Septum-diagonal Band Complex Research Articles

Immunohistochemical localization of phospholipase D1 in rat central nervous system

Phospholipase D (PLD) is one of the intracellular signal transduction enzymes and plays an important role in a variety of cellular functions. We investigated the distribution of PLD isozyme, PLD1 in the rat brain and spinal cord using an immunological approach. Western blot analysis showed the presence of PLD1 protein in all tissues studied, with significantly higher levels in the brainstem and spinal cord, which was correlated with the results obtained from PLD activity assay. Prominent and specific signals of PLD1 were observed in many functionally diverse brain areas, including the olfactory bulb, medial septum–diagonal band complex, cerebral cortex, brainstem, cerebellum, and spinal cord. In the brainstem, the red nucleus, substantia nigra, interpeduncular nucleus, cranial motor nuclei (trigeminal motor, abducent, facial, and hypoglossal), sensory cranial nerve nuclei (spinal trigeminal, vestibular, and cochlear), as well as nuclei of the reticular formation, all showed intense immunoreactivity. Purkinje cells and deep cerebellar nuclei of the cerebellum were also labeled intensely. However, no significant labeling was found in the thalamus, epithalamus, and basal ganglia. Although many of the PLD1 immunoreactive cells were neurons, PLD1 was also expressed in glial cells such as presumed astrocytes and tanycytes. These findings suggest that PLD1 may play an important role in the central nervous system of the adult rat.

Tyrosine kinase A, galanin and nitric oxide synthase within basal forebrain neurons in the rat

Cholinergic basal forebrain neurons appear to play a key role in cognition and attention. In rat, basal forebrain neurons express multiple proteins including the high-affinity signal transducing tyrosine kinase A receptor for nerve growth factor, the neuropeptide galanin and nitric oxide synthase, a marker for the novel neurotransmitter nitric oxide. The present study was undertaken to define the relationship between neurons expressing each of these markers within the medial septum-vertical limb of the diagonal band, horizontal limb of the diagonal band and nucleus basalis in colchicine pre-treated rats. Tyrosine kinase A-immunopositive neurons were seen throughout all subfields of the basal forebrain. In contrast, nitric oxide synthase- and galanin-immunoreactive neurons were mainly distributed within the septal-diagonal band complex. Co-localization experiments revealed that virtually all nitric oxide synthase-positive neurons (visualized by nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry) also contained tyrosine kinase A, whereas, many fewer tyrosine kinase A neurons were nicotinamide adenine dinucleotide phosphate-diaphorase positive within the medial septum-vertical limb of the diagonal band. Within the horizontal limb of the diagonal band, numerous nicotinamide adenine dinucleotide phosphate-diaphorase neurons expressed tyrosine kinase A, whereas only a small number of tyrosine kinase A neurons contained nicotinamide adenine dinucleotide phosphate-diaphorase. Within the nucleus basalis very few neurons were nicotinamide adenine dinucleotide phosphate-diaphorase reactive, and a minor number contained tyrosine kinase A. Additional co-localization experiments revealed minor percentages of neurons containing nicotinamide adenine dinucleotide phosphate-diaphorase and galanin immunoreactivity within the various subfields of the basal forebrain. Within the horizontal limb of the diagonal band minor numbers of nicotinamide adenine dinucleotide phosphate-diaphorase-reactive perikarya displayed galanin. Similarly, only a few galanin-containing neurons expressed nicotinamide adenine dinucleotide phosphate-diaphorase. The existence of tyrosine kinase A, nitric oxide synthase and galanin within select neuronal subgroups of the cholinergic basal forebrain suggests that these perikarya are responsive to a complex set of chemical signals. A greater understanding of the chemical signature of the cholinergic basal forebrain neurons will provide the insight required to develop novel pharmacological approaches aimed at preventing or slowing the degenerative processes that effect these neurons in aging and pathologic disorders.

Evidence of the direct projection from the medial septum-diagonal band complex to the pontine nuclei in rat demonstrated by means of anterograde transport of dextran biotin

A monoclonal antibody directed against rat brain choline acetyltransferase (ChAT) was used to stain cholinergic nerve cells within the brain and spinal cord of macaques. ChAT immunoreactivity was seen in motor neurons of the brainstem and spinal cord, in large neurons of the striatum, and in large neurons in the basal forebrain (medial septum-diagonal band-nucleus basalis complex); each of these groups of neurons is believed to be cholinergic. The ability to visualize cholinergic neurons in the nervous system of primates provides a new approach to the study of cholinergic systems in health and disease.

Plasticity of galaninergic fibers following neurotoxic damage within the rat basal forebrain: initial observations.

Galanin immunoreactive fibers hypertrophy and hyperinnervate remaining cholinergic basal forebrain neurons within the septum-diagonal band complex in Alzheimer's disease. The present investigation determined whether a similar hyperinnervation of galanin immunoreactive fibers occurs following intraparenchymal injections of ibotenic acid within the cholinergic medial septum or diagonal band nucleus in young adult rats. Sections through the medial septum and the diagonal band were either concurrently immunostained for galanin and the low-affinity p75 neurotrophin receptor (an excellent marker of cholinergic basal forebrain neurons) or single stained for choline acetyltransferase. Following chemical lesion, an increase in the density of galanin immunoreactivity was seen within the medial septum on the lesion, as opposed to the contralateral control side. In contrast, within diagonal band-lesioned animals, the increase in galanin immunoreactivity was low to moderate. In either lesion paradigm we did not observe hyperinnervation of remaining cholinergic basal forebrain neurons. In fact, there was no correlation between the galanin hypertrophy and the amount of cholinergic cell loss. We hypothesize that galanin hyperinnervation within the cholinergic basal forebrain may provide a protective effect by down-regulating acetylcholine release following brain insult.

Recovery of ChAT immunoreactivity in axotomized rat cholinergic septal neurons despite reduced NGF receptor expression.

Previous studies have suggested that target-derived nerve growth factor (NGF) is essential for the survival of cholinergic basal forebrain neurons. Thus, axotomy of septohippocampal neurons in adult rats resulting in the withdrawal of target-derived NGF caused a dramatic loss of choline acetyltransferase (ChAT)-immunoreactive neurons in the medial septum-diagonal band complex. We have recently shown that this loss of immunolabelled neurons does not indicate cell death, since many septohippocampal cholinergic neurons recover their immunoreactivity for ChAT after a long survival time despite disconnection from target-derived neurotrophins. One possibility would be that these surviving ChAT-immunoreactive neurons have gained access to other, probably local, NGF sources. Here we provide evidence that the recovery of ChAT immunoreactivity after axotomy is not accompanied by a similar recovery of NGF receptor expression in these neurons. In situ hybridization for p75NTR mRNA and trkA mRNA 6 months after bilateral fimbria-fornix transection revealed a substantial loss of labelled cells. In addition, there was a persisting loss of p75NTR-immunoreactive and NGF-immunoreactive medial septal neurons. Cholinergic neurons in controls did not express NGF mRNA, but were heavily immunostained for NGF protein due to receptor-mediated uptake. These data suggest that at least some cholinergic septohippocampal neurons re-express ChAT either independently of NGF or with a reduced need for NGF.

Afferents to the seizure-sensitive neurons in layer III of the medial entorhinal area: a tracing study in the rat.

Neurons in layer III of the medial entorhinal area (MEA) in the rat are extremely vulnerable to local injections of amino-oxyacetic acid and to experimentally induced limbic seizures. A comparable specific pathology has been noted in surgical specimens from patients with temporal lobe epilepsy. Efforts to understand this preferential neuronal vulnerability led us to study the neural input to this layer in the rat. Iontophoretic injection of the retrograde tracer fast blue, aimed at layer III of the MEA, resulted in retrogradely labeled neurons in the presubiculum in all the injected hemispheres. The nucleus reuniens thalami, the anteromedial thalamic nucleus, the ventral portion of the claustrum (endopiriform nucleus), the dorsomedial parts of the anteroventral thalamic nucleus, and the septum-diagonal band complex were labeled less frequently. In only one experiment, retrogradely labeled neurons were observed in the ventrolateral hypothalamus and in the brainstem nucleus raphe dorsalis. Since projections from claustrum to the entorhinal cortex has not been studied in the rat with modern sensitive anterograde tracing techniques, iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin were placed into the ventral portion of the claustrum. Anterogradely labeled fibers in the entorhinal area proved not to be confined to the MEA, since a prominent projection distributed to the lateral entorhinal area as well. In both areas, the densest terminal labeling was present in layers IV-VI, whereas layer III appeared to be only sparsely labeled. The present data indicate that of all potential afferents only those from the presubiculum distribute preferentially to layer III of the MEA. This, in turn, suggests a potentially important role of the presubiculum in the seizure-related degeneration of neurons in layer III of the MEA.

Identification of distinct GABAA-receptor subtypes in cholinergic and parvalbumin-positive neurons of the rat and marmoset medial septum-diagonal band complex.

GABAA-receptor heterogeneity is based on a multiplicity of subunits (alpha 1-6, beta 1-4, gamma 1-4, delta, rho 1-2) encoded by distinct genes. Flexibility in GABAergic signal transduction and allosteric modulation is expected to arise from the differential assembly of subunits into receptor subtypes. The aim of the present study was to investigate the potential diversity of receptor subtypes expressed by defined neuron populations, as identified by their neurotransmitter phenotype. To this end we have determined immunohistochemically the subunit repertoire of cholinergic and GABAergic neurons in the basal forebrain of rat and marmoset monkey, focusing on the medial septum-diagonal band complex. Co-localization of the GABAA-receptor subunits alpha 1, alpha 3, beta 2, beta 3, and gamma 2 with markers of cholinergic and GABAergic neurons (choline acetyltransferase and parvalbumin, respectively) was assessed by double- and triple immunofluorescence staining. The results reveal that cholinergic neurons in the rat basal forebrain are typically characterized by the subunit combination alpha 3/beta 3/gamma 2, whereas most of the parvalbumin-positive GABAergic neurons express either the subunit combination alpha 1/beta 2/gamma 2 or the combination alpha 1/alpha 3/beta 2/gamma 2. A similar pattern was observed in marmoset monkey, with GABAA-receptors containing the alpha 1-subunit being associated with parvalbumin-positive cells, but never with cholinergic neurons. Thus, the expression of distinct subunit repertoires by cholinergic and GABAergic neurons points to a functional specialization which is conserved across species. These subunit combinations are likely to correspond to different receptor subtypes, and may reflect the engagement of cholinergic and GABAergic neurons in distinct neuronal circuits in the basal forebrain.

Light and electron microscopic study of m2 muscarinic acetylcholine receptor in the basal forebrain of the rat.

The m2 muscarinic acetylcholine receptor gene is expressed at high levels in basal forebrain, but the paucity of information about localization of the encoded receptor protein has limited the understanding of cellular and subcellular mechanisms involved in cholinergic actions in this region. The present study sought to determine the cellular localization of m2 protein, its relationship to cholinergic neurons, and its pre- and postsynaptic distribution in the rat medial septum-diagonal band complex using immunocytochemistry with polyclonal rabbit antibodies and a newly developed rat monoclonal antibody specific to the m2 receptor. Light microscopic colocalization studies demonstrated that m2 was present in a subset of choline acetyltransferase immunoreactive neurons, in choline acetyltransferase-negative neurons, and in more neuropil elements than was choline acetyltransferase. Intraventricular injections of 192 IgG-saporin, an immunotoxin directed to the low-affinity nerve growth factor receptor, resulted in depletion of choline acetyltransferase-immunoreactive neurons in the medial septum-diagonal band complex, whereas m2 immunoreactivity in neurons and in the neuropil was unchanged. By electron microscopy, m2 receptor in medial septum-diagonal band complex was localized to the plasmalemma of a small population of small to medium-sized neurons, and it was also found in dendrites, axons, and axon terminals in the neuropil. Neurons expressing m2 immunoreactivity received synaptic contacts from unlabelled axon terminals. A small distinct subpopulation of large neurons, unlabelled by m2 immunoreactivity, received synaptic contacts from m2-immunoreactive terminals. Thus, m2 receptor is situated to mediate the local effects of acetylcholine on basal forebrain cholinergic and noncholinergic neurons and, also, at both pre- and postsynaptic sites.

Topographical projections from the medial septum-diagonal band complex to the hippocampus: a retrograde tracing study with multiple fluorescent dyes in rats

Direct projections from the medial septum-diagonal band complex (MS-DB) to the hippocampus were studied in rats using the retrograde labeling method with fluorescent dyes. In the first series of experiments, the distribution patterns of retrogradely labeled cells in the MS-DB were examined in sagittal sections, following simultaneous injection of two different fluorescent dyes separately into the ventral hippocampus (VHPC) and the dorsal hippocampus (DHPC). It was found that the DHPC received fiber terminals mainly from the rostral half of the MS-vertical limb of the diagonal band (VDB) and the core part of the horizontal limb of the diagonal band (HDB), whereas the VHPC received fiber terminals largely from the caudal half part of the MS-VDB and the marginal part of the HDB. The marginal part appears to form a ‘C’ and not a complete circle around the core part. In the second series of experiments, the distribution patterns of retrogradely labeled cells in the MS-DB were investigated in sagittal sections, following simultaneous injection of three or two different fluorescent dyes separately into the subfields of the DHPC. The CA1 received fiber terminals mainly from the HDB and VDB, while the hilus received fiber terminals mainly from the MS. The CA2–CA3 received fiber terminals almost equally from the MS, VDB and HDB. The present results reveal distinctive topographical organizations of the septohippocampal connections.

Histaminergic Modulation of Hippocampal Acetylcholine Release In Vivo

In order to elucidate the modulatory role of the histaminergic neural system in the cholinergic neural system, the acetylcholine release from the CA1-CA3 region in the hippocampus of anesthetized rats was studied by an in vivo microdialysis method coupled with HPLC-electrochemical detection. The mean value for the basal acetylcholine release was 0.98 +/- 0.04 pmol/20 min. The acetylcholine release was increased to 172% of the basal level when an electrical stimulation at 200 microA was applied to the tuberomammillary nucleus. An administration of alpha-fluoromethylhistidine (100 mg/kg i.p.) blocked the electrically evoked release of histamine both from the septal-diagonal band complex and the hippocampus, and abolished the electrically evoked release of acetylcholine from the hippocampus. Zolantidine (5 mg/kg i.p.) attenuated the increase in the electrically stimulated acetylcholine release, but pyrilamine (5 mg/kg i.p.) did not attenuate the increase in the acetylcholine release. These drugs showed no significant effect on the basal acetylcholine release. An administration of (R)-alpha-methylhistamine (5 mg/kg i.p.) caused a decrease in the acetylcholine release to 48.7% of the basal level, whereas thioperamide (5 mg/kg i.p.) caused an increase in the acetylcholine release 60 min after the injection. These results suggest that the histaminergic system may contribute to the modulation of the activity of the septohippocampal cholinergic system, mainly through H2 receptors.

Co-localization of nitric oxide synthase and NGF receptor in neurons in the medial septal and diagonal band nuclei of the rat

The relationship of neurons that express nitric oxide synthase type I (NOS-I), the synthetic enzyme of the free radical nitric oxide (NO), with cells that contain the low affinity p75 nerve growth factor receptor (NGFr) was examined in the basal forebrain region of the rat using double immunohistofluorescence. NOS and NGFr were found to be co-localized in a neuronal population that displayed a selective distribution. Double immunopositive neurons were evident in the medial septum and in rostral levels of the diagonal band nuclei where the vast majority (more than 80%) of NOS-immunoreactive cells were also NGFr-positive; the two cell populations were separate at more caudal levels of the basal forebrain. These findings support the chemical heterogeneity of septal and basal forebrain neurons which contain NGFr and indicate that in the rat a subset of neurons of the septum-diagonal band complex may release NO.

A low-affinity nerve growth factor receptor antibody is internalized and retrogradely transported selectively into cholinergic neurons of the rat basal forebrain

The mechanism by which nerve growth factor transduces its signal in responsive cells is yet to be clearly defined. However, it has been suggested that the internalization of nerve growth factor, the first step in the retrograde flow of nerve growth factor, is a property of the high-affinity receptors, p140 trka. Here we show that when a monoclonal antibody (MC 192), which immunoprecipitates p75 NGFR (the low-affinity 75,000 mol. wt nerve growth factor receptor protein) and not p140 trka, was administered into the dorsal hippocampal formation of the rats, it was internalized and retrogradely transported to the cell bodies residing in the medial septum-diagonal band complex. The topographic organization and the localization of these neurons containing retrogradely transported p75 NGFR antibody were strikingly similar to those nerve cells immunostained for choline acetyltransferase in the immediately-adjacent section, indicating that the neurons which contained p75 NGFR antibody were cholinergic neurons. A double-label immunocytochemistry confirmed this conclusion. On the other hand, none of the parvalbumin-positive GABAergic neurons contained retrogradely transported p75 NGFR antibody. Moreover, in contrast to specific transport of p75 NGFR antibody into cholinergic neurons, when wheat germ agglutinin-colloidal gold was injected into the hippocampus at the same levels, it was taken up and retrogradely transported into both choline acetyltransferase-positive cholinergic and parvalbumin-immunoreactive GABAergic neurons in the medial septum-diagonal band complex.

Origin of noradrenergic projections to GnRH perikarya-containing areas in the medial septum-diagonal band and preoptic area

The purpose of the present study was to identify the sites of origin of the noradrenergic fibers that project to areas containing gonadotropin-releasing hormone (GnRH) perikarya since norepinephrine (NE) is known to influence the activity of GnRH neurons. Fluorescent retrograde tracers were used in combination with immunohistochemistry for dopamine-β-hydroxylase (DBH) and GnRH. Small volumes of either Fluoro-gold (FG) or Fluoro-Ruby (FR) were pressure injected into areas that contain the largest number of GnRH cell bodies, i.e., the medial septum-diagonal band complex or preoptic area. Retrogradely labeled neurons were observed ipsilaterally in the following noradrenergic cell groups: A2 (in the nucleus tractus solitarii), A1 (in the ventrolateral medulla) and locus coeruleus. Approximately 8% of all DBH-positive neurons within the A2-cell group were retrogradely labeled, while 12% of DBH-ir neurons in the A1-group were double-labeled. Only a few retrogradely labeled DBH-ir neurons were observed in the locus coeruleus (<1%). Double-labeled neurons were not organized into discrete cell groups, but were dispersed among other NE-neurons within the A2- and A1-cell groups. The highest concentrations of double-labeled neurons were located in the central one-third of both the A2 and A1 cell groups. The results suggest that most noradrenergic terminals in the region of the GnRH perikarya in the medial septum-diagonal band/rostral preoptic area originate from ipsilateral neurons in areas A1 and A2. These data support the view that the medullary cell groups A1 and A2, and not the locus coeruleus, are the primary sites of origin of the noradrenergic component that is crucial for the direct regulation of GnRH perikarya.

Acetylcholinesterase activity and post-lesional plasticity in the hippocampus of young and aged rats

Applying quantitative microscopic histochemistry, the activity of acetylcholinesterase was determined in the various layers of the rat hippocampus at three different levels along the rostrocaudal extent. Two age groups of animals were examined: young adults (two to three months old) and aged subjects (26 months old). Young adults were divided into controls, and animals killed eight and 35 days following bilateral ibotenate lesioning of the medial septum-diagonal band complex. Aged rats were divided into controls and animals 35 days post-lesion. Analysis of variance revealed that the mean acetylcholinesterase activities of the entire hippocampus of individuals were not significantly different between young and aged rats when averaged across controls and 35 days post-lesion. There was a significant decrease of acetylcholinesterase activity (−52%) in young adults eight days post-lesion as compared to controls, but a significant increase (+63%) took place until 35 days post-lesion as compared to eight days post-lesion. Significantly lower activities existed, however, in young (−22%) and aged rats (−18%) 35 days post-lesion as compared to controls. This decrease in mean activity was not age dependent. As acetylcholinesterase is considered to be a good cholinergic indicator in the hippocampus, the results suggest a homotypic collateral sprouting from spared cholinergic afferents following ibotenate lesion of the medial septum-diagonal band complex in young and aged rats. Based on the data obtained, it is reasonable to assume that there was no difference in the post-lesional plasticity of neuronal acetylcholinesterase between young adult and aged rats.

State-dependent and state-independent effects of thyrotropin-releasing hormone on medial septum neuronal activity in the brain slices of waking and hibernating ground squirrels

Effects of thyrotropin-releasing hormone on spontaneous activity and responses to medial forebrain bundle stimulation were tested in the units of the medial septum-diagonal band complex in slices taken from the brain of hibernating and waking ground squirrels. Administration of thyrotropin-releasing hormone (0.1 μM) into the flow of incubating medium increased the frequency of spontaneous activity of all the medial septum-diagonal band complex neurons in hibernating ground squirrels and of the majority of neurons in the waking ground squirrels. However, in the septal slices of hibernating ground squirrels this increase was significantly more pronounced. In addition, the neuropeptide slightly increased the frequency of bursts in the majority of cells with rhythmic burst activity. The excitatory influence of thyrotropin-releasing hormone on the units was preserved in conditions of synaptic blockade. In neurons from other structures (lateral septum, medial preoptic area, hippocampus) in the brain slices of both hibernating and waking ground squirrels, thyrotropin-releasing hormone did not usually affect the level of spontaneous discharges. When studying the responses of the medial septum-diagonal band complex neurons to electrical stimulation of medial forebrain bundle it was found that application of thyrotropin-releasing hormone (0.1 μM) led to the disappearance of responses in 50 and 44% of units in the hibernating and waking ground squirrels, respectively; in the rest of the neurons a disturbance of stability and probability of responses was observed. The existence of a modulatory thyrotropin-releasing hormone system which participates post-, and, probably, presynaptically in the regulation of the medial septum-diagonal band complex neuronal activity is suggested. The role of thyrotropin-releasing hormone and of medial septum-diagonal band complex in the neural control of hibernation/euthermic waking cycle is discussed.

Studies related to the use of colchicine as a neurotoxin in the septohippocampal cholinergic system

Colchicine has been shown to be neurotoxic to cholinergic neurons in the medial septum 1 week following intracerebroventricular injections. The experiments described here were designed to examine the selectivity of this effect over a longer time course, and to examine the role of axoplasmic transport in the neurotoxic effect. As previously reported, 1 week after intracerebroventricular injections of colchicine, the numbers of choline acetyltransferase (ChAT)-immunoreactive neurons in the medial septum-diagonal band complex (MSDB) were reduced to 38% of control; this reduction was stable 2 and 3 weeks post injection. Injections of colchicine placed into the body of the fornix produced similar results. GAD-immunoreactive somata, the other major population of neurons in the MSDB, were unaffected 3 weeks following colchicine, as previously reported 1 week following similar injections. The normal AChE staining pattern in the hippocampus, particularly the dentate gyrus, was depleted following either ICV or intrafornical injections of colchicine. This depletion was more severe with longer survival times. Injections of lumicolchicine, an isomer of colchicine which does not bind tubulin, had no effect on ChAT-immunoreactive neurons in the MSDB or on AChE staining in the hippocampus. Injections of colchicine, but not of lumicochicine, partially blocked the retrograde transport of the fluorescent dye Fluoro-Gold from the hippocampus to the MSDB. In addition, the content of NGF in the hippocampus rose 84% above control values 2 weeks following colchicine and remained elevated at three weeks. Together these results indicate that colchicine is selectively toxic for cholinergic neurons in the septohippocampal system, and suggest that the alkaloid's neurotoxic effects work via the blockade of axoplasmic transport.

Muscarinic M 2 receptor mRNA expression and receptor binding in cholinergic and non-cholinergic cells in the rat brain: A correlative study using in situ hybridization histochemistry and receptor autoradiography

The goal of the present study was to identify the cells containing mRNA coding for the m2 subtype of muscarinic cholinergic receptors in the rat brain. In situ hybridization histochemistry was used, with oligonucleotides as hybridization probes. The distribution of cholinergic cells was examined in consecutive sections with probes complementary to choline acetyltransferase mRNA. Furthermore, the microscopic distribution of muscarinic cholinergic binding sites was examined with a non-selective ligand ([ 3H] N-methylscopolamine) and with ligands proposed to be M 1selective ([ 3H]pirenzepine) or M 2-selective ([ 3H]oxotremorine-M). The majority of choline acetyltransferase mRNA-rich (i.e. cholinergic) cell groups (medial septum-diagonal band complex, nucleus basalis, pedunculopontine and laterodorsal tegmental nuclei, nucleus parabigeminalis, several motor nuclei of the brainstem, motoneurons of the spinal cord), also contained m2 mRNA, strongly suggesting that at least a fraction of these receptors may be presynaptic autoreceptors. A few groups of cholinergic cells were an exception to this fact: the medial habenula and some cranial nerve nuclei (principal oculomotor, trochlear, abducens, dorsal motor nucleus of the vagus). Furthermore, m2 mRNA was not restricted to cholinergic cells but was also present in many other cells throughout the rat brain. The distribution of m2 mRNA was in good, although not complete, agreement with that of binding sites for the M 2 preferential agonist [ 3H]oxotremorine-M, but not with [ 3H]pirenzepine binding sites. Regions where the presence of [ 3H]oxotremorine-M binding sites was not correlated with that of m2 mRNA are the caudate-putamen, nucleus accumbens, olfactory tubercle and islands of Calleja. The present results strongly suggest that the M 2 receptor is expressed by a majority of cholinergic cells, where it probably plays a role as autoreceptor. However, many non-cholinergic neurons also express this receptor, which would be, presumably, postsynaptically located. Finally, comparison between the distribution of m2 mRNA and that of the proposed M 2-selective ligand [ 3H]oxotremorine-M indicates that this ligand, in addition to M 2 receptors, may also recognize in certain brain areas other muscarinic receptor populations, particularly M 4.

Colchicine-induced cholinergic denervation of the hippocampus elicits sympathetic ingrowth

Sympathetic neurons from the peripheral nervous system invade the hippocampus following destruction of its septal inputs. It is thought that sympathetic ingrowth is due to the loss of cholinergic innervation since damage to the medial septum-diagonal band complex (MSDB) or its major efferent bundle, the fimbria-fornix, is required to induce ingrowth. The MSDB provides the major source of cholinergic fibers projecting to the hippocampus; however, non-cholinergic (e.g. GABAergic) neurons are also present in the MSDB and project to the hippocampus. Thus, the role of cholinergic denervation in sympatho-hippocampal sprouting cannot be directly tested by non-specific lesion techniques. In the present study, colchicine, which has been demonstrated to be specifically toxic to cholinergic neurons in the medial septum, was injected into each lateral ventricle of female Sprague-Dawley rats. Following colchicine-induced degeneration of cholinergic septohippocampal neurons, coarse, branched fibers immunoreactive for dopamine-beta-hydroxylase were observed predominantly in the dentate gyrus, on both sides of the granule cell layer, with increasing density as survival time increased. These results support the hypothesis that the invasion of the hippocampal formation by sympathetic fibers results from cholinergic denervation.

Seasonal changes in methionine-enkephalin immunoreactivity in the brain of a hibernator, Spermophilus columbianus

To identify the actual location of central endogenous opioid systems which may be involved in regulating the hyberbation cycle, differences in the pattern of central methionine-enkephalin (Met-EK) immunoreactivity were compared between hibernating (body temperature, T b = 7 °C) and non-hibernating ( T b = 37 °C) Columbia ground using the peroxidase-antiperoxidase technique. In non-hibernating animals, Met-EK-immunoreactive perikya were observed in telencephalic (putamen, caudate,, nucleus, medial septum-diagonal band complex, amygdala) and diencephalic (periventricular hypothalamic nucleus, lateral hypothalamic area) regions; whereas immunoreactive fibers were found in the lateral septum, stria terminalis nucleus, various hypothalamic areas, arcuate nucleus, median eminence, thalamic intralaminar, periventricular nucleus and lateral habenular nucleus. Compared to the non-hiberbnating animal, a marked increase in the number of Met-EK-immunoreactive fibers was found in the lateral septal nucleus, the periventricular nucleus, the intralaminar thalamus and the paraventricular hypothalamus of hibernating ground squirrels. Since these changes in immunoreactivity were not observed in the artificially induced hypothermic ground squirrels ( T b = 7 °C), it is unlikely that the dissimilarity in immunoreactivity between animals from different hibernating phases is due to differences in their T b. In combination with our previous studies, these results tend to suggest that hibernation maybe be brought about an increase in endogenous opioid activity, especially in the lateral septal region.

Distribution of the rat medial septum-diagonal band complex neurons projecting to the hippocampus demonstrated by the fluorescent retrograde labeling method

Distribution of the rat medial septum-diagonal band complex neurons projecting to the hippocampus demonstrated by the fluorescent retrograde labeling method