effects on cholinergic neurons. We have measured the effects of nerve growth factor on septal cholinergic neurons in three-dimensional reaggregating cell cultures, a system which closely simulates the cellular environment in situ. Septal cells obtained from 15-day-old mouse embryos were dissociated into a single cell suspension and then allowed to reaggregate in culture in a rotary incubator shaker. After 17 days in culture, half of the reaggregates from a flask were sonicated for measurement of choline acetyltransferase activity, and the remaining reaggregates were processed for acetylcholinesterase histochemistry. Addition of nerve growth factor to medium containing septal reaggregates resulted in greater than a three-fold increase in choline acetyltransferase activity and in the number of acetylcholinesterase-positive cells, as well as an enhancement in the staining of acetylcholinesterase-positive fibers. All of these effects of nerve growth factor could be neutralized by antibodies to nerve growth factor. In order to evaluate the possible role of endogenous hippocampal-derived nerve growth factor, antiserum to nerve growth factor was added to the culture media containing septal-hippocampal coaggregates. After 21 days in culture, the presence of nerve growth factor antibodies did not qualitatively affect the pattern or density of cholinergic fibers observed. Synapse formation between cholinergic axons and hippocampal target cells was still in evidence as revealed by electron microscopy. However, there was a modest decrease in choline acetyltransferase activity (20%) and cholinergic cell number (30%) when compared with coaggregates grown in culture medium either without nerve growth factor antiserum or with non-immune serum. The magnitude of these effects was markedly less than the effects observed when exogenous nerve growth factor was added to septal cells grown alone in reaggregate culture. These results suggest that nerve growth factor may play a role during central cholinergic development, but that additional trophic mechanisms are likely to be required.     

Neurotransmitter characteristics of brain grafts: striatal and septal tissues form the same laminated input to the hippocampus

In previous experiments we have studied the development of grafts of embryonic septal tissues implanted alongside the hippocampal formation of neonatal rats. In the present study we examined intracerebral implants of corpus striatum, a brain region that contains acetylcholinesterase-positive cells and does not normally project to the hippocampal formation, in order to evaluate the possibility that neurotransmitter identity may be involved in mechanisms guiding patterns of afferent growth and connectivity. Implant cavities were made in the entorhinal cortices of neonatal rat recipients and 3-6 days later embryonic striatal tissues were grafted to these preformed cavities. Implants were examined with acetylcholinesterase histochemistry one month after implantation. Grafts of embryonic striatal tissues did not survive implantation when the implant was introduced at the same time as the cavity was made. Grafts of corpora striata containing acetylcholinesterase-positive neurons were found in 7 of 11 rats in the delayed implant paradigm and, in all but one of these animals, acetylcholinesterase was present within those terminal laminae in the ipsilateral hippocampus and dentate gyrus that normally receive cholinergic input from the septal area. These findings suggest that cues underlying the development of specific connections between native (and implanted) septal efferents and hippocampal target neurons may be recognized by ingrowing acetylcholinesterase-reactive fibers from striatal implants.     

Interleukin-6 improves the survival of mesencephalic catecholaminergic and septal cholinergic neurons from postnatal, two-week-old rats in cultures

Interleukin-6 (human recombinant) supported the survival of cultured mesencephalic, catecholaminergic and septal cholinergic neurons from postnatal, two-week-old (P13-P15) rats. Significantly, more catecholaminergic neurons, stained by monoclonal anti-tyrosine hydroxylase antibody, were found in cultures supplemented with interleukin-6 at a concentration of 5 ng/ml than in cultures not treated with interleukin-6. The optimal dose used was 50 ng/ml. The survival effect of interleukin-6 on postnatal rat, tyrosine hydroxylase-positive neurons was observed both in cultures using serum-containing and serum-free medium. Contents of dopamine and noradrenaline in cultures with interleukin-6 were also larger than in control cultures. Interleukin-6 also increased the survival of cultured embryonic (E17) rat midbrain tyrosine hydroxylase-positive neurons. The effect on these neurons was, however, smaller, and the optimal dose of interleukin-6 was nearly 5 ng/ml. Interleukin-6 also supported the survival of cultured postnatal (P13) rat septal cholinergic neurons, visualized by acetylcholinesterase staining. The concomitant addition of mouse nerve growth factor (100 ng/ml) and interleukin-6 (50 ng/ml) had a synergetic effect on the survival of acetylcholinesterase-positive neurons in culture. Our data suggest that the survival of cultured tyrosine hydroxylase-positive, mesencephalic, and acetylcholinesterase-positive, septal neurons from postnatal two-week-old rats was supported by interleukin-6, just as there was a different dose dependency of interleukin-6 on the cultured postnatal neurons compared with embryonic neurons.     

Guidance of acetylcholinesterase-containing fibres by target tissue in co-cultured brain slices

Slices of various brain regions were prepared from newborn and from 7-day old rats and co-cultured in different combinations. In the majority of co-cultures of septal and hippocampal slices, acetylcholinesterase-positive fibres originating in the septal nuclei invaded the adjacent hippocampal slice. A similar pattern of hippocampal ingrowth by acetylcholinesterase-positive fibres occurred with slices prepared from the nucleus basalis of Meynert and from spinal cord. Septal neurones also projected to cortical slices, an effect which even occurred in the presence of their natural target tissue. In contrast to these massive projections to brain areas which in situ receive cholinergic inputs, no significant acetylcholinesterase-positive fibre ingrowth was observed in tissues which lack major cholinergic afferents in situ (hypothalamus, substantia nigra and cerebellum). These results indicate that under our culture conditions, acetylcholinesterase-positive fibres selectively invade cholinergic target areas. This effect is independent of the brain area from which the cholinergic neurones were derived.     

Colocalization of gamma-aminobutyric acid and acetylcholinesterase in rodent cortical neurons

We have previously demonstrated that neurons of the rat cerebral cortex which stain positively for acetylcholinesterase are not likely to be cholinergic since they do not colocalize with choline acetyltransferase immunoreactivity [Levey, Rye, Wainer, Mufson and Mesulam (1984) Neuroscience 9, 9-22]. These noncholinergic acetylcholinesterase-positive cells were similar in morphology to cortical neurons which localize gamma-aminobutyric acid or glutamate decarboxylase immunoreactivity. In order to investigate the possibility that the two substances may be colocalized to the same cortical neurons, gamma-aminobutyric acid immunohistochemistry and acetylcholinesterase histochemistry were combined in single sections of rat cerebral cortex. We found that 18% of gamma-aminobutyric acid-immunoreactive cortical neurons are also acetylcholinesterase-positive, and about 36% of acetylcholinesterase-positive cells are gamma-aminobutyric acid-immunoreactive. Neurons which colocalized both substances were multipolar and bipolar neurons in cortical laminae II-VI and were observed in every cortical area examined. The possibility that gamma-aminobutyric acid-immunoreactive/acetylcholinesterase-positive cortical neurons may be postsynaptic targets of cholinergic afferents to the cerebral cortex is discussed.     

Transmitter phenotype is a major determinant in the specificity of synapses formed by cholinergic neurons transplanted to the hippocampus

Embryonic habenular or striatal cholinergic tissues were transplanted to the hippocampal formation of adult rats. The connectivity of these grafts with the host hippocampal formation was analysed using acetylcholinesterase histochemistry and immunocytochemistry with a monoclonal antibody to choline acetyltransferase. Both graft types produced laminar arrangements of acetylcholinesterase-positive fibers in the hippocampal formation that closely resembled the native pattern of cholinergic innervation. In addition, graft-derived choline acetyltransferase-immunoreactive synapses were found in the host hippocampal formation. These synapses were formed on non-immunoreactive dendritic structures and were similar to the types of cholinergic synapses found in the hippocampal formation of normal animals. These data indicate that the cholinergic transmitter phenotype is a major determinant of whether a neuron will form typical cholinergic synapses with hippocampal targets.     

Recovery of hippocampal cholinergic activity by transplantation of septal neurons in AF64A treated rats

Embryonic septal neurons were transplanted into the hippocampus of adult rats which had received lateral-ventricular administration of AF64A, a cholinergic neurotoxin, and the effects on hippocampal cholinergic activity were studied. One week after AF64A administration, we injected dissociated septal cell suspension into the dorsal hippocampus, unilaterally. About 3 months after the transplantation, acetylcholine (ACh)-rich septal grafts formed extensive acetylcholinesterase (AChE)-positive fibers into the host hippocampus, recovering choline acetyltransferase (ChAT) level only in the grafted side. These results indicate that septal implants can produce a partial recovery of the cholinergic activity in the chemically damaged hippocampus.     

Nerve growth factor increases choline acetyltransferase but not survival or fiber outgrowth of cultured fetal septal cholinergic neurons

Neurons dissociated from the septal area of fetal rat brains were grown in culture. Cholinergic neurons were identified by immunocytochemical visualization of choline acetyltransferase and cytochemical demonstration of acetyl cholinesterase. Choline acetyltransferase immunocytochemistry stained cell bodies and proximal processes while acetylcholinesterase cytochemistry visualized the entire neuron. Choline acetyltransferase-positive neurons could only be identified in cultures grown under conditions that produced the maximal choline acetyltransferase activity, measured biochemically. All of the choline acetyltransferase-positive neurons were double stained for acetylcholinesterase while only 6% of the acetylcholinesterase-positive cells were choline acetyltransferase negative in these cultures. These results indicate that acetylcholinesterase is a reliable marker for cholinergic cells in cultures of dissociated septal neurons. Being the more sensitive method, acetylcholinesterase staining was therefore used to identify cholinergic cells in cultures with choline acetyltransferase levels insufficient for immunocytochemical visualization of this enzyme. Addition of nerve growth factor or antibodies to nerve growth factor to the medium did not affect the number of cholinergic neurons surviving in culture. Furthermore, nerve growth factor and anti-nerve growth factor failed to influence the general morphological appearance and the number of processes of these neurons. However, nerve growth factor elevated the biochemically measured activity of choline acetyltransferase up to two-fold. The nerve growth factor-mediated increase in choline acetyltransferase activity was dose dependent with an ED50 of 10 ng/ml (4 X 10(-10) M). The increase was highly specific for nerve growth factor. It was blocked by anti-nerve growth factor, and epidermal growth factor, insulin and other control proteins failed to exert a similar effect. Nerve growth factor had to be present for at least 3 days in the culture medium to increase choline acetyltransferase activity, suggesting that the increase was due to an elevated choline acetyltransferase synthesis rather than to an activation of the enzyme.     

Nerve-growth-factor-dependent and cell-density-independent survival of septal cholinergic neurons in culture from postnatal rats

We have established a primary neuronal cell culture technique from the postnatal (P11 to P15) rat CNS to study the nerve growth factor (NGF) response to basal forebrain cholinergic neurons. The survival of septal cholinergic neurons in culture was monitored both by the determination of choline acetyltransferase activity and by counting acetylcholinesterase-positive cells. Cells obtained from postnatal septal regions were found to require a plentiful oxygen supply during the dissociation of the cells. NGF-mediated survival of the septal cholinergic neurons was similarly observed in the cultures by using different plating cell densities up to 12.5 X 10(5) cells/cm2. These results suggest that the promotion by NGF of cell survival in culture is independent of plating cell density.     

Acetylcholinesterase on the dendrites of central cholinergic neurons: an electron microscopical study in the ferret

A combination of choline acetyltransferase immunohistochemistry and acetylcholinesterase histochemistry was used to characterize the ultrastructural distribution of acetylcholinesterase in identified cholinergic and non-cholinergic neurons in the ferret brain. Previous studies have shown that most of the cholinergic input to the brain arises from choline acetyltransferase-positive neurons found in the neostriatum, basal forebrain and dorsolateral pontine tegmentum. In all these cells intense staining for acetylcholinesterase was localized within the cisternae of the rough endoplasmic reticulum, in the nuclear envelope and Golgi apparatus, and along the plasma membranes of the soma and dendrites. In contrast, the distribution of acetylcholinesterase in non-cholinergic neurons was restricted mainly to the cisternae of the endoplasmic reticulum and the nuclear envelope. Since previous studies have associated high levels of acetylcholinesterase staining with non-cholinergic neurons in the locus coeruleus and substantia nigra zona compacta, these areas were examined as well. The ultrastructural localization of acetylcholinesterase in the principal locus coeruleus neurons was as observed in typical non-cholinergic neurons. On the other hand, the distribution of acetylcholinesterase in the principal cells of the zona compacta of the substantia nigra was more like that found in cholinergic neurons. In conclusion, the subcellular distribution of acetylcholinesterase in the principal cholinergic neurons of the brain follows a characteristic pattern which, with one exception, is different from that of acetylcholinesterase-positive non-cholinergic neurons.     

Parasympathetic innervation of cutaneous blood vessels by vasoactive intestinal polypeptide-immunoreactive and acetylcholinesterase-positive nerves: histochemical and experimental study on rat lower lip

The distribution and origin of perivascular acetylcholinesterase-active and vasoactive intestinal polypeptide-immunoreactive nerve fibers were studied in the rat lower lip by means of acetylcholinesterase histochemistry and vasoactive intestinal polypeptide immunohistochemistry. The perivascular nerve fibers stained intensely with both histochemical techniques and were widely distributed on small arteries and arterioles of the lower lip, especially in the transitional zone between the hairy skin and the mucous membrane. The distributions of the two types of fibers were very similar and most of them showed overlapping coloration, on consecutive staining for vasoactive intestinal polypeptide and acetylcholinesterase. Both acetylcholinesterase-positive and vasoactive intestinal polypeptide-immunoreactive fibers were completely lost on removal of the otic ganglion, while they were not affected by sympathetic ganglion removal or sensory nerve sectioning. In the otic ganglion, most cells exhibited acetylcholinesterase activity, and about 60% of the cells showed light to heavy vasoactive intestinal polypeptide immunoreactivity. These findings indicate that vessels in the rat lip are innervated by parasympathetic fibers originating from the otic ganglion and support the view that vasoactive intestinal polypeptide is present in cholinergic neurons. This may suggest the possible control by the parasympathetic nervous system of cutaneous blood vessels through vasoactive intestinal polypeptide-containing cholinergic neurons, in general or at least in the facial area.     

Enhanced graft survival in the hippocampus following selective denervation

The trophic effects of denervation on the survival of fetal cholinergic neuronal cell suspensions grafted to the hippocampal formation of the rat were assessed in the present study. Young adult female rats were injected with cell suspensions of neurons obtained from the fetal basal forebrain region into the hippocampal formation simultaneously with (or without) a fimbria-fornix transection, which removes the hippocampal cholinergic afferents. Four to six months later, one group of grafted animals was evaluated histochemically for: transplant volume; number of acetylcholinesterase-positive cells, and size of acetylcholinesterase-positive cells in the graft. A parallel study was conducted to determine the total number and size of the acetylcholinesterase-positive cells in the septal-diagonal band-substantia innominata complex of the adult rat, to match with the cell survival and growth in the grafts. A second group of grafted rats was taken in parallel for biochemical analysis of choline acetyltransferase activity in the grafted hippocampus. The transplant volume in the rats with fimbria-fornix transection was greater than twice the volume seen in animals without fimbria-fornix lesion. In addition, the number of acetylcholinesterase-positive cells in the transplant was twice as great in the denervated animals as in the non-denervated ones. However, the number of acetylcholinesterase-positive cells per mm3 of graft volume did not differ between the two groups, suggesting that the trophic effect of the denervation was not specific for the cholinergic neurons, but affected the entire grafted tissue. The hippocampal choline acetyltransferase activity of the animals that received the fimbria-fornix lesion simultaneously with transplantation was about three times higher than that of the rats that received grafts but no simultaneous fimbria-fornix transection. A control experiment with animals that received an aspirative lesion of the retrosplenial cortex, transecting the perforant path input, revealed no enhancing effect of hippocampal choline acetyltransferase activity over non-lesioned grafted animals. Thus, the denervation-enhancing effects of the fimbria-fornix lesion appear to be selective and not the result of a general wound-induced mechanism. These results strongly support the contention that neurotrophic factors are released as a result of denervation in the adult hippocampal formation, and that these neurotrophic factors can support survival and growth of central cholinergic neurons. However, the factors involved do not appear to be specific for the cholinergic neurons, but rather have their trophic effects on many types of cells.     

Distribution and co-localization of choline acetyltransferase and p75 neurotrophin receptors in the sheep basal forebrain: implications for the use of a specific cholinergic immunotoxin

The basal forebrain cholinergic system is involved in different forms of memory. To study its role in social memory in sheep, an immunotoxin, ME20.4 immunoglobulin G (IgG)-saporin, was developed that is specific to basal forebrain cholinergic neurons bearing the p75 neurotrophin receptor. The distribution of sheep cholinergic neurons was mapped with an antibody against choline acetyltransferase. To assess the localization of the p75 receptor on basal forebrain cholinergic neurons, the distribution of p75 receptor-immunoreactive neurons with ME20.4 IgG was examined, and a double-labeling study with antibodies against choline acetyltransferase and p75 receptor was undertaken. The loss of basal forebrain cholinergic neurons and acetylcholinesterase fibers in basal forebrain projection areas was assessed in ewes that had received intracerebroventricular injections of the immunotoxin (50, 100 or 150 microg) alone, as well as, in some of the ewes treated with the highest dose, with bilateral immunotoxin injections in the nucleus basalis (11 microg/side). Results indicated that choline acetyltransferase- and p75 receptor-immunoreactive cells had similar distributions in the medial septum, the vertical and horizontal limbs of the band of Broca, and the nucleus basalis. The double-labeling procedure revealed that 100% of the cholinergic neurons are also p75 receptor positive in the medial septum and in the vertical and horizontal limbs of the band of Broca, and 82% in the nucleus basalis. Moreover, 100% of the p75 receptor-immunoreactive cells of these four nuclei were cholinergic. Combined immunotoxin injections into ventricles and the nucleus basalis produced a near complete loss (80-95%) of basal forebrain cholinergic neurons and acetylcholinesterase-positive fibers in the hippocampus, olfactory bulb and entorhinal cortex. This study provides the first anatomical data concerning the basal forebrain cholinergic system in ungulates. The availability of a selective cholinergic immunotoxin effective in sheep provides a new tool to probe the involvement of basal forebrain cholinergic neurons in cognitive processes in this species.     

Septal cholinergic neurons from postnatal rat can survive in the dissociate culture conditions in the presence of nerve growth factor

The survival effect by nerve growth factor (NGF) on the cholinergic neurons of postnatal rat septal neurons in culture was examined. When the septal neurons from 10 to 12-day-old rats were cultured without NGF, the activities of choline acetyltransferase gradually decreased during the period of cultivation. The addition of NGF to the culture prevented the decline of activities. And, the number of acetylcholinesterase-positive neurons in culture with NGF was found to be more than that without NGF, after 5 days in culture. These results suggest that NGF promotes the survival of septal cholinergic neurons from postnatal rats in culture.     

脑内注射Aβ_(25-35)未见对大鼠隔-海马胆碱能系统的毒性作用

本文运用ＣｈＡＴ 免疫组织化学方法和ＡＣｈＥ组织化学方法观察了Ａβ２５ ３５对大鼠隔 海马胆碱能系统的影响。结果如下：Ａβ２５ ３５注射入内侧隔核，存活１４ ｄ，内侧隔核ＣｈＡＴ 阳性细胞和其支配靶区海马ＡＣｈＥ 阳性纤维的形态和数量未见明显变化；Ａβ２５ ３５注射入海马，存活１４ ｄ，注射点附近ＡＣｈＥ纤维和同侧内侧隔核ＣｈＡＴ 阳性细胞也未见明显变化。上述结果显示，在目前实验条件下，脑内注射Ａβ２５ ３５对大鼠隔 海马胆碱能系统没有明显影响。结合文献讨论，本文作者认为：Ａβ对中枢胆碱能系统是否具有毒性作用还有待进一步探索。     

The effect of epileptiform discharges evoked by intrahippocampal injection of kainic acid on cholinergic and catecholaminergic hippocampal afferents

The influence of epileptiform seizures evoked by intrahippocampal injection of kainic acid on morphological changes of hippocampus and related brain regions was analyzed in rabbits using catecholamine histofluorescence, monoamine oxidase, acetylcholinesterase and Nissl staining methods. It was found that kainic acid induced generalized electroencephalographic seizures and a disappearance of hippocampal neurons. These effects did not affect the volume of neurons in septum and locus coeruleus. In the injected hippocampus, kainic acid destroyed hippocampal pyramidal cells and induced some sprouting of catecholamine, acetylcholinesterase-positive and monoamine oxidase-positive nerve fibers near the injection site.These results indicate that intrahippocampal kainic acid injection does not provoke a retrograde, transsynaptic degeneration in the medial septum and locus coeruleus, the brain regions which innervate the hippocampus.     

Choline acetyltransferase-immunoreactive neurons intrinsic to rodent cortex and distinction from acetylcholinesterase-positive neurons

Cholinergic neurons intrinsic to rat cortex were studied using a sensitive method for the localization of choline acetyltransferase immunoreactivity, acetylcholinesterase histochemistry, combined localization of choline acetyltransferase and acetylcholinesterase, and combined localization of choline acetyltransferase and retrogradely transported horseradish peroxidase-wheat germ agglutinin. Choline acetyltransferase immunoreactivity was localized predominantly in small bipolar cortical neurons within the upper layers of isocortex, while small multipolar neurons were the predominantly stained cell type in allocortical regions. Acetylcholinesterase histochemistry demonstrated mainly small polymorphic cells scattered throughout all cellular layers in all cortices. Combined staining for choline acetyltransferase and acetylcholinesterase resulted in localization of the markers in different cell populations; choline acetyltransferase-immunoreactive neurons did not contain detectable acetylcholinesterase and acetylcholinesterase-positive neurons did not contain detectable immunoreactivity to choline acetyl-transferase. Some possible connections of the cortical choline acetyltransferase-immunoreactive cells were studied in rats which had received injections of horseradish peroxidase-wheat germ agglutinin into either cortex or brainstem. The choline acetyltransferase-immunoreactive cells were frequently admixed with cells labeled with the retrograde marker; however, no double-labeled cells were observed.It was concluded that cortical cholinergic cells are not visualized by acetylcholinesterase histochemistry, and are likely to be involved in local circuitry.     

Hypothalamic melanin-concentrating hormone projections to the septo-hippocampal complex in the rat

Melanin-concentrating hormone (MCH) and neuropeptide glutamic acid-isoleucine (NEI) are expressed in neurons that are located mainly in the hypothalamus and project widely throughout the rat central nervous system. One of the main targets of melanin-concentrating hormone is the hippocampal formation, although the exact origin of the projections is unknown. By using injections of the retrograde tracer True Blue into the hippocampus, together with immunohistochemical analysis, we observed retrogradely labeled melanin-concentrating hormone-containing neurons in the lateral hypothalamic area, incerto-hypothalamic area, perifornical area, the periventricular nucleus of the hypothalamus, and in the internuclear area (between the dorsomedial and ventromedial nuclei of the hypothalamus), as well as a few retrogradely labeled and melanin-concentrating hormone-immunoreactive cells in the supramammillary nucleus. The afferents from the lateral hypothalamic area were confirmed using injection of the anterograde tracer biotinylated dextran amine, which enabled us to use histochemical analysis in order to visualize fibers and terminals in the hippocampal formation. In the medial septal nucleus, we found cholinergic neurons that are also putatively innervated by melanin-concentrating hormone immunoreactive fibers and project to the hippocampal formation. Finally, using two different protocols for immunoperoxidase, we were able to show GABAergic basket cells presumably innervated by melanin-concentrating hormone-immunoreactive fibers in the hippocampal formation. On the basis of the data collected herein, we hypothesize that the MCH/NEI projections from hypothalamic nuclei participate in spatial memory and learning through direct and indirect pathways. These pathways would enable the animal to organize its exploratory behavior during foraging.     

Retrograde cell changes in medial septum and diagonal band following fimbria-fornix transection: quantitative temporal analysis

Complete unilateral fimbria-fornix transections, including the overlying cingulate cortex, were administered to female rats. At time points from 1 day to 6 weeks, the septal-diagonal band region was examined using acetylcholinesterase histochemistry, Cresyl Violet cell staining, and choline acetyltransferase biochemistry. As early as 1 day following the transection a decrease in acetylcholinesterase positive cell body staining was observed in the medial septum; however, no loss of Nissl-stained neurons was measured in Cresyl Violet stained sections until 1 week after the lesion. Maximal loss of acetylcholinesterase-positive cells, as visualized after irreversible acetylcholinesterase inhibition, was measured at 1 week, and no further change was observed at time points up to 6 weeks after operation. The loss of acetyltransferase-positive cells was greatest in the medial septal area (-65%) and the vertical limb of the diagonal band (-55%). Little cell loss was measured in the horizontal limb of the diagonal band. This is consistent with the known projections of these cell bodies. Remaining acetylcholinesterase-positive cell bodies in the medial septum had shrunk by about 20% (measured as the diameter along the major axis). A marked neuronal cell loss (about 50%) was demonstrable in the medial septum and vertical limb of the diagonal band in the Cresyl Violet-stained sections, too. A pile-up of acetylcholinesterase-stained material was observed in the dorsal-lateral quadrant of the septal area just proximal to the lesion at 1 day following transection. This pile-up occurred in the medial septum and diagonal band area up to 1 week following the transection, and had nearly disappeared by 2 weeks post-transection. Choline acetyltransferase biochemical activity, measured in samples of whole septum, decreased significantly at 1 day but subsequently returned to control levels. By 2 weeks following transection, an increase in acetylcholinesterase-positive stained fibers was observed in the dorsal-lateral quadrant of the septum, ipsilateral to the lesion relative to the contralateral septum. This response, which was interpreted as sprouting from the lesioned axons proximal to the transection, probably accounted for the rise in choline acetyltransferase biochemical activity in the whole septum following the reduction on the first day.