Jack H. Jhamandas

Efferent projections from the parabrachial nucleus demonstrated with the anterograde tracer Phaseolus vulgaris leucoagglutinin

Efferent projections from the parabrachial complex (PBN) were studied in the rat using the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L). Projections to the hypothalamus (ventromedial, dorsomedial, paraventricular, and supraoptic nuclei) originate primarily in the lateral PBN (1PBN). The amygdalar central nucleus (ACE) receives strong projections from all parts of the PBN although the external 1PBN projects primarily to the lateral ACE. Whereas the projections to the lateral bed nucleus of the stria terminalis, median preoptic nucleus, diagonal band of Broca, and lateral preoptic area originate primarily from the 1PBN, those to the insular cortex arise from the medial PBN (mPBN). The mPBN projects to the ventral posteromedial thalamus and the 1PBN and mPBN project to the zona incerta. Descending projections from the mPBN and Kölliker-Fuse area target the commissural nucleus tractus solitarius (NTS); the mPBN projects to the more rostral NTS. Similarly, the caudal parvicellular reticular formation (RF) receives projections from the mPBN and 1PBN, whereas input to the rostral RF arises from the former. All compartments of the PBN project to the ventrolateral medulla, although the projections arising from the 1PBN are densest. Finally, the raphe nuclei and periaqueductal gray receive some projections from most PBN divisions. These pathways provide a potential means whereby autonomic information can be relayed through the PBN to other structures important in regulating autonomic functions.

Principles underlying new methods for chronic neural recording.

Chronic recording is possible from nerve fibers which have grown through holes in an insulating medium (regeneration electrodes) or which are enclosed by an insulating sheath (cuff electrodes). Use of three electrodes in a balanced configuration permits good rejection of electromyographic (EMG) signals and other sources of electrical interference (fluorescent lights, 60 Hz signals from the mains, etc.). Equations are derived and tested for predicting the amplitude and form of the signals expected for a given cuff length and diameter. These equations can be used to design electrode units optimally for a given application. Finally, the use of transformers permits the neural signals to be carefully matched to the recording apparatus and further optimizes the neural signal-to-noise and signal-to-EMG ratios. Use of these methods in several physiological and clinical applications, as well as potential abuses, are discussed.

Branching projections of catecholaminergic brainstem neurons to the paraventricular hypothalamic nucleus and the central nucleus of the amygdala in the rat.

In this study, we have employed triple fluorescent-labelling to reveal the distribution of catecholaminergic neurons within three brainstem areas which supply branching collateral input to the central nucleus of the amygdala (CNA) and the hypothalamic paraventricular nucleus (PVN): the ventrolateral medulla (VLM), the nucleus of the solitary tract (NTS) and the locus coeruleus (LC). The catecholaminergic identity of the neurons was revealed by immunocytochemical detection of the biosynthetic enzyme, tyrosine hydroxylase. The projections were defined by injections of two retrograde tracers, rhodamine- and fluorescein-labelled latex microspheres, in the CNA and PVN, respectively. In the VLM and NTS, the greatest incidence of neurons which contained both retrograde tracers was found at the level of the area postrema. These neurons were mainly located within the confines of the A1/C1 (VLM) and A2 (NTS) catecholaminergic neuronal groups. Double-projecting neurons in the LC (A6) were distributed randomly within the nucleus. It was found that 15% in the VLM, 10% in the NTS and 5% in the LC of the retrogradely labelled cells projected via branching collaterals to the PVN and CNA. One half of these neurons in the VLM and NTS were catecholaminergic, in contrast to the LC where virtually all double-retrogradely labelled neurons revealed tyrosine hydroxylase immunoreactivity. In the other brainstem catecholaminergic cell groups (A5, A7, C3), no catecholaminergic neurons were found that supplied branching collaterals to the CNA and PVN. Our results indicate that brainstem neurons may be involved in the simultaneous transmission of autonomic-related signals to the CNA and the PVN. Catecholamines are involved in these pathways as chemical messengers. Brainstem catecholaminergic and non-catecholaminergic neurons, through collateral branching inputs may provide coordinated signalling of visceral input to rostral forebrain sites. This may lead to a synchronized response of the CNA and PVN for the maintenance of homeostasis.

Stable long-term recordings from cat peripheral nerves

A procedure has been developed for the stable long-term recording of nerve signals in unanesthetized mammals, which should have wide application in basic research on the nervous system and also in clinical areas such as the derivation of control signals for powered prostheses. Methods are fully described for constructing devices consisting of (1) Silastic nerve cuffs containing three or more electrodes, (2) coiled leads insulated with Silastic which extend from the cuffs to an integrated circuit socket, (3) a vitreous carbon transcutaneous connector which surrounds the integrated circuit socket and makes a good interface with the skin. Neural activity has been recorded from mammalian nerves for many months during normal behaviour. The peak-to-peak amplitude and latency of the recorded compound action potentials remain stable and may continue at a constant level more or less indefinitely. A tripolar recording configuration between a central lead and the two end leads, which are connected together, permits good rejection of EMG signals from surrounding muscles. The amplitude of single unit potentials increases as the square of the conduction velocity of the nerve fibre. Thus, the largest nerve fibres will dominate the signals recorded during behaviour. The reasons for premature termination of a few experiments are given together with methods for overcoming these problems. For example, platinum-iridium electrodes remain relatively stable, whereas silver wires tend to fracture after being in an animal for several months. This and other relationships are discussed which permit an optimal design of nerve cuffs for a given recording situation.

Angiotensin II may mediate excitatory neurotransmission from the subfornical organ to the hypothalamic supraoptic nucleus: an anatomical and electrophysiological study in the rat

In the rat, it has been proposed that angiotensin II (AII) neurons in the subfornical organ, a midline circumventricular structure, participate in the activation of hypothalamic neurosecretory neurons and promote a rise in plasma vasopressin and oxytocin. In this study, we observed AII-immunoreactive fibers coursing throughout the supraoptic nucleus as well as in other magnocellular cell groups of the hypothalamus. Moreover, following retrograde transport of Fast blue deposited within the supraoptic nucleus, cell counts in our best case revealed that 40% of AII-immunoreactive neurons in subfornical organ contained Fast blue, and 46% of the retrogradely labeled subfornical organ cells contained AII. In separate electrophysiological studies, post-stimulus histograms from 18 of 28 supraoptic neurons displayed a 30-55% reversible reduction in the excitation evoked by an electrical stimulus in the subfornical organ during local pressure applications of 100 microM to 1 mM saralasin. In 2 of 14 other cells, tubocurare (100 microM) produced only a 10% reduction in subfornical organ excitation. These observations indicate that AII may mediate an excitatory input to supraoptic neurons from the subfornical organ.

HIV-1 Vpr Causes Neuronal Apoptosis and In Vivo Neurodegeneration

Despite the introduction of highly active antiretroviral therapy, dementia caused by human immunodeficiency virus-1 (HIV-1) infection remains a devastating and common neurological disorder. Although the mechanisms governing neurodegeneration during HIV-1 infection remain uncertain, the HIV-1 accessory protein, viral protein R (Vpr), has been proposed as a neurotoxic protein. Herein, we report that Vpr protein and transcript were present in the brains of HIV-infected persons. Moreover, soluble Vpr caused neuronal apoptosis, involving cytochrome c extravasation, p53 induction, and activation of caspase-9 while exerting a depressive effect on whole-cell currents in neurons (p < 0.05), which was inhibited by iberiotoxin. Vpr-activated glial cells secreted neurotoxins in a concentration-dependent manner (p < 0.001). Transgenic (Tg) mice expressing Vpr in brain monocytoid cells displayed the transgene principally in the basal ganglia (p < 0.05) and cerebral cortex (p < 0.01) compared with hindbrain expression. Vpr was released from cultured transgenic macrophages, which was cytotoxic to neurons and was blocked by anti-Vpr antibody (p < 0.05). Neuronal injury was observed in Tg animals compared with wild-type littermates, chiefly affecting GAD65 (p < 0.01) and vesicular acetylcholine transferase (p < 0.001) immunopositive neuronal populations in the basal ganglia. There was also a loss of subcortical synaptophysin (p < 0.001) immunoreactivity as well as an increase in activated caspase-3, which was accompanied by a hyperexcitable neurobehavioral phenotype (p < 0.05). Thus, HIV-1 Vpr caused neuronal death through convergent pathogenic mechanisms with ensuing in vivo neurodegeneration, yielding new insights into the mechanisms by which HIV-1 injures the nervous system.

Interaction between hypothalamic dorsomedial nucleus and the suprachiasmatic nucleus determines intensity of food anticipatory behavior

Food anticipatory behavior (FAA) is induced by limiting access to food for a few hours daily. Animals anticipate this scheduled meal event even without the suprachiasmatic nucleus (SCN), the biological clock. Consequently, a food-entrained oscillator has been proposed to be responsible for meal time estimation. Recent studies suggested the dorsomedial hypothalamus (DMH) as the site for this food-entrained oscillator, which has led to considerable controversy in the literature. Herein we demonstrate by means of c-Fos immunohistochemistry that the neuronal activity of the suprachiasmatic nucleus (SCN), which signals the rest phase in nocturnal animals, is reduced when animals anticipate the scheduled food and, simultaneously, neuronal activity within the DMH increases. Using retrograde tracing and confocal analysis, we show that inhibition of SCN neuronal activity is the consequence of activation of GABA-containing neurons in the DMH that project to the SCN. Next, we show that DMH lesions result in a loss or diminution of FAA, simultaneous with increased activity in the SCN. A subsequent lesion of the SCN restored FAA. We conclude that in intact animals, FAA may only occur when the DMH inhibits the activity of the SCN, thus permitting locomotor activity. As a result, FAA originates from a neuronal network comprising an interaction between the DMH and SCN. Moreover, this study shows that the DMH–SCN interaction may serve as an intrahypothalamic system to gate activity instead of rest overriding circadian predetermined temporal patterns.

Chemically defined collateral projections from the pons to the central nucleus of the amygdala and hypothalamic paraventricular nucleus in the rat

Triple fluorescence labelling was employed to reveal the distribution of chemically identified neurons within the pontine laterodorsal tegmental nucleus and dorsal raphe nucleus which supply branching collateral input to the central nucleus of the amygdala and hypothalamic paraventricular nucleus. The chemical identity of neurons in the laterodorsal tegmental nucleus was revealed by immunocytochemical detection of choline-acetyltransferase or substance P; in the dorsal raphe nucleus, the chemical content of the neurons was revealed with antibody recognizing serotonin. The projections were defined by injections of two retrograde tracers, rhodamine-and fluorescein-labelled latex microspheres, in the central nucleus of the amygdala and paraventricular nucleus, respectively. Neurons projecting to both the central nucleus of the amygdala and the paraventricular nucleus were distributed primarily within the caudal extensions of the laterodorsal tegmental nucleus and dorsal raphe nucleus. Approximately 11% and 7% of the labelled cells in the laterodorsal tegmental nucleus and dorsal raphe nucleus projected via branching collaterals to the paraventricular nucleus and central nucleus of the amygdala. About half of these neurons in the laterodorsal tegmental nucleus were cholinergic, and one-third were substance-P-ergic; in the dorsal raphe nucleus, approximately half of the neurons containing both retrograde tracers were serotonergic. These results indicate that pontine neurons may simultaneously transmit signals to the central nucleus of the amygdala and paraventricular nucleus and that several different neuroactive substances are found in the neurons participating in these pathways. This coordinated signalling may lead to synchronized responses of the central nucleus of the amygdala and paraventricular nucleus for the maintenance of homeostasis. Interactions between different neuroactive substances at the target site may serve to modulate the responses of individual neurons.

Compound action potentials recorded from mammalian peripheral nerves following ligation or resuturing.

1. Cat hind limb peripheral nerves were fitted with cuff recording electrodes, and their distal portions were later cut and ligated to prevent regeneration. The compound action potential amplitude initially declined with a time constant between 1 and 2 months and then remained relatively unchanged for periods of more than a year. Similar but smaller changes were observed in the conduction velocity of the nerves which also stabilized after a few months. 2. In nerves that were cut and resutured to their distal stumps or sutured directly to nearby muscles, a recovery was observed. The time course was well fitted by an initial exponential decay with a similar time constant to that above, followed by an exponential recovery with a longer time constant (3‐4 months). Nerve conduction, muscle potentials and twitch tension often recovered to control values, even when the amplitude of the nerve compound action potential remained depressed. 3. Thus, nerve fibres survive axotomy for long periods of time and continue to conduct action potentials, even if unable to regenerate to appropriate end‐organs. When regeneration is permitted, a fraction of nerve fibres may reinnervate nearly all end‐organs. The diameter and conduction velocity of these nerve fibres presumably increase toward control values, while other fibres remain subnormal in these parameters. 4. Factors in the design of cuff electrodes which determine the amplitude of compound action potentials are described in an Appendix.

Proteolytic processing of SDF-1α reveals a change in receptor specificity mediating HIV-associated neurodegeneration

Proteolytic cleavage of constitutively expressed proteins can generate peptides with novel bioactive properties. Matrix metalloproteinase (MMP)-2 cleaves the 4 amino-terminal residues of the chemokine, stromal cell-derived factor (SDF)-1α, yielding a highly neurotoxic molecule, SDF(5-67), which fails to bind to its cognate receptor, CXCR4. Herein, we detected SDF(5-67) in brain monocytoid cells of HIV-infected persons, particularly in those with HIV-associated dementia. SDF(5-67) activated cell type-specific expression of proinflammatory genes including IL-1β, TNFα, indoleamine 2′,3′-dioxygenase (IDO), and IL-10 in both astrocytic and monocytoid cells (P < 0.05). Unlike SDF-1α, SDF(5-67) caused neuronal membrane perturbations with ensuing neurotoxicity and apoptosis (P < 0.05) through engagement of an inducible receptor. CXCR3 antagonists and siRNA-mediated knockdown of CXCR3 inhibited SDF(5-67)-stimulated neurophysiological changes, neuronal death, and neuroimmune activation (P < 0.05). Moreover SDF(5-67) bound directly to CXCR3 in a competitive manner, mediated by its amino terminus. In vivo neuroinflammation, neuronal loss, and neurobehavioral abnormalities caused by SDF(5-67) (P < 0.05) were prevented by a CXCR3 antagonist. These studies reveal additive neuropathogenic properties exerted by a proteolytically cleaved chemokine as consequences of a change in receptor specificity, culminating in neurodegeneration.