Perception of species-specific vocalizations in rats : role of the cholinergic septo-hippocampal pathway and aging

The effect of a chemical lesion of the cholinergic septo-hippocampal pathway induced by ethylcholine aziridinium (AF64A) on brain potentials evoked by species-specific vocalization containing informations of high biological relevance was studied in young adult (10 months) and aged (24 months) rats by means of neocortical electroencephalographic recordings. In control rats, the perception of a rats vocalization in a life endangering situation (lasting 0.8 s) initiated an evoked potential followed by a late positive slow wave (LPSW)-complex and a direct current shift with a duration of up to 16 s. Four months after treatment with AF64A (2 nmol into each of the lateral ventricles), the main negative component of the initial acoustic evoked potential (peak latency of about 60 ms after stimulus onset) was reduced (P = 0.04) both in young adult and aged rats. Further changes included a decrease of the late positive wave amplitude in young adult rats (P = 0.001) and a shorter duration of the LPSW-complex in aged rats (P = 0.03). AF64A induced also changes in specific components revealed by Principle Component Analysis, but only in the group of young rats. A decrease in the slow wave component (factor 1, 3000–4000 ms after stimulus onset ; P = 0.02) was observed. Age per se affected the late positive potential shifts as indicated by a shorter latency of the late positive wave (P = 0.03).

A detailed analysis of the major neurotransmitter markers proved an almost exclusive reduction of the activity of choline acetyltransferase in the ventral and dorsal hippocampus (up to 60%), which was equal in young adult and aged rats. The irreversible loss in choline acetyltransferase activity, which was restricted to the hippocampus, was associated by a slight reduction in serotonergic function.

The present data suggest that the complex cognitive and emotional processes initiated by species-specific vocalization appear to be affected by aging. Furthermore, as a consequence of a cholinergic deficit in the hippocampus, the integration processes essential for the recognition of the biological meaning of a species-specific vocalization are considerably disturbed. These findings provide an experimental basis for studying disturbances in the perceptual response to stimuli of high emotional value in patients with hypocholinergic function as in Alzheimers disease.     

Pure Schwann cell suspension grafts promote regeneration of the lesioned septo-hippocampal cholinergic pathway

Regeneration of central nervous system (CNS) axons has been studied in the cholinergic septo-hippocampal system using various ‘bridges’ able to support fiber growth. In this study, a pure Schwann cell (Sc) suspension labeled with bisbenzimide (Hoechst 33342) was grafted in the lesioned septo-hippocampal pathway. At 2 weeks post-grafting, acetyl-cholinesterase (AChE)-positive fibers invaded the graft and grew in association with the Hoechst-labeled Sc, some of which expressed the low-affinity nerve growth factor receptor (NGF-R). At 2 months and 4 months post-grafting, the dorsal hippocampus was reinnervated with an apparently normal innervation pattern. Analysis of fiber growth in the hippocampus at four months post-grafting revealed a significant increase of reinnervation in the grafted animals (2 mm) compared to the non-grafted ones. No difference was observed in the number of cholinergic septal neurons expressing the NGF-R. These results demonstrate that a Sc suspension grafted into the lesioned septo-hippocampal system, integrates well into the host tissue, and supports axonal CNS outgrowth, implying that Sc by themselves provide an adequate environment for regeneration to occur.     

Denervation supersensitivity for benzodiazepine receptors in the rat substantia nigra

The injection of kainic acid into the substantia nigra causes, 3 weeks after treatment, a 40% decrease in the total number of binding sites for [³H]diazepam with an increase in the dissociation constant. This decrease was restored to approximately normal by the subsequent injection of kainic acid into the striatum, homolateral to the lesioned substantia nigra. The injection of kainic acid into the striatum of intact animals failed to modify the number of [³H]diazepam binding sites but increased the K_D. The results indicate that benzodiazepine binding sites in the substantia nigra are partly located on kainic acid sensitive elements (probably interneurons) and, partly, on kainic acid resistant ones. These binding sites become supersensitive after degeneration of striato-nigral pathways. The possible role of GABAergic denervation of the substantia nigra in the development of benzodiazepine binding sites supersensitivity is discussed.     

Denervation supersensitivity to serotonin in rat forebrain: Single cell studies

To investigate the development of denervation supersensitivity to serotonin (5-hydroxytryptamine, 5-HT) in the amygdala (AMYG) and the ventral lateral geniculate nucleus (vLGN), single cell recordings, microiontophoretic, histochemical and biochemical techniques were used in the present study. 5-HT projections to the vLGN and the AMYG were destroyed by 5,7-dihydroxytryptamine (5,7-DHT, a relatively selective toxin for 5-HT neurons) injected directly into the lateral ventricle or the ascending 5-HT pathway in the ventromedial tegmentum area. Enhanced responsiveness of cells to the inhibitory effect of microiontophoretically applied 5-HT (ionto-5-HT) began to develop within 24 h and approached a maximum 7 days after 5,7-DHT pretreatment. In general, the time courses for the reduction in both the density of 5-HT fluorescent varicosities and synaptosomal 5-HT uptake activity paralleled the time course for the development of denervation supersensitivity to 5-HT. During the first 2 days after 5,7-DHT, the enhanced sensitivity was selective for 5-HT; responses to D-lysergic acid diethylamide (LSD), norepinephrine (NE) and gamma-aminobutyric acid (GABA) were unchanged. Seven or more days after 5,7-DHT there was a marked increase of the responsiveness of neurons in the vLGN and the AMYG to both 5-HT and LSD (a 5-HT agonist which is not a substrate for the high affinity 5-HT uptake system). At these later times, the responsiveness of cells in the AMYG to NE and to a lesser extent GABA was also increased. In contrast to the marked supersensitivity seen after 5,7-DHT induced denervation, chronic administration of parachlorophenylalanine, a 5-HT synthesis inhibitor, failed to induce 5-HT supersensitivity.     

Stress-induced immobility in rats with cholinergic supersensitivity

Immobility during forced swimming or after mild footshock (1 mA for 2 sec) was observed in five groups of rats. The Flinders Sensitive Line (FSL) of rats, known to be more sensitive to cholinergic agonists, exhibited the greatest degree of immobility in the forced swim test. Rats chronically treated with, and subsequently withdrawn from, either scopolamine (2 mg/kg, once daily) or amitriptyline (10 mg/kg, once daily) were also significantly more immobile than either a control group treated chronically with isotonic saline or the Flinders Resistant Line (FRL) of rats in the forced swim test. Similar trends were observed for locomotor depression in the open field following exposure to footshock. Receptor binding studies indicated significantly greater concentrations of muscarinic acetylcholine receptors in the hippocampus of the scopolamine, and amitriptyline, withdrawn rats. These findings indicate that rats with increased cholinergic sensitivity are more sensitive to the immobility-inducing effects of mild stressors. Thus, they may prove to be useful models for studying the relationship between affective disorders and the cholinergic system.     

The role of the septo-hippocampal cholinergic projection in T-maze rewarded alternation

Administration of 192IgG-saporin, a cholinergic neurotoxin, to the medial septum destroys the cell bodies from which the septo-hippocampal cholinergic projection originates, leading to reductions in both hippocampal acetylcholinesterase (AChE) and choline acetyltransferase (ChAT). Despite reports that 192IgG-saporin-induced cholinergic loss leads to post-operative impairments in acquisition and performance of spatial memory tasks, a number of other reports have described intact spatial memory performance following these lesions. Factors that might account for these different outcomes include variations in toxin injection sites or volumes, and post-operative testing at times that might permit regeneration of damaged neuronal processes. We, therefore, assessed the effects of intraseptal microinjection of 192IgG-saporin, in rats, on the post-operative retention of pre-operatively acquired discrete-trial rewarded alternation in the T-maze. This design allowed us to assess the effects of the lesion 7 days post-surgery, at which point, at best, incomplete neuronal regeneration would have been expected to have occurred. The lesion led to a profound loss of hippocampal AChE staining, and a clear inflammatory response, as assessed by proliferation of OX42-stained macrophages in the medial septum and diagonal band nuclei, but there was no impairment in spatial working memory.     

Stain-dependent differences between the septo-hippocampal cholinergic system and hippocampal size

The activity of choline acetyltransferase is over twice as high in the hippocampus of Wistar Kyoto (WKY) than in Brown Norway (BN) rats, and this is paralleled by a comparable difference in acetylcholinesterase staining intensity within the hippocampal formation. However, the size of the whole hippocampus is smaller in WKY than in BN rats. There are no strain differences in the activities of the neurotransmitter-synthesizing enzymes: tyrosine hydroxylase in the septum and glutamic acid decarboxylase in the hippocampus. The findings indicate the existence of strain-dependent inverse relationship between the septo-hippocampal cholinergic system and the size of the hippocampus.     

Genetic variation in the morphology of the septo-hippocampal cholinergic and GABAergic systems in mice: II. Morpho-behavioral correlations

We investigated the contribution of the septo-hippocampal cholinergic and GABAergic system to spatial and nonspatial aspects of learning and memory that had previously been found to correlate with the extent of the hippocampal intra- and infrapyramidal mossy fiber projection in different inbred mouse strains. The following cholinergic and GABAergic markers were measured in the septi and hippocampi of male mice: the number of cholinergic and parvalbumin-containing neurons in the medial septum/vertical limb of the diagonal band of Broca (MS/vDB), the number of septo-hippocampal cholinergic and GABAergic projection neurons, the density of cholinergic fibers in different hippocampal subfields, and the density of muscarinic receptors (predominantly M1 and M2) in the hippocampus. In addition, animals were behaviorally tested for spatially dependent and activity-dependent variables in a water maze and spatial and nonspatial working and reference memory in different experimental set-ups in an eight-arm radial maze. Using only those variables for which significant strain differences were obtained, we looked for covariations between behavior and neuroanatomy. The density of cholinergic fibers in the dentate gyrus was significantly correlated with activity-dependent learning in the water maze, whereas the number of septo-hippocampal cholinergic projection neurons correlated with spatial and, to a lesser extent, also with nonspatial aspects of radial maze learning. Only weak correlations were found between receptor densities and behavioral traits. From these data we conclude that variations in the septo-hippocampal cholinergic system, like variations in the mossy fiber projection, entail functional consequences for different types of maze learning in mice. © 1996 Wiley-Liss, Inc.     

Altered NGF response but not release in the aged septo-hippocampal cholinergic system

An important aspect of aging and Alzheimer's disease (AD) pathology includes the degeneration of basal forebrain cholinergic neurons (BFCNs), possibly due to disrupted nerve growth factor (NGF) signaling. Previous studies on disrupted NGF signaling have focused on changes in retrograde transport. This study focuses on two other possible mechanisms for loss of trophic support: diminished release of NGF from hippocampal neurons or diminished TrkA receptor response of BFCNs to NGF. We measured NGF levels in the effluent of hippocampal slices from young and aged rats in response to potassium chloride and glutamate. We found that release of NGF was not altered in aged hippocampal slices compared to slices from young controls. To measure the in situ response of the BFCNs to NGF, we injected NGF intraparenchymally into the right hippocampus of young and aged rats. Injections of cytochrome C served as controls. Fifteen minutes post-administration, a dramatic increase in TrkA immunoreactivity was found in the cell bodies of medial septal neurons. We found that this rapid response was blunted in aged rats compared to young adult controls. To determine whether retrograde transport was necessary for this rapid response, we injected colchicine prior to NGF injection. The NGF-induced upregulation was not blocked by colchicine, suggesting that this acute response was not dependent on classical retrograde transport. Since cholinergic degeneration coupled with altered levels of NGF and TrkA receptors are also seen in human aging and AD, the loss of acute responsivity to NGF in the BFCNs may also play a role in these processes.     

The stress-induced response of the septo-hippocampal cholinergic system. A vectorial outcome of psychoneuroendocrinological interactions

Considerable data have emerged which strongly indicate that the septohippocampal cholinergic system is involved in the adaptive response to stress. Neurotransmitter regulatory mechanisms in cholinergic synaptic terminals of this part of the limbic system undergo adaptive changes in response to stress and recover slowly after stress. The initial stress-induced response is characterized by activation of hippocampal cholinergic terminals within minutes, as indicated by a rapid and transient elevation in high affinity choline uptake and increased newly synthesized acetylcholine release. The response of this cholinergic system to stress is influenced by both neuronal and hormonal stimuli. Among the several neuronal systems converging in the septum, terminals of the dopaminergic mesolimbic system have been found to be selectively involved in the early response to stress. Pharmacological interference with dopaminergic neurotransmission, with agonist and antagonist treatments, revealed that changes in the tonic inhibitory influence of septal dopaminergic terminals can modulate the response of hippocampal cholinergic terminals to stress. A similar activation of hippocampal cholinergic terminals as after short-term stress was observed after treatments with a large dose of either adrenocorticotropic hormone or corticosterone. Furthermore, glucocorticoids and not adrenocorticotropic hormone can directly enhance acetylcholine release, but only from excited terminals. This indicates that stress-induced activation of the septo-hippocampal system may occur secondary to, but not directly by, increased levels of pituitary-adrenocortical hormones. Yet, it is possible that under stressful conditions the increased glucocorticoid levels may modulate the activity of the stimulated hippocampal cholinergic terminals. Together the findings support the notion that the stress-induced response of the septo-hippocampal cholinergic system represents an integrated output of converging neuronal and hormonal stimuli which convey signals of stress to this limbic brain region.     

Denervation supersensitivity of 5-HT-1c receptors in rat choroid plexus

Serotonin (5-HT)-stimulated phosphoinositide hydrolysis in the choroid plexus is mediated by the 5-HT-1c receptor. The current study demonstrates that treatment of rats with the serotonergic neurotoxin, 5,7-dihydroxytryptamine, caused a marked depletion of 5-HT and a supersensitive 5-HT-1c mediated phosphoinositide hydrolysis response. These data suggest that the 5-HT-1c site in choroid plexus receives tonic serotonergic input.     

Serotonergic excitation of sympathetic preganglionic neurons: a microiontophoretic study

The effects of microiontophoretically applied serotonin on the extracellularly recorded discharges of sympathetic preganglionic neurons (SPNs) were studied in anesthetized cats. Thoracic SPNs were identified on the basis of constancy of antidromic activation and collision. Low ejecting currents of serotonin (5-30 nA) invariably excited spontaneously active SPNs. Serotonin also excited the vast majority of quiescent SPNs, as well as neurons brought to discharge threshold by the excitatory amino acid L-glutamate. A population of SPNs was identified which was insensitive to the excitatory effects of both serotonin and L-glutamate. Iontophoretic or intravenous administration of the putative serotonin antagonists methysergide and metergoline blocked the excitatory effects of serotonin on SPNs. The blockade of the serotonin-induced excitation was not associated with a local anesthetic action of methysergide or metergoline. Methysergide and metergoline also reduced the firing rate of SPNs in intact but not in spinal animals. These data provide strong evidence to support the contention that serotonergic neurons provide a tonic excitatory input to SPNs.     

Denervation supersensitivity to glutamate in the nucleus tractus solitarii after removal of the nodose ganglion

Ifl-glutamate (l-Glu) is the transmitter released from cardiovascular vagal afferent nerve fibers in the nucleus tractus solitarii (NTS), then interruption of those afferents should lead to denervation supersensitivity tol-Glu in the NTS. Therefore, we sought to determine if dose-related changes in arterial pressure (AP) and heart rate (HR) elicited byl-Glu microinjected into the NTS evolve after removal of the left nodose ganglion in rats. Twelve rats served as unoperated controls; and eight were studied 5 days, eleven 10 days, and nine 15 days after ganglionectomy. Each rat was anesthetized with halothane (1.5–2.0%) and cannulated for recording AP and HR. After exposure of the brainstem, vehicle orl-Glu (3, 30, 75, 150, 300 and 1500 pmol/50 nl) was microinjected alternately into the right and left dorsomedial NTS. In control animals, and in animals 5 days and 15 days after ganglionectomy, the lowest dose ofl-Glu that produced a significant fall of mean AP (−10±2, −5±2, −6±1 mmHg, respectively) was 30 pmoles. The threshold dose with injections on the lesioned side 10 days after ganglionectomy was 3 pmol (−8±2 mmHg). In rats studied at 10 days, but not in control, 5 or 15 day rats, the dose-responses for injections made on the left were shifted significantly to the left of those made on the right (P < 0.05). In control, 5, and 15 day rats there were no significant differences between dose-related responses elicited from right and left NTS. Responses of HR did not differ between groups of animals or sides of injection. These results suggest that supersensitivity tol-Glu evolves after nodose ganglionectomy and further support a role forl-Glu as a transmitter of cardiovascular afferents in the NTS.     

Genetic variation in the morphology of the septo-hippocampal cholinergic and GABAergic system in mice. I. Cholinergic and GABAergic markers

In the present study, variations of cholinergic and GABAergic markers in the medial septum/vertical limb of the diagonal band of Broca (MS/vDB) and the hippocampus of eight different inbred mouse strains were investigated. By means of immunocytochemistry against the acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT), the cholinergic neurons were visualized and the number of ChAT-positive neuronal profiles in the MS/vDB was counted. Cholinergic and GABAergic septo-hippocampal projection neurons were detected with a combined retrograde tracing and immunocytochemical approach. In order to quantify the cholinergic innervation of various hippocampal subregions, we estimated the density of acetylcholinesterase (AChE)-containing fibers as visualized by AChE histochemistry. Additionally, the densities of muscarinic receptors (mainly the subtypes M1 and M2) in different hippocampal areas of seven inbred strains were measured by means of quantitative receptor autoradiography. We found significant strain differences for the number of ChAT-positive neurons in the MS/vDB; in the numbers of cholinergic septo-hippocampal projection neurons; in the density of cholinergic fibers in hippocampal subfields CA3c, CA1, and in the dentate gyrus; and in the density of muscarinic receptors in the hippocampus. In contrast, the GABAergic component of the septo-hippocampal projection did not differ between the strains investigated. The number of ChAT-reactive neurons in the MS/vDB was not correlated with either hippocampal cholinergic markers. This might be attributed to different collateralization of cholinergic neurons or to different projections of these neurons to other brain regions. These results show a strong hereditary variability within the septo-hippocampal cholinergic system in mice. In view of the role of the cholinergic system in learning and memory processes, strain differences in cholinergic markers might be helpful in explaining behavioral variation. © 1996 Wiley-Liss, Inc.     

Cholinergic and non-cholinergic septo-hippocampal projections: a double-label horseradish peroxidase-acetylcholinesterase study in the rabbit

The existence of a massive cholinergic projection from cells in the medical septal nucleus (MS) and nucleus of the diagonal band (DB) to the hippocampal formation has been recognized for some time. However, the actual percentages of cholinergic and non-cholinergic neurons in the MS and DB which project to the hippocampus have not been reported. A procedure which combines horseradish peroxidase (HRP) and acetylcholinesterase (AChE) histochemistry in the same tissue was used to determine these percentages in the rabbit. Less than 50% of the neurons in the MS and DB which were labeled with reaction product following an HRP injection into the dorsal hippocampus also stained for AChE. Moreover, 70% of all neurons containing HRP reaction product were located in the DB, but neurons in the DB could not be differentiated from those in the MS on the basis of size or morphology. These data are taken to indicate that much of the MS-DB hippocampal projection is not cholinergic. Substance P is suggested as another possible transmitter within this anatomical system.     

脑内注射乙酰胆碱受体抗体IgG对大鼠隔-海马胆碱能系统的损害

目的　探讨乙酰胆碱受体抗体 ( Ach Rab) Ig G对大鼠隔 -海马胆碱能系统的损害及对认知功能的影响。方法　 Ach Rab阳性 Ig G或健康人 Ig G注入大鼠一侧隔 -斜角带核区 ,跳台试验测试大鼠的学习记忆功能 ,乙酰胆碱酯酶 ( Ach E)组织化学法测定颞叶、海马 Ach E阳性纤维 ,免疫组化测定隔 -斜角带核区胆碱乙酰转移酶( Ch AT)阳性神经元。结果　实验组大鼠错误次数 ( 7.8± 1..5 )较对照组 ( 3.6± 1.3)明显增加 ;实验组大鼠颞叶及海马各区 Ach E纤维面密度均明显低于对照组 ( P<0 .0 1) ;实验组注射侧 Ch AT阳性细胞数 ( 30 .4 5± 5 .4 1)明显少于实验组非注射侧 ( 5 4 .0 0± 7.5 9)和对照组注射侧 ( 6 3.5 3± 5 .12 ) ;实验组非注射侧明显少于对照组非注射侧 ( 6 8.35± 4 .72 )。结论　内侧隔 -斜角带核区定向注射 Ach Rab Ig G可使胆碱能神经元损害 ,进而导致胆碱能系统功能障碍。因而认为 MG认知功能障碍与中枢胆碱能系统损害有关