Synaptic responses of guinea pig cingulate cortical neurons in vitro.

1. Intracellular recordings were made from layer V/VI neurons of the guinea pig anterior cingulate cortex to investigate postsynaptic potentials (PSPs) evoked by electrical stimulation of the subcortical white matter (forceps minor). 2. Four distinct types of PSPs were recorded (at the resting potential) under normal physiological conditions; 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)-sensitive excitatory postsynaptic potentials (EPSPs) were followed by bicuculline- or picrotoxin-sensitive depolarizing or hyperpolarizing inhibitory postsynaptic potentials (IPSPs), which were further followed by phaclofen-sensitive, long-lasting hyperpolarizing postsynaptic potentials (LPSPs). The average times-to-peak for the EPSP, depolarizing and hyperpolarizing IPSPs, and LPSP were 10, 22, 28, and 146 ms, respectively. 3. In the presence of CNQX and bicuculline, high-intensity electrical stimulation elicited a longer lasting EPSP with a time-to-peak of 21 ms. The amplitude and duration of the EPSP decreased with membrane hyperpolarization and increased with membrane depolarization. The EPSP was reversibly abolished by D,L-2-amino-5-phosphonovaleric acid (D,L-APV). 4. The bicuculline- or picrotoxin-sensitive depolarizing and hyperpolarizing IPSPs and the phaclofen-sensitive LPSP were markedly suppressed by CNQX, suggesting that glutamate (Glu) and/or aspartate nerve terminals project to GABAergic interneurons, and that the GABAergic interneurons are activated mainly by non-N-methyl-D-aspartate (non-NMDA) receptors. 5. In the presence of picrotoxin, the average reversal potential for the compound EPSP was 0 mV, which was similar to that (-6 mV) for the Glu-induced depolarization. In a solution containing D,L-APV at low concentrations, the average reversal potentials for the depolarizing and hyperpolarizing IPSPs and for the early and late components of the gamma-aminobutyric acid (GABA)-induced responses were -62, -72, -70, and -61 mV, respectively. Thus the value for the depolarizing IPSP was similar to that for the late response to GABA, whereas the value for the hyperpolarizing IPSP was almost the same as that for the early response to GABA. The average reversal potential of -90 mV for the LPSP was similar to -93 mV for the baclofen-induced hyperpolarization and to -94 mV for the spike afterhyperpolarization. 6. Application of phaclofen decreased the interspike interval of the spontaneous firing and reversed the increase in the interspike interval after subcortical stimulation. This result indicates that, even in a slice preparation, the anterior cingulate neurons are under tonic inhibitory control exerted by spontaneously active GABAergic interneurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in cortical noradrenergic axon terminals of locus coeruleus neurons in aged F344 rats.

The noradrenergic innervations and noradrenaline contents of the frontal cortex in two age groups (9 and 25 months) of male F344 rats have been quantified by electrophysiological and biochemical methods. In the electrophysiological study, the percentage of locus coeruleus (LC) neurons activated antidromically from the frontal cortex decreased with age. In contrast, the percentage of LC neurons showing multiple antidromic latencies, which suggests axonal branching of individual LC neurons, increased markedly between 9 and 25 months in the frontal cortex. In the biochemical study, we found no significant difference in noradrenaline levels in the cortical terminal fields of LC neurons during aging. These results suggest that LC neurons give rise to axonal branches to retain noradrenaline levels in their target fields in the aged brain. Our findings show that LC neurons preserve a strong capability for remodeling their axon terminals even in the aged brain.     

A model of the electrophysiological properties of thalamocortical relay neurons.

1. A model of the electrophysiological properties of single thalamocortical relay neurons in the rodent and cat dorsal lateral geniculate nucleus was constructed, based in part on the voltage dependence and kinetics of ionic currents detailed with voltage-clamp techniques. The model made the simplifying assumption of a single uniform compartment and incorporated a fast and transient Na+ current, INa; a persistent, depolarization-activated Na+ current, INap; a low-threshold Ca2+ current, I(T); a high-threshold Ca2+ current, IL; a Ca(2+)-activated K+ current, IC; a transient and depolarization-activated K+ current, IA; a slowly inactivating and depolarization-activated K+ current, IK2; a hyperpolarization-activated cation current, Ih; and K+ and Na+ leak currents IKleak and INaleak. 2. The effects of the various ionic currents on the electrophysiological properties of thalamocortical relay neurons were initially investigated through examining the effect of each current individually on passive membrane responses. The two leak currents, IKleak and INaleak, determined in large part the resting membrane potential and the apparent input resistance of the model neuron. Addition of IA resulted in a delay in the response of the model cell to a depolarizing current pulse, whereas addition of IK2, or IL combined with IC, resulted in a marked and prolonged decrease in the response to depolarization. Addition of Ih resulted in a depolarizing "sag" in response to hyperpolarization, whereas addition of IT resulted in a large rebound Ca2+ spike after hyperpolarization. Finally, addition of INap resulted in enhancement of depolarization. 3. The low-threshold Ca2+ spike of rodent neurons was successfully modeled with the active currents I(T), IL, IA, IC, and IK2. The low-threshold Ca2+ current I(T) generated the low-threshold Ca2+ spike. The transient K+ current IA slowed the rate of rise and reduced the peak amplitude of the low-threshold Ca2+ spike, whereas the slowly inactivating K+ current IK2 contributed greatly to the repolarization of the Ca2+ spike. Activation of IL during the peak of the Ca2+ spike led to activation of IC, which also contributed to the repolarization of the Ca2+ spike. Reduction of any one of the K+ currents resulted in an increase in the other two, thereby resulting in substantially smaller changes in the Ca2+ spike than would be expected on the basis of the amplitude of each ionic current alone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution and synaptic contacts of the cortical terminals arising from neurons in the rat ventromedial thalamic nucleus

Injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin within the ventromedial thalamic nucleus resulted in many filled fibres in the frontal areas of rat cerebral cortex. The fibres were restricted to the upper part of layer I except in a small area of motor cortex where terminals were also found in deeper layers. Terminals were also seen in the striatum, in parts of the mesencephalic reticular formation and occasionally in the contralateral ventromedial nucleus. There is some topographical order in the projection with medial and dorsal areas well represented in medial cortex while lateral parts of ventromedial nucleus are more directly related to the cortical area that receives the ventrolateral thalamic nucleus projection. Electron microscopic examination showed the terminals in layer I of cortex making synaptic contact with dendritic spines and small dendritic profiles that showed a very dense postsynaptic specialization. Neurons in the ventromedial nucleus could be antidromically driven from electrode positions along strips of cortex which could not be easily related to any known organizational pattern in the cortex. Thalamic neurons responding antidromically to only one stimulation site were more common when the stimulation was within motor cortical areas, suggesting that in this region a more restricted pattern of termination is the rule.     

Cytology, synaptology and immunocytochemistry of commissural neurons and their putative axonal terminals in the dorsal cochlear nucleus of the rat

The first binaural integration within the auditory system responsible for sound localization depends upon commissural neurons that connect the two symmetrical cochlear nuclei. These cells in the deep polymorphic layer of the rat dorsal cochlear nucleus were identified with the electron microscope after injection of the retrograde tracer, Wheat Germ Agglutinin conjugated to Horseradish Peroxydase, into the contralateral cochlear nucleus. Commissural neurons are multipolar or bipolar with an oval to fusiform shape. Few commissural neurons, most inhibitory but also excitatory, connect most of the divisions of the rat cochlear nuclei. The most common type is a glycinergic, sometimes GABAergic, moderately large cell. Its ergastoplasm is organized into peripheral stacks of cisternae, and few axo-somatic synaptic boutons are present. Another type of commissural neuron is a medium-sized, spindle-shaped cell, glycine and GABA-negative, with sparse ergastoplasm and synaptic coverage. A giant, rare type of commissural neuron is glycine-positive and GABA-negative, with short peripheral stacks of ergastoplasmic cisternae. It is covered with synaptic boutons, many of which contain round synaptic vesicles. Another rare type of commissural neuron is a moderately large cell, oval to fusiform in shape, immunonegative for both glycine and GABA, and contacted by many axo-somatic boutons. It contains large dense mitochondria and numerous dense core vesicles of peptidergic type. Some labelled boutons, mostly inhibitory and probably derived from commissural neurons, contact pyramidal, cartwheel, giant and tuberculo-ventral neurons. The prevalent inhibition of electrical activity in a cochlear nucleus observed after stimulation of the contralateral cochlear nucleus may be due to commissural inhibitory terminals which contact excitatory neurons such as pyramidal and giant cells. Other inhibitory commissural terminals which contact inhibitory neurons such as cartwheel and tuberculo-ventral neurons, may explain the stimulation of electrical activity in the DCN after contralateral stimulation.     

Aging,cortical injury and Alzheimer's disease-like pathology in the guinea pig brain

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized histopathologically by the abnormal deposition of the proteins amyloid-beta (Aβ) and tau. A major issue for AD research is the lack of an animal model that accurately replicates the human disease, thus making it difficult to investigate potential risk factors for AD such as head injury. Furthermore, as age remains the strongest risk factor for most of the AD cases, transgenic models in which mutant human genes are expressed throughout the life span of the animal provide only limited insight into age-related factors in disease development. Guinea pigs (Cavia porcellus) are of interest in AD research because they have a similar Aβ sequence to humans and thus may present a useful non-transgenic animal model of AD. Brains from guinea pigs aged 3–48 months were examined to determine the presence of age-associated AD-like pathology. In addition, fluid percussion-induced brain injury was performed to characterize mechanisms underlying the association between AD risk and head injury. No statistically significant changes were detected in the overall response to aging, although we did observe some region-specific changes. Diffuse deposits of Aβ were found in the hippocampal region of the oldest animals and alterations in amyloid precursor protein processing and tau immunoreactivity were observed with age. Brain injury resulted in a strong and sustained increase in amyloid precursor protein and tau immunoreactivity without Aβ deposition, over 7 days. Guinea pigs may therefore provide a useful model for investigating the influence of environmental and non-genetic risk factors on the pathogenesis of AD.     

Topographical arrangement of thalamocortical neurons in the centrolateral nucleus (CL) of the cat,with special reference to a spino-thalamo-motor cortical path through the CL

Summary In the centrolateral nucleus of the thalamus (CL) in the cat, a topographical arrangement of the thalamocortical projection neurons was demonstrated by utilizing retrograde axonal transport of horseradish peroxidase (HRP). Following injections of HRP into the medial or lateral areas of the anterior sigmoid gyrus (ASG), HRP-labeled neurons were located medially or laterally in the caudal levels of the CL, respectively; neurons in the central areas of the CL were labeled after injections of HRP into the rostral areas of the middle suprasylvian or the lateral gyrus.It was also shown by means of the combined HRP and Fink-Heimer method (Blomqvist and Westman, 1975) that the spinothalamic fibers terminated around CL neurons which were labeled with HRP injected into the lateral areas of the ASG. Hence, the caudolateral aspects of the CL were considered to represent a relay of the spino-thalamo-motor cortical paths.     

On the development of non-pyramidal neurons and axons outside the cortical plate: The early marginal zone as a pallial anlage

Summary The development of non-pyramidal neurons was studied in the pallium of albino rats using autoradiography after thymidine labelling (determination of birth dates), Golgi impregnations (differentiation of dendrites and axons) and electron microscopy including 3D-reconstructions (cytoplasmic differentiation and early synaptogenesis).The marginal zone appears between E13 and E14 and contains glial cells, axons and preneurons from the beginning. The latter can be identified by structural criteria (contacts, cytoplasm, nuclei). The first vertically oriented pyramidal neurons (cortical plate) appear within the marginal zone not before E16, separating its contents into a superficial (lamina I) and a deep portion (intermediate and subventricular zone). Since this old neuronal population of lamina I and the subcortical pallial region can be followed until adulthood, it is proposed to call the early marginal zone a pallial anlage. It can be demonstrated that during the whole period of neuron production (until E21) non-pyramidal neurons are added to all parts of the pallial anlage.The structural differentiation of non-plate neurons is described. Neurons form specific, desmosome-like contacts with axonal growth cones already on E14. Typical synapses (vesicle aggregations) have been observed two days later. In lamina I two types of neurons develop: horizontal neurons (Cajal-Retzius cells) and multipolar neurons (small spiny stellate cells). Subcortical pallial neurons retain mostly their clear horizontal orientation. Only neurons situated very close to the lower border of the cortex show dendritic branches extending into lamina VI. Axons appearing early in the neocortex originate not only from subcortical regions, but also from neurons of the paleopallium, the archicortex, the limbic cortex and the neighbouring neocortex. The tangential growth of the neocortex, as estimated from E14 onwards causes a strong dilution of the elements of the pallial anlage until adulthood.The classification of neurons outside the cortical plate and the fate of the total pallial anlage are discussed. As a consequence of these observations some modifications of the terminology of the Boulder Committee are proposed.     

Intracellular evidence for incompatibility between spindle and delta oscillations in thalamocortical neurons of cat.

Recent studies have revealed that the thalamus does not only generate spindle oscillations (7-14 Hz), but that it also participates in the genesis of a slower (less than 4 Hz) rhythm within the frequency range of delta waves on the electroencephalogram. In thalamic cells, delta is an intrinsic oscillation consisting of low-threshold spikes alternating with afterhyperpolarizing potentials. It is known from electroencephalographic recordings in humans and animals that slow or delta waves prevail during late sleep stages, whereas spindle oscillations are characteristic for the early stages of sleep. We studied the dependence of spindles and delta oscillations on membrane potential, as well as the effects of spindles on delta oscillations, in thalamocortical neurons of cats under urethane anesthesia and in cerveau isolé preparations (low collicular transections). Spindles appeared at membrane potentials between -55 and -65 mV, whereas delta oscillations occurred by bringing the membrane potential between -68 and -90 mV. Spindles either evoked by cortical stimulation or occurring spontaneously in cerveau isolé preparations prevented delta oscillations. This effect was probably due to the increase in membrane conductance associated with spindles. Barbiturates also blocked delta activity in thalamocortical neurons, probably through the same mechanism. A certain degree of incompatibility between spindles and delta rhythms in thalamocortical cells may explain the prevalence of these two types of oscillations during different stages of sleep with synchronization of the electroencephalogram.     

Transneuronal transport of herpes simplex virus from the cervical vagus to brain neurons with axonal inputs to central vagal sensory nuclei in the rat.

The recent introduction of live viruses as intra-axonal tracing agents has raised questions concerning which central neurons are transneuronally labelled after application of the virus to peripheral organs or peripheral nerves. Since the central connections of the vagus nerve have been well described using conventional neuronal tracing agents, we chose to inject Herpes Simplex Virus Type 1 into the cervical vagus of the rat. After survival times of up to 3 days the rat brains were processed immunohistochemically using a polyclonal antiserum against herpes simplex virus. Two days after injection of the virus we observed viral antigen in the area postrema and in the nucleus tractus solitarius and the dorsal motor nucleus of the vagus (dorsal vagal complex), principally ipsilaterally. At this survival time the viral antigen in the dorsal vagal complex was largely confined to glial cells. After 3 days the viral antigen was localized both in glia and in nerve cells within the dorsal vagal complex and in brain regions previously demonstrated, using conventional tracing procedures, to contain neurons with axonal projections to the dorsal vagal complex. This was true for medullary, pontine, midbrain and hypothalamic regions and for telencephalic regions including the amygdala, the bed nucleus of the stria terminalis, and the insular and medial frontal cortices. Many of the nerve cells containing viral antigen were displayed in a Golgi-like manner, with excellent visualization of the dendritic tree. Axonal processes, in contrast, were not visualized. We used co-localization studies to confirm previous findings concerning monoamine neurotransmitter-related antigens present in medullary and pontine neurons projecting to the dorsal vagal complex. After 3 days there were many Herpes Simplex Virus Type 1-containing glial cells along the intra-medullary course of the vagal rootlets. However, no viral antigen was found in brain regions containing neurons whose axons pass through the region of glial cell-labelled rootlets. Glial cells containing viral antigen were particularly numerous in brain regions known to receive an input from neurons in the area postrema and the dorsal vagal complex. Taken together with our observation concerning the early appearance of viral antigen within glial cells in the dorsal vagal complex, this suggests that when the virus reaches the axon terminal portion it is transferred to nearby glial cells and possibly enters central neurons by way of these structures.     

Relationship between postsynaptic NK1 receptor distribution and nerve terminals innervating myenteric neurons in the guinea‐pig ileum

The amounts of neurokinin 1 (NK₁) receptor immunolabelling on the membranes of myenteric cell bodies at appositions with tachykinin‐immunoreactive nerve terminals, other nerve terminals, and glial cells were compared at the ultrastructural level using pre‐embedding, double‐label immunocytochemistry. NK₁ receptor immunoreactivity was revealed using silver‐intensified, 1 nm gold, and tachykinin‐immunoreactive nerve terminals were revealed using diaminobenzidine. The density of NK₁ receptor immunolabelling (silver particles per length of cell membrane) on the membrane at appositions with tachykinin‐immunoreactive nerve terminals was not significantly different from that at appositions with other (nonimmunoreactive) nerve terminals or with glial cells. Synaptic specializations (“active zones”) were present at a small proportion of the appositions between NK₁ receptor‐immunoreactive cell bodies and tachykinin‐immunoreactive or other nerve terminals. The density of NK₁ receptor immunolabelling at synaptic specializations was lower than that at regions of appositions where no synaptic specializations were present. The presence of NK₁ receptor on the cell surface in areas not directly apposed to tachykinin‐containing nerve terminals suggests that tachykinins that diffuse away from their site of release may still exert an action via NK₁ receptors. Although NK₁ receptors do not appear to be targetted to particular sites on the surfaces of myenteric nerve cell bodies and proximal dendrites, they are reduced in density at regions of the membrane‐forming synaptic specializations. Anat Rec 263:248–254, 2001. © 2001 Wiley‐Liss, Inc.     

Comparison of hypocretin/orexin and melanin-concentrating hormone neurons and axonal projections in the embryonic and postnatal rat brain

Hypocretin/orexin (H/O) and melanin-concentrating hormone (MCH) are peptide neuromodulators found in separate populations of neurons located within the lateral and perifornical hypothalamic regions. H/O has been linked to sleep-wakefulness regulation and to the sleep disorder narcolepsy, and both systems have been implicated in energy homeostasis, including the regulation of food intake. In the present study we compared the development of H/O and MCH-expressing neuronal populations with in situ hybridization and immunohistochemistry on adjacent sections in the embryonic and postnatal rat brain. We found that MCH mRNA and protein were present in developing neurons of the hypothalamus by embryonic day 16 (E16), whereas H/O mRNA and protein were not detected until E18. We also identified previously undescribed populations of MCH-immunoreactive cells in the lateral septum, paraventricular hypothalamic nucleus, lateral zona incerta, and ventral lateral geniculate nucleus that may play a specific role in the development of these regions. MCH immunoreactive axonal processes were also evident earlier than H/O stained fibers and at the time H/O immunoreactive processes were first identified in the hypothalamus at E20, extensive MCH axonal fiber systems were already present in many brain regions. Interestingly, however, the density of axonal fibers immunoreactive for H/O in the locus coeruleus reached peak levels at the same developmental age (P21) as MCH immunoreactive axons in the diagonal band of Broca (DBB). The peak of axon density coincided with the developmental stage at which adult patterns of feeding and sleep-waking activity become established. The present results demonstrate developmental differences and similarities between the MCH and H/O systems that may relate to their respective roles in feeding and sleep regulation.     

Neuroprotective effects of ebselen are associated with the regulation of Bcl-2 and Bax proteins in cultured mouse cortical neurons

There is little information available on the mechanisms underlying the neuroprotective actions of the organoselenium compound ebselen. In this study, we sought to determine the relationship between alterations in the expression of Bcl-2 and Bax proteins and intracellular levels of calcium and the protective effects of ebselen with a concentration range of 0.01-20 microM against glutamate toxicity in cultured mouse cortical neurons. Pretreatment with ebselen at moderate doses (4-12 microM), but not at lower or higher doses, significantly improved glutamate-induced suppression of cell viability. Pretreatment with ebselen (8 microM) also prevented apoptotic alterations, completely reversed the suppression of Bcl-2 expression, and significantly inhibited Bax overexpression, but did not alter elevated intracellular concentrations of calcium induced by glutamate. Pre-, co-, and post-treatment with ebselen (8 microM) had similar potency in improving the decreased viability of glutamate-exposed cells. These results indicate that the neuroprotective effects of ebselen at low doses are associated with the regulation of Bcl-2 and Bax proteins but appear to be independent of glutamate-mediated elevation of intracellular calcium, suggesting that different mechanisms are involved in the actions of low and high dose regimens. Ebselen may be an effective agent used for early treatment of acute brain injuries.     

Plasticity in the intrinsic excitability of cortical pyramidal neurons.

During learning and development, the level of synaptic input received by cortical neurons may change dramatically. Given a limited range of possible firing rates, how do neurons maintain responsiveness to both small and large synaptic inputs? We demonstrate that in response to changes in activity, cultured cortical pyramidal neurons regulate intrinsic excitability to promote stability in firing. Depriving pyramidal neurons of activity for two days increased sensitivity to current injection by selectively regulating voltage-dependent conductances. This suggests that one mechanism by which neurons maintain sensitivity to different levels of synaptic input is by altering the function relating current to firing rate.     

Localization of fast MEG waves in patients with brain tumors and epilepsy

It was investigated if single dipole analysis of spontaneous fast waves (>8 Hz) can be used to determine the location of the epileptic focus. Automatic dipole analysis was applied to MEG data of 25 patients with intracranial tumors and epilepsy. The frequency range of 8-50 Hz was divided into standard EEG bands. MEG results were overlaid on the MRI scans of the patients. Dipoles describing fast wave fields were located in the parietal/occipital cortex, and not at tumor border zones. In the cases that the dipoles were lateralized there was no clear preference to be located ipsi or contralateral to the tumor. However the generators of epileptic activity in these patients are thought to be located in the border areas of the tumors. Therefore it seems unlikely that the dipole locations describing fast waves are related to the epileptic zones in patients with brain tumors and epilepsy. A remarkable finding is that lateralized dipoles tend to be located in the right hemisphere and not in the left hemisphere. This appears to reflect an asymmetry of possibly normal background activity.