Kim H. Harris

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.

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.

Parabrachial nucleus projection to the amygdala in the rat: Electrophysiological and anatomical observations

The amygdala, an important limbic forebrain centre, is the recipient of projections from a number of autonomic brainstem nuclei including the pontine parabrachial nucleus. This study examined the influence of electrical stimulation of the parabrachial nucleus on the excitability of amygdala neurons and their response to two cardiovascular stimuli, namely baroreceptor activation and the administration of systemic angiotensin II. We also defined the chemical identity of some amygdala neurons that receive parabrachial nucleus projections by combining the transport of the anterograde tracer Phaseolus vulgaris leucoagglutinin injected into the parabrachial nucleus with immunocytochemical labelling of neurotensin and galanin profiles within the amygdala. In urethane-anesthetized rats, stimulation of parabrachial nucleus evoked four basic types of synaptic responses in amygdala cells: (1) a short duration (< 100 ms) excitation in 75 of 167 neurons, (2) a longer duration (> 100 ms) excitatory response in 36 neurons, (3) an inhibitory response in 32 cells, and (4) more complex responses consisting of excitation-inhibition or inhibition-excitation sequences in the remainder of the cells. Thirty-seven of 72 amygdala neurons activated synaptically by parabrachial nucleus stimulation also responded to baroreceptor activation or intravenous angiotensin II. Anatomical data revealed the presence of Phaseolus vulgaris leucoagglutinin labelled terminals predominantly within the lateral, medial, and capsular subdivisions of the central nucleus of amygdala. Phaseolus vulgaris leucoagglutinin varicosities and boutons were observed apposed to the neurotensin and galanin neuronal perikarya within the central nucleus of amygdala. The electrophysiological results provide a framework whereby parabrachial nucleus efferents influence the activity of amygdala neurons that are responsive to cardiovascular stimuli. Furthermore, the anatomical data indicate that a portion of the parabrachial nucleus input is directed toward galanin and neurotensin neurons within the central nucleus of amygdala.

Single Transmembrane Domain Insulin-Like Growth Factor-II/Mannose-6-Phosphate Receptor Regulates Central Cholinergic Function by Activating a G-Protein-Sensitive, Protein Kinase C-Dependent Pathway

The insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor is a single-pass transmembrane glycoprotein that plays an important role in the intracellular trafficking of lysosomal enzymes and endocytosis-mediated degradation of IGF-II. However, its role in signal transduction after IGF-II binding remains unclear. In the present study, we report that IGF-II/M6P receptor in the rat brain is coupled to a G-protein and that its activation by Leu27IGF-II, an analog that binds rather selectively to the IGF-II/M6P receptor, potentiates endogenous acetylcholine release from the rat hippocampal formation. This effect is mediated by a pertussis toxin (PTX)-sensitive GTP-binding protein and is dependent on protein kinase Cα (PKCα)-induced phosphorylation of downstream substrates, myristoylated alanine-rich C kinase substrate, and growth associated protein-43. Additionally, treatment with Leu27IGF-II causes a reduction in whole-cell currents and depolarization of cholinergic basal forebrain neurons. This effect, which is blocked by an antibody against the IGF-II/M6P receptor, is also sensitive to PTX and is mediated via activation of a PKC-dependent pathway. These results together revealed for the first time that the single transmembrane domain IGF-II/M6P receptor expressed in the brain is G-protein coupled and is involved in the regulation of central cholinergic function via the activation of specific intracellular signaling cascades.

Fucoidan inhibits cellular and neurotoxic effects of β-amyloid (Aβ) in rat cholinergic basal forebrain neurons

The deposition of β‐amyloid protein (Aβ), a 39–43 amino acid peptide, in the brain and a loss of cholinergic neurons in the basal forebrain are pathological hallmarks of Alzheimers disease (AD). Seaweeds consumed in Asia contain Fucoidan, a sulfated polysaccharide. Fucoidan has been known to exhibit various biological actions, such as an anti‐inflammatory and antioxidant action. In this study, using whole‐cell patch clamp recordings we examined the effects of Fucoidan on Aβ‐induced whole‐cell currents in acutely dissociated rat basal forebrain neurons. We further investigated whether Fucoidan is capable of blocking Aβ neurotoxicity in primary neuronal cultures. In dissociated cells, bath application of Aβ25−35 (1 µm) caused a reduction of the whole‐cell currents by 16%. Fucoidan, in a dose‐dependent manner, blocks the Aβ25−35 reduction of whole‐cell currents. Exposure of Aβ25−35 (20 µm) or Aβ1−42 (20 µm) to rat cholinergic basal forebrain cultures for 48 h resulted in 40–60% neuronal death, which was significantly decreased by pretreatment of cultures with Fucoidan (0.1–1.0 µm). Fucoidan also attenuated Aβ‐induced down‐regulation of phosphorylated protein kinase C. Aβ1−42‐induced generation of reactive oxygen species was blocked by prior exposure of cultures to Fucoidan. Furthermore, Aβ activation of caspases 9 and 3, which are signaling pathways implicated in apoptotic cell death, is blocked by pretreatment of cultures with Fucoidan. These results show that Fucoidan is able to block Aβ‐induced reduction in whole‐cell currents in basal forebrain neurons and has neuroprotective effects against Aβ‐induced neurotoxicity in basal forebrain neuronal cultures.

Changes in blood volume and pressure induce c-fos expression in brainstem neurons that project to the paraventricular nucleus of the hypothalamus

Immunohistochemistry for c-fos was combined with retrograde tracing techniques to study the effects of acute reductions in arterial blood pressure due to hemorrhage (HEM) in conscious rats on activated neurons in the brainstem nucleus of the tractus solitarius (NTS) or ventrolateral medulla (VLM) which project to the paraventricular nucleus (PVN) of the hypothalamus. In an attempt to separate blood pressure effects from those associated with changes in blood volume, a similar approach was used to study the effects of drug-evoked hypotension using peripheral infusions of sodium nitroprusside (NP). Few differences were found in patterns or numbers of activated neurons (Fos-immunoreactive) in the NTS or VLM after HEM or NP treatment; only in the NTS at the level of the area postrema were significantly higher numbers of neurons that expressed Fos found in NP rats. In addition, a large proportion of PVN-projecting neurons in the NTS and VLM was activated whereas many activated neurons in the NTS and VLM did not project to the PVN. These results show that a decrease in blood pressure leads to the activation of NTS and VLM neurons but that a change in blood volume does not activate significantly greater numbers of neurons in these areas that project to the PVN or to other targets. Whereas substantial numbers of neurons in the NTS and VLM appear to transmit cardiovascular information to the PVN, many others likely transmit this information to other central targets.

Expression of c-fos Protein in Rat Brain Elicited by Electrical and Chemical Stimulation of the Hypothalamic Paraventricular Nucleus

The functional connectivity of the paraventricular nucleus of the hypothalamus (PVN) was studied by assessing the expression of the immediate early gene, c-fos, after unilateral stimulation of this structure in urethane-anesthetized rats. Electrical stimulation for 1 h (10 s on, 10 s off; 15-40 microA at 20 Hz) was accompanied by increases in mean arterial pressure (13-29 mm Hg). In these animals, ipsilateral increases in numbers of neurons with Fos-like immunoreactivity (FLI) were immunohistochemically demonstrated in the insular cortex, lateral septum, medial amygdala, hypothalamus, lateral division of the parabrachial nucleus (PBN) of the pons and the nucleus of the tractus solitarius (NTS) and ventrolateral medulla (VLM). Numbers of cells with FLI were quantitated in five areas known for their roles in autonomic function: arcuate nucleus, ventromedial hypothalamus, lateral PBN, NTS (at three levels) and VLM (caudal and rostral). In each case, stimulation of the PVN led to significant differences in number of neurons with FLI on the side ipsilateral to the stimulation compared to the contralateral side. To eliminate effects associated with stimulation of fibers of passage in the vicinity of the PVN, the results after electrical stimulation were compared to those obtained in animals in which the PVN was chemically stimulated unilaterally with the excitatory amino acid L-glutamate (5 one-minute infusions of 50 nl, 0.5 M glutamate over 1 h). Mean arterial pressure was increased after each injection (7-13 mm Hg), and significant differences in numbers of neurons with FLI between sides were maintained in all five areas except the NTS caudal to, and at, the level of the area postrema. An increase in neurons with FLI in the piriform cortex of all animals including controls may be due to injury-induced activation of target neurons from the PVN. These data illustrate that electrical and chemical stimulation of the PVN leads to simultaneous activation of neurons in many targets. All of the target areas studied receive direct projections from the PVN, although multisynaptic projections may also contribute to activation of target neurons.

Characterization of the parabrachial nucleus input to the hypothalamic paraventricular nucleus in the rat.

The brainstem parabrachial nucleus (PBN) is viewed as an increasingly important site for the transfer of autonomic‐related information to more rostral structures in the forebrain including the hypothalamus. In this study, we examined electrophysiologically in vivo and anatomically the nature of PBN input to the hypothalamic paraventricular nucleus (PVN) and particularly to the vasopressin‐and oxytocin‐secreting magnocellular neurosecretory cells within this nucleus.

Neurons in the rat medulla oblongata containing neuropeptide Y-, angiotensin II-, or galanin-like immunoreactivity project to the parabrachial nucleus.

Projections from the medulla to the parabrachial complex of the rat were examined for their content of neuropeptide Y-, angiotensin II- or galanin-like immunoreactivity using combined retrograde tracing and immunohistochemical techniques. Rhodamine-labelled latex microspheres were stereotaxically injected into discrete nuclei of the parabrachial complex. After survival of two to five days, colchicine (100 micrograms in 10 microliters saline) was injected into the cisterna magna. One day later, rats were perfused and the brainstems were prepared for visualization of the retrograde tracer and immunoreactivity of one of the three peptides. Retrograde labelling verified that the area postrema, nucleus of the tractus solitarius, caudal spinal nucleus of the trigeminal nerve, parvocellular reticular nucleus, and ventrolateral medulla including the rostral ventrolateral medulla and nucleus paragigantocellularis project to the lateral parabrachial and Kölliker-Fuse nuclei. While most projections were primarily ipsilateral, a small proportion of the projections from the ventrolateral medulla was bilateral. Neurons containing neuropeptide Y-like immunoreactivity were found in the caudal and intermediate nucleus of the tractus solitarius, dorsal to the lateral reticular nucleus and in the nucleus paragigantocellularis. After bilateral microsphere injections into the lateral parabrachial and Kölliker-Fuse nuclei, double-labelled neurons were found dorsal to the lateral reticular nucleus of caudal and intermediate medullary levels, at the ventral surface of the medulla at intermediate levels and in the nucleus paragigantocellularis at rostral levels. Neurons with angiotensin II-like immunoreactivity were observed at the dorsomedial border of the caudal and intermediate nucleus of the tractus solitarius, in the area postrema and in the lateral reticular nucleus and nucleus paragigantocellularis. Of these neurons, small numbers in the nucleus of the tractus solitarius and ventrolateral medulla also projected to the lateral parabrachial and Kölliker-Fuse nuclei. Neurons containing galanin-like immunoreactivity were found in the caudal nucleus of the tractus solitarius, the area postrema, the spinal trigeminal nucleus, the raphe nuclei (pallidus and obscurus), the nucleus paragigantocellularis and dorsal to the lateral reticular nucleus. Of these cells, double-labelled neurons were found in the commissural and medial subdivisions of the caudal nucleus of the tractus solitarius and in the rostral ventrolateral medulla including the ventral surface and the nucleus paragigantocellularis. The results suggest that neuropeptide Y, angiotensin II and galanin may serve as neurochemical messengers in pathways from the medulla to the parabrachial complex. The location of double-labelled neurons suggests that the information relayed by these neurons is related to autonomic activity.

Hypotension induces fos immunoreactivity in NADPH-diaphorase positive neurons in the paraventricular and supraoptic hypothalamic nuclei of the rat

Double staining for Fos and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-D) was used to study the distribution of activated neurons that synthesize nitric oxide in the paraventricular (PVN) and supraoptic nuclei (SON) following hypotensive stimulation in conscious rats. Fos was detected in many magno- and parvocellular NADPH-D positive neurons in response to haemorrhage or drug-evoked hypotension using i.v. infusions of sodium nitroprusside. However, quantitative analysis did not reveal any differences in the number of Fos positive PVN neurons following either mode of stimulation. These results suggest that a subpopulation of hypothalamic NADPH-D positive neurons is activated following hypotensive challenge. This activation of NADPH-D neurons may occur indirectly through other CNS structures that influence the excitability of hypothalamic SON and PVN. Furthermore, the lack of a difference in activated neurons within the PVN following either haemorrhage or nitroprusside infusion suggests that while a drop in blood pressure causes activation of neurons that produce nitric oxide, a decrease in blood volume, which accompanies haemorrhage, does not.