Restricted diet delays accelerated ageing and genomic stress in DNA-repair-deficient mice.

Mice deficient in the DNA excision-repair gene Ercc1 (Ercc1∆/-) show numerous accelerated ageing features that limit their lifespan to 4-6 months. They also exhibit a 'survival response', which suppresses growth and enhances cellular maintenance. Such a response resembles the anti-ageing response induced by dietary restriction (also known as caloric restriction). Here we report that a dietary restriction of 30% tripled the median and maximal remaining lifespans of these progeroid mice, strongly retarding numerous aspects of accelerated ageing. Mice undergoing dietary restriction retained 50% more neurons and maintained full motor function far beyond the lifespan of mice fed ad libitum. Other DNA-repair-deficient, progeroid Xpg-/- (also known as Ercc5-/-) mice, a model of Cockayne syndrome, responded similarly. The dietary restriction response in Ercc1∆/- mice closely resembled the effects of dietary restriction in wild-type animals. Notably, liver tissue from Ercc1∆/- mice fed ad libitum showed preferential extinction of the expression of long genes, a phenomenon we also observed in several tissues ageing normally. This is consistent with the accumulation of stochastic, transcription-blocking lesions that affect long genes more than short ones. Dietary restriction largely prevented this declining transcriptional output and reduced the number of γH2AX DNA damage foci, indicating that dietary restriction preserves genome function by alleviating DNA damage. Our findings establish the Ercc1∆/- mouse as a powerful model organism for health-sustaining interventions, reveal potential for reducing endogenous DNA damage, facilitate a better understanding of the molecular mechanism of dietary restriction and suggest a role for counterintuitive dietary-restriction-like therapy for human progeroid genome instability syndromes and possibly neurodegeneration in general.

Arc expression identifies the lateral amygdala fear memory trace.

Memories are encoded within sparsely distributed neuronal ensembles. However, the defining cellular properties of neurons within a memory trace remain incompletely understood. Using a fluorescence-based Arc reporter, we were able to visually identify the distinct subset of lateral amygdala (LA) neurons activated during auditory fear conditioning. We found that Arc-expressing neurons have enhanced intrinsic excitability and are preferentially recruited into newly encoded memory traces. Furthermore, synaptic potentiation of thalamic inputs to the LA during fear conditioning is learning-specific, postsynaptically mediated and highly localized to Arc-expressing neurons. Taken together, our findings validate the immediate-early gene Arc as a molecular marker for the LA neuronal ensemble recruited during fear learning. Moreover, these results establish a model of fear memory formation in which intrinsic excitability determines neuronal selection, whereas learning-related encoding is governed by synaptic plasticity.

Glutamate receptor subunits at mossy fiber-unipolar brush cell synapses: light and electron microscopic immunocytochemical study in cerebellar cortex of rat and cat.

The present study provides a survey of the immunolocalization of ionotropic glutamate receptor subunits throughout the rat and cat cerebellar cortex, with emphasis on the unipolar brush cell (UBC), a hitherto neglected cerebellar cell that is densely concentrated in the granular layer of the vestibulocerebellum and that forms giant synapses with mossy fibers. An array of nine previously characterized antibodies has been used, each of which stained a characteristic profile of cerebellar cells. The UBCs of both rat and cat were strongly immunostained by an antibody against the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) receptor subunits, GluR2 and GluR3; were moderately immunostained by a monoclonal antibody to kainate receptor subunits, GluR5/6/7; were weakly immunostained by antibodies to NR1 subunits; and were not stained by antibodies to GluR1, GluR4, GluR6/7, KA-2, and NR2A/B. Postsynaptic densities of the giant mossy fiber-UBC synapses were GluR2/3, GluR5/6/7, and NR1 immunoreactive. The other cerebellar neurons were all immunolabeled to some extent with the GluR2/3 and NR1 antibodies. In addition, Purkinje cells were immunopositive for GluR1 and GluR5/6/7; granule cells were immunopositive for GluR5/6/7, GluR6/7, KA-2, and NR2A/B. The Golgi-Bergmann glia was densely stained by GluR1 and GluR4 antibodies, whereas astrocytes of the granular layer were stained by the GluR4 antiserum. Data provided herein may guide further electrophysiological and pharmacological studies of cerebellar cells in general and the UBCs in particular.

Expression of the low affinity neurotrophin receptor p75 in spinal motoneurons in a transgenic mouse model for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a lethal neurodegenerative disorder involving motoneuron loss in the cortex, brainstem and spinal cord, resulting in progressive paralysis. Aberrant neurotrophin signalling via the low affinity neurotrophin receptor p75 has been suggested to be involved in the motoneuron death by the activation of apoptotic pathways. In order to investigate the involvement of neurotrophin receptor p75 in the amyotrophic lateral sclerosis related motoneuron degeneration process, we have studied the expression of this receptor in the spinal cord of transgenic mice carrying a mutated human Cu, Zn superoxide dismutase gene. Mutations in the superoxide dismutase gene are one of the genetic causes for familiar amyotrophic lateras sclerosis and human superoxide dismutase-1 transgenic mice develop symptoms and pathology similar to those in human amyotrophic lateras sclerosis. Our study shows that in these mice, spinal motoneurons, which normally do not contain the neurotrophin receptor p75 receptor, express this receptor during the progress of the disease. Expression of the neurotrophin receptor p75 receptor coincides with the expression of activating transcription factor 3, a member of the activating transcription factor/cyclic AMP family of stress transcription factors. Only a minority of these spinal motoneurons actually showed co-expression of neurotrophin receptor p75 with caspase-3 activity, suggesting that expression of the neurotrophin receptor p75 receptor is not directly related to the execution phase of the apoptosis process.

The unipolar brush cells of the mammalian cerebellum and cochlear nucleus: cytology and microcircuitry.

The unipolar brush cell (UBC) is a novel type of small neuron that is characterized by sets of morphological and chemical phenotypes. UBCs occur in the granular layer of the mammalian cerebellar cortex, particularly in folia of the vestibulocerebellum, and in the granule cell domains of the dorsal cochlear nucleus. The UBC is characterized by a single dendrite that terminates with a brush-like tip of dendrioles. The soma, the dendritic stem, and especially the dendrioles emit short, non-synaptic appendages. The dendrioles represent the main synaptic apparatus of the UBC and articulate tightly with a single mossy fiber rosette forming a glomerular array characterized by an extraordinarily extensive synaptic contact. Electron microscopic and electrophysiological observations indicate that the unusual synaptic ultrastructure may produce entrapment of neurotransmitter in the synaptic cleft. While ionotropic glutamate receptors are enriched in correspondence of the postsynaptic density, metabotropic glutamate receptors are situated extrasynaptically and are particularly enriched at the appendages, which usually do not bear synaptic junctions. Some of the UBCs receive their input from choline acetyltransferase-positive mossy rosettes originating from the vestibular nuclei, suggesting that ACh and glutamate are co-released at these synapses. The UBC brush occupies a glomerulus where granule cell dendrites are intermixed with the UBC dendrioles, both of which receive synapses from the same mossy fiber rosette and portions of the Golgi axonal plexus. In addition, the dendrioles are presynaptic to granule cell dendrites, forming dendrodendritic contacts that display features of excitatory synapses. Branches of the UBC axon in the granular layer bear large endings resembling mossy fibers. The UBCs may represent an extraordinary device for feedforward, excitatory links along the mossy fiber pathways of cerebellum and dorsal cochlear nucleus.

Cholinergic innervation and receptors in the cerebellum.

We have studied the source and ultrastructural characteristics of ChAT-immunoreactive fibers in the cerebellum of the rat, and the distribution of muscarinic and nicotinic receptors in the cerebellum of the rat, rabbit, cat and monkey, in order to define which of the cerebellar afferents may use ACh as a neurotransmitter, what target structures are they, and which cholinergic receptor mediate the actions of these pathways. Our data confirm and extend previous observations that cholinergic markers occur at relatively low density in the cerebellum and show not only interspecies variability, but also heterogeneity between cerebellar lobules in the same species. As previously demonstrated by Barmack et al. (1992a,b), the predominant fiber system in the cerebellum that might use ACh as a transmitter or a co-transmitter is formed by mossy fibers originating in the vestibular nuclei and innervating the nodulus and ventral uvula. Our results show that these fibers innervate both granule cells and unipolar brush cells, and that the presumed cholinergic action of these fibers most likely is mediated by nicotinic receptors. In addition to cholinergic mossy fibers, the rat cerebellum is innervated by beaded ChAT-immunoreactive fibers. We have demonstrated that these fibers originate in the pedunculopontine tegmental nucleus (PPTg), the lateral paragigantocellular nucleus (LPGi), and to a lesser extent in various raphe nuclei. In both the cerebellar cortex and the cerebellar nuclei these fibers make asymmetric synaptic junctions with small and medium-sized dendritic profiles. Both muscarinic and nicotinic receptor could mediate the action of these diffuse beaded fibers. In the cerebellar nuclei the beaded cholinergic fibers form a moderately dense network, and could in principle have a significant effect on neuronal activity. For instance, the cholinergic fibers arising in the PPTg may modulate the excitability of the cerebellonuclear neurons in relation to sleep and arousal (e.g. McCormick, 1989). Studies on the distribution of cholinergic markers in the cerebellum have proven valuable besides the issue whether cholinergic mechanism play a role in the cerebellar circuitry, because they illustrate a complexity of the cerebellar anatomy that extends beyond its regular trilaminar and foliar arrangement. For instance, AChE histochemistry has been shown to preferentially stain the borders of white matter compartments (the 'raphes', Voogd, 1967), and therefore is useful in topographical analysis of the cortico-nuclear and olivocerebellar projections (Hess and Voogd, 1986; Tan et al., 1995; Voogd et al., 1996; see Voogd and Ruigrok, 1997, this Volume). ChAT-immunoreactivity, at least in rat, appears to be a good marker to outline the morphological heterogeneity of mossy fibers, and m2-immunocytochemistry could be used to label (subpopulations of) Golgi cells, subsets of mossy fibers and, in the rabbit, a specific subset of Purkinje cells (Jaarsma et al., 1995).

Light-microscopic distribution and parasagittal organisation of muscarinic receptors in rabbit cerebellar cortex.

Recent studies on the effects of intrafloccular injections of muscarinic agonists and antagonists on compensatory eye movements in rabbit, indicate that muscarinic receptors may play a modulatory role in the rabbit cerebellar circuitry. It was previously demonstrated by Neustadt et al. (1988), that muscarinic receptors in rabbit cerebellar cortex are distributed into alternating longitudinal zones of very high and very low receptor density. In the present study, the zonal and cellular distribution of muscarinic receptors in the rabbit cerebellar cortex is investigated in detail using in vitro ligand autoradiography with the non-selective high-affinity antagonist [3H]quinuclidinyl benzilate (QNB), and the M2-specific antagonist [3H]AF-DX384, and immunocytochemistry with a monoclonal antibody specific for the cloned m2 muscarinic receptor protein. [3H]QNB and [3H]AF-DX384 binding sites and m2-immunoreactivity had similar overall distributions: dense labeling occurred in the dendritic arbors of a subset of Purkinje cells that are organized into parasagittal bands. A high level of muscarinic receptor labeling was also observed in a thin substratum of the molecular layer immediately above the Purkinje cell layer of the vestibulo-cerebellar lobules, i.e. the nodulus, the ventral uvula and the flocculus. Labeling in this stratum was associated with densely packed fibres, which were putatively identified as parallel fibres. Also Golgi cells, which were localized in part in the molecular layer, and a subset of mossy fibre rosettes, primarily concentrated in lobule VI, were immunoreactive for the m2 receptor. The parasagittal band of labeled Purkinje cell dendrites were most prominent in the anterior lobe (lobules I-V), in crus 1 and 2, in the flocculus, the ventral paraflocculus and the rostral folium of the nodulus. In other lobules, only infrequent Purkinje cells contained muscarinic receptors. The parasagittal organisation of muscarinic receptors differed from that of zebrin I, a Purkinje cell-specific protein which is often used as a marker of parasagittal parcelation of the cerebellar cortex. In the anterior lobe, however, there was a partial correspondence between muscarinic receptor and zebrin I bands. In the flocculus the distribution of muscarinic-receptor-positive Purkinje cells was related to the distinct white matter compartments as revealed with acetylcholinesterase (AChE) histochemistry. Muscarinic receptor-containing Purkinje cells were located primarily in the floccular zone 1, which is implicated in the control of eye movements about a horizontal axis. In order to relate the distribution of muscarinic receptor labeling to that of cholinergic nerve terminals, [3H]QNB binding sites and sodium-dependent [3H]hemicholinium-3 binding were compared. Sodium-dependent [3H]hemicholinium-3 binding sites mainly occurred in the granule cell layer of the vestibulo-cerebellum, which corresponds well with the distribution of the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT). However, sodium-dependent [3H]hemicholinium binding complemented, rather than co-localized with, muscarinic receptors which were primarily distributed in the molecular layer of the lobules of the vestibulo-cerebellar lobules. Their functional significance is puzzling, since their distribution does not correspond to that of markers of cholinergic innervation.

A novel non-perfusion Timm method for human brain tissue.

A simple modification of the Timm sulphide-silver method for unfixed brain tissue is described. Instead of perfusing animals with a sodium sulphide solution, sulphide treatment was performed by exposure to mounted frozen sections to H2S gas. The staining pattern in the rat brain obtained with this modification is similar to that With the Neo-Timm method, primarily yielding staining of the neuropil of telencephalic structures. Because perfusion is not required, the modified Timm method can also be applied to postmortem human tissue. Application of the modified Timm stain to the human tissue is exemplified on the hippocampus with special attention to the non-mossy fiber staining.

Glutamate dehydrogenase improves binding of [3H]CGP39653 to NMDA receptors in the autoradiographic assay.

The novel high-affinity competitive NMDA receptor antagonist, CGP39653, is employed as radioligand to autoradiographically label the NMDA receptor in rat and human brain. Glutamate dehydrogenase (GlDH; E.C.1.4.1.3) was added to the incubation buffer to degrade residual endogenous L-glutamate, which was not entirely removed from the section after the prewashing step and interfered with [3H]CGP39653 binding. At 20 nM [3H]CGP39653, GLDH increased specific binding as much as 5 times, depending on the dose of GlDH and the presence of NAD+, and hydrazine. Scatchard plots of binding data revealed that this increase was due to a decrease of the KD from 148 nM to 33 nM in the absence and the presence of GlDH, respectively. Addition of GlDH in the NMDA receptor autoradiographic assay may be of importance in quantitative studies with human brain tissue which may contain variable levels of endogenous L-glutamate.

Presence of apolipoprotein E immunoreactivity in degenerating neurones of mice is dependent on the severity of kainic acid-induced lesion.

Apolipoprotein E (apoE) is a major apolipoprotein in the central nervous system (CNS) that may play a role in various CNS disorders. ApoE is primarily localised in astrocytes, but neuronal apoE mRNA expression has been demonstrated in normal and diseased human brain, as well as in ischaemic rat brain. To obtain further insight into the role of apoE in neuronal degeneration in the CNS and conditions of neuronal apoE localisation, we have investigated in mice the distribution of apoE following neuronal injury induced by kainic acid (n=35, 25 or 35 mg kainic acid/kg BW). Consecutive series of brain sections were immunostained for apoE and markers for astroglia (GFAP) and microglia/macrophage cells (CR3). Degenerating neurones were identified with a silver-degeneration staining technique. The intensity and cellular distribution of apoE-immunoreactivity (apoE-ir) was dependent on the severity of neuronal injury. Mice that developed mild neuronal degeneration, restricted to a subset of neurones in the hippocampus, showed increased apoE-ir in astrocytes concomitant with increased GFAP-ir and mild microgliosis. In these mice, no neuronal apoE-ir was detected. In contrast, mice developing severe neuronal injury in the hippocampus - frequently also showing degeneration in other brain regions including cortex, thalamus, striatum and amygdala - showed intense apoE-ir in degenerating neurones. Surrounding the lesion, apoE-ir was increased in neuropil recurrently whereas GFAP-ir astrocytes disappeared. Thus, in mice apoE accumulates in degenerating neurones in conditions of severe neuronal injury putatively in association with disruption of the glial network.

Localization of NMDA and AMPA receptors in rat barrel field.

The aim of this study was to assess the distribution of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-S-methyl-4-isoxazole propionic acid (AMPA) receptors in the barrel field of rat primary somatosensory (SI) cortex using light-microscopic in vitro autoradiography. NMDA receptors were labeled with the competitive antagonist [3H]CGP39653 or with [3H]glycine in the presence of strychnine, and AMPA receptors with [3H]AMPA. In the SI cortex high densities of the NMDA receptor occurred in the supragranular layers and in layer Va. In layer IV high NMDA receptor densities were specifically confined to the barrel hollows. The AMPA binding sites showed less intralaminar variation and no apparent density differences between the barrel hollows and sides in layer IV. It can be concluded that the distribution of NMDA (but not AMPA) receptors in the rat barrel field shows a strong coincidence with the zone of termination of the specific sensory afferents from the ventral posterior nucleus of the dorsal thalamus.

Reduction of adenosine A1-receptors in the perforant pathway terminal zone in Alzheimer hippocampus.

The cells of origin of the perforant pathway are destroyed in Alzheimer's disease (AD). In rat the adenosine A1-receptors are specifically localized on the perforant path terminals in the molecular layer of the dentate gyrus. In the present study the density of A1-receptors in the hippocampus of Alzheimer's disease (AD) patients (n = 9) and non-dement controls (n = 3) has been investigated autoradiographically with [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]CPDPX) as the ligand probe. In AD hippocampi binding of [3H]CPDPX was greatly reduced in the outer two thirds of the dentate gyrus molecular layer, likely due to the degeneration of the perforant path. Binding of [3H]CPDPX was not significantly altered in other parts of the AD hippocampus, e.g. the CA1 and the CA3, in spite of a pronounced cellular pathology and reduced N-methyl-D-aspartate (NMDA) receptor densities, assessed as strychnine insensitive [3H]glycine autoradiography. This contrasts with the presumed localization on dendrites of pyramidal neurons of A1 receptors within the CA1 and the CA3.

Induction of c-Jun immunoreactivity in spinal cord and brainstem neurons in a transgenic mouse model for amyotrophic lateral sclerosis.

Transgenic mice carrying amyotrophic lateral sclerosis (ALS)-linked superoxide dismutase 1 (SOD1) mutations develop a motoneuron disease resembling human ALS. c-Jun is a transcription factor frequently induced in injured neurons. In this study we have examined the distribution of c-Jun-immunoreactivity in the brainstem and spinal cord of transgenic SOD1 mice with a glycine 93 alanine (G93A) mutation. In non-transgenic littermates c-Jun immunostaining was predominantly situated in motoneurons. The number of c-Jun immunoreactive motoneuron was reduced in SOD1(G93A) mice due to pronounced loss of motoneurons. In SOD1(G93A) mice, however, c-Jun-immunoreactivity was strongly induced in neurons in the intermediate zone (Rexed's laminae V-VIII and X) of the spinal cord and throughout the brainstem reticular formation. These findings are of interest since increased levels of c-jun also have been found in the intermediate zone of the spinal cord of ALS patients. This c-Jun may be involved in the neurodegenerative processes both in ALS and in motoneuron disease in SOD1(G93A) mice.

N-acetylaspartate and N-acetylaspartylglutamate levels in Alzheimer's disease post-mortem brain tissue.

The tissue concentrations of two related amino acid derivatives, N-acetylaspartate (NAA) and N-acetylaspartylglutamate (NAAG) were determined in autopsy hippocampus, amygdala, cerebellar cortex and olfactory bulb of Alzheimer's disease patients and age-matched non-demented controls, using reverse-phase HPLC and fluorescence detection after precolumn derivatisation with the fluorophore 2-aminoanthracene. In Alzheimer's disease, NAA and NAAG concentrations were significantly reduced in the hippocampus (by 38 and 24%) and the amygdala (by 28 and 22%), but not in the olfactory bulb and the cerebellar cortex. These results indicate that the concentrations of NAA and NAAG are selectively decreased in brain areas affected by pathology in Alzheimer's disease.

Detection of autoantibodies to ribonucleoproteins by counter-immunoelectropheresis, immunoblotting and RNA-immunoprecipitation: comparison of results.

Counterimmunoelectropheresis (CIE), immunoblotting (IB) and RNA-immunoprecipitation (RNA-IP) were compared for their potential to detect antibodies to U1RNP, Sm and SS-B in sera from 50 patients with SLE and 18 with MCTD. Anti-SS-B were detected in 6 SLE-sera (12%) and one MCTD-serum (6%) by all three techniques. Anti-Sm were present in 7 SLE-sera (14%) by RNA-IP and IB; 3 of these sera showed identity with a-Sm and 4 with a-U1RNP by CIE. Antibodies precipitating U1RNA were present in 8 SLE sera (16%); 4 of these sera were positive for anti-U1RNP by CIE (8%) and 4 sera recognized one or more of the U1RNA-associated proteins by IB (8%). All MCTD-sera were positive for anti-U1RNP by RNA-IP including one serum negative by IB and CIE. RNA-IP allowed the detection of antibodies precipitating U1-U2RNA in 4 sera; in IB, the U2RNA specific protein B" was recognized by 3 sera. RNA-IP appears to be a sensitive method for detecting anti-URNP's.

Time course and distribution of neuronal degeneration in the dentate gyrus of rat after adrenalectomy: a silver impregnation study.

Recently, Sloviter et al. reported that adrenalectomy (ADX) of young adult rats after 3 months led to a selective loss of granule neurons in the dentate gyrus (DG) and that this loss could be prevented by low doses of corticosterone. In the present study, the ADX-induced neuronal degeneration was investigated in Wistar rats, using a silver impregnation method for degenerating neurons. To examine the time course and distribution of the ADX-induced degeneration, young adult male rats were allowed to survive 2, 3, and 5 days and 1, 2, and 3 weeks after ADX. Argyrophilic neurons were present in the dentate granule cell layer on the second day following ADX. Three days after ADX, the number of argyrophilic granule neurons was much more abundant, and it increased gradually with longer post-ADX survival times. Argyrophilia was specifically confined to dentate granule cells and was accompanied by the occurrence of pyknotic nuclei as observed in adjacent cresyl violet-stained sections. There were significant differences between individual rats in quantity of argyrophilia. About one fifth of the ADX rats showed sporadic or no argyrophilia, in spite of plasma corticosterone levels below the detection limit (10 ng/mL). Sham-operated rats and ADX rats receiving corticosterone (10 mg/L) or dexamethasone (15 mg/L) in their drinking water did not display any argyrophilic neurons in the dentate gyrus. The distribution of the argyrophilia within the DG was highly characteristic with the highest number of degenerating cells in the hidden blade of the middle and the temporal thirds of the DG.(ABSTRACT TRUNCATED AT 250 WORDS)

Cerebellar choline acetyltransferase positive mossy fibres and their granule and unipolar brush cell targets: a model for central cholinergic nicotinic neurotransmission.

A subset of cerebellar mossy fibres is rich in choline acetyltransferase, the rate-limiting enzyme for the synthesis of acetylcholine. These choline acetyltransferase-positive mossy fibres are concentrated in the vestibulocerebellum and originate predominantly from the medial vestibular nucleus. The granular layer of the vestibulocerebellum is also enriched in unipolar brush cells, an unusual type of small neuron that form giant synapses with mossy fibres. In this immunocytochemical light and electron microscopic study, we explored whether choline acetyltransferase-positive mossy fibres innervate unipolar brush cells of the rat cerebellum. We utilized monoclonal antibodies to rat choline acetyltransferase of proven specificity, and immunoperoxidase procedures with 3,3'-diaminobenzidine tetrahydrochloride as the chromogen. A high density of choline acetyltransferase-positive fibres occurred in the nodulus and ventral uvula, where they showed an uneven, zonal distribution. Immunostained mossy fibre rosettes contained high densities of round synaptic vesicles and mitochondria. They formed asymmetric synaptic junctions with dendritic profiles of both granule cells and unipolar brush cells. The synaptic contacts between choline acetyltransferase-immunoreactive mossy fibres and unipolar brush cells were very extensive, and did not differ from synapses of choline acetyltransferase-negative mossy fibres with unipolar brush cells. Analysis of a total area of 1.25 mm2 of the nodulus from three rats revealed that 14.2% of choline acetyltransferase-immunoreactive mossy fibre rosettes formed synapses with unipolar brush cells profiles. Choline acetyltransferase-positive rosettes accounted for 21.7% of the rosettes forming synapses with unipolar brush cells. Thus, the present data demonstrate that unipolar brush cells are innervated by a heterogeneous population of mossy fibres, and that some unipolar brush cells receive cholinergic synaptic input from the medial vestibular nucleus. The ultrastructure of these synapses is compatible with the possibility that choline acetyltransferase-positive mossy fibres co-release acetylcholine and glutamate. As the granular layer of the vestibulocerebellum contains nicotinic binding sites, the choline acetyltransferase-positive mossy fibres may be a model for studying nicotinic neurotransmission in the CNS.

Rat hippocampal lactate efflux during electroconvulsive shock or stress is differently dependent on entorhinal cortex and adrenal integrity.

The role of the entorhinal cortex and the adrenal gland in rat hippocampal lactate formation was assessed during and after a short-lasting immobilization stress and electroconvulsive shock (ECS). Extracellular lactate was measured on-line using microdialysis and enzyme reactions (a technique named lactography); in some rats, unilateral lesions of the entorhinal cortex were made or the bilateral adrenal glands were removed. The stress-evoked increase in hippocampus lactate was not altered either ipsi- or contralateral to an entorhinal cortex lesion. The response to ECS was attenuated only in the hippocampus ipsilateral to the entorhinal cortex lesion. Removal of bilateral adrenal glands caused some delay in the increase in hippocampal lactate after ECS and a major reduction in the stress-evoked lactate response. These results indicate that (1) the entorhinal cortex is activated by ECS, thereby activating hippocampal lactate efflux and presumably metabolism, and (2) the adrenal gland is essential in the response to stress and, to a minor extent, in the ECS-altered hippocampal metabolism.

Mesencephalic cuneiform nucleus and its ascending and descending projections serve stress-related cardiovascular responses in the rat.

The aim of the present study was to explore the neuroanatomic network that underlies the cardiovascular responses of reticular formation origin in the region of the cuneiform nucleus (CNF). The study was performed in urethane anesthetized male Wistar rats. The left iliac artery was supplied with a catheter for the measurement of systemic blood pressure. Low intensity electrical stimulation of the mesencephalic reticular formation (MRF) in the vicinity of the CNF always resulted in pressor and bradycardiac responses, whereas stimulation in the parabrachial nucleus (PB) and Kölliker-Fuse nucleus (KF) led to a pressor response and a small tachycardiac response. The cuneiform area may be placed in the center of a circuit that serves a specific autonomic response pattern to stress: parallel activation of the sympathetic (pressor response) and parasympathetic limb (bradycardia). The efferent connections of the effective stimulation sites in the MRF and the CNF area, were investigated by anterograde tracing with the lectin Phaseolus vulgaris leucoagglutine (PHA-L). The CNF sends descending fibers to the gigantocellular reticular nuclei (GI), the motor nucleus of the vagus (DMNV) and nucleus tractus solitarius (NTS). These projections are probably involved in the bradycardiac response to stimulation. The descending pathway to the NTS/DMNV and GI may therefore be the parasympathetic limb of the circuit. Furthermore, the CNF sends ascending fibers to limbic forebrain areas and descending fibers to the PB-KF complex. The KF in its turn projects to the rostroventrolateral medullary nucleus (RVLM) and the intermediolateral cell column (IML). These latter projections are partly involved in producing the pressor response and thereby represent the sympathetic limb of the circuit. Accordingly, the transection of the descending fibers from the CNF to the PB-KF complex resulted in a decreased pressor and an increased bradycardiac response. This suggests that a baroreceptor reflex-induced bradycardia which results from blood pressure increase can be excluded as the origin of the stimulation-induced bradycardia, and that the pressor and bradycardiac responses are two independent moieties. It cannot be excluded that ascending fibers from the CNF are also involved in producing the pressor response. On the basis of the present physiological and neuroanatomical study, a brain circuit has been proposed in which the cuneiform nucleus has a central position. The described brain circuit may serve a passive coping strategy to novel, painful or threatening stimuli during which the animals show orientation/attention or freezing behavior accompanied by a bradycardiac and pressor response.