John S. Kelly

Neuropeptide Y innervation of the rodent pineal gland and cerebral blood vessels

Neuropeptide Y (NPY)-immunoreactivity has been shown to be present in sympathetic nerve fibres in the rat pineal gland and a dense network of NPY-containing nerve fibres demonstrated to innervate the rat circle of Willis. The NPY content of the major rabbit intracranial arteries was determined by radioimmunoassay and maximal levels found in the anterior cerebral arteries. After bilateral superior cervical ganglion (SCG) removal, no NPY was detectable in the rat pineal gland; however, significant NPY-immunoreactive nerve fibres remained throughout the rat vertebrobasilar arteries, and 47% of the assayable NPY was still present. Neither intraventricular 6-hydroxydopamine (6-OHDA) nor the combination of 6-OHDA treatment and SCG removal resulted in any further loss of NPY. In conclusion, the NPY innervation of the pineal gland originates exclusively from the peripheral sympathetic nervous system. In contrast the caudal portion of the rat circle of Willis contains NPY fibres which are resistant to sympathectomy.

The effect of sympathectomy on calcitonin gene-related peptide levels in the rat trigeminovascular system

The effect of sympathectomy on the calcitonin gene-related peptide (CGRP) level in the rat primary trigeminal sensory neurone was investigated. Six weeks after bilateral removal of the superior cervical ganglion there was a 70% rise in the CGRP content of the iris and the pial arteries, a 34% rise in the concentration in the trigeminal ganglion but no change in the brainstem. The CGRP rise in both end organs suggests that this phenomenon may be common to all peripheral organs receiving combined sensory and sympathetic innervations. The lack of any rise in the brainstem CGRP content raises the possibility that this process spares central terminations. In contrast, the level of neuropeptide Y, a peptide mainly contained in sympathetic terminals, fell to 35% of control values in the iris and pial arteries whilst the trigeminal ganglion and brainstem concentrations remained unchanged. The possible relevance of these observations to the clinical syndrome of postsympathectomy pain (sympathalgia) is discussed. There are similarities between the delayed onset of the human pain state and the delayed rise in sensory peptides after sympathectomy.

Excitatory amino acids in the hippocampus: synaptic physiology and pharmacology

Abstract Although more than 20 years have elapsed since Curtis, Krnjevic, Phillis and Watkins 1,2 showed that the acidic amino acids glutamate and aspartate produce excitation of central neurones, until recently the electrophysiological and pharmacological evidence for the role of these substances as neurotransmitters has been weak. However, during the past three years the situation has changed dramatically with the development of new antagonists for the excitatory amino acids 3 , the refinement of the in-vitro brain slice preparation and the improvements in intracellular recording techniques 4 . In the hippocampus, a brain structure well suited to in-vitro studies and where excitatory pathways have long been thought to use an amino acid transmitter, the application of these new compounds and techniques has been particularly successful. Thus it has been possible to use vertebrate neurones for the first time to measure synaptic currents, to compare the reversal potential of the EPSPs and of the excitatory amino acids, and to analyse pharmacological antagonism at the intracellular level. The synaptic receptors have been partially characterized while the exact nature of the endogenous excitatory transmitter must still await the development of more selective antagonists.

Passive membrane properties of neurones in the dorsal raphe and periaqueductal grey recorded in vitro

A slice preparation of the rat mesencephalon containing the dorsal and medial raphe nucleus, the periaqueductal grey, the superior colliculi and the reticular formation is described. Intracellular recordings showed marked differences in the passive membrane properties of neurones of the dorsal raphe. Serotonin-containing neurons were characterized by a high membrane input resistance, a very long time constant and by the presence of membrane rectification only in a very hyperpolarized (less than -120 mV) region of their voltage-current relationship. In most of the neurones in and around the dorsal raphe area a brief pulse of depolarizing current was followed by a pronounced after-hyperpolarization, which appeared to be mediated by the activation of a Ca2+ dependent-K+ conductance.

Neuroleptics decrease calcium-activated potassium conductance in hippocampal pyramidal cells

Intracellular recordings were made from pyramidal CA1-neurones of the hippocampal slice preparation. Bath application of a wide variety of neuroleptics was found to depress the slow afterhyperpolarization, which is mediated in these neurons by a calcium-dependent potassium conductance occurring following a burst of spikes. The depression of this conductance took place in the presence of calcium spikes of normal amplitude and duration, and except in the case of trifluoperazine, without alteration in resting membrane potential or input resistance.

Excitable properties of insect neurones in culture: A developmental study

Abstract The passive and excitable electrical properties of cockroach neurones growing in vitro have been investigated using intracellular recording techniques. The resting membrane potentials of the neurones are similar to those of their in vivo counterparts but the input resistances and membrane capacitive properties are more typical of embryonic insect neurones. During the first 12 days of growth in vitro the neurones exhibit delayed rectification in response to the injection of depolarising current steps. After this period “all or none” action potentials can be evoked by depolarising pulses in approximately half of the neurones tested. These spikes are abolised by 1 μM tetrodotoxin but are unaffected by 5 mM Co2+. Spontaneous excitatory activity develops in approx 25% of the neurones after 3 weeks in culture.

Spontaneous electrical activity induced by herpes virus infection in rat sensory neuron cultures

Dissociated cultures of rat dorsal root ganglion neurons were infected with a syncytial strain of herpes simplex virus type 1. Over 90% of neurons in infected cultures were spontaneously active and fired action potentials which, on membrane potential hyperpolarization, were replaced by depolarizing events similar to excitatory postsynaptic potentials. Amplitude analysis of these events produced populations described by the sum of several unitary events with Gaussian rather than binomial or Poisson distributions. Such spontaneous activity was blocked by tetrodotoxin but not by low calcium high magnesium solutions containing cadmium. Simultaneous recording from pairs of spontaneously active neurons revealed excitatory connexions between cells. It is suggested that virus-induced fusion of nerve cell processes induces electrical coupling between sensory neurons, and that the resulting electrical network supports spontaneous activity.

The possible modulation of the development of rat dorsal root ganglion cells by the presence of 5-HT-containing neurones of the brainstem in dissociated cell culture

Reliable methods for coculturing dissociated rat brainstem cells together with dorsal root ganglion (DRG) cells have been established. The cells were characterized using autoradiographic, morphological, immunocytochemical and electrophysiological techniques. Light-level microscopy showed the cocultures to be extensively invaded by serotonin (5-HT)-containing neuronal processes and intense clustering of 5-HT-containing varicosities to occur in the vicinity of large round phase-bright neurones thought to be DRGs. Rather extensive fine ramification of the neuronal processes throughout the culture dish were visualized using scanning electron microscopy. Intracellular recording showed the brainstem neurones to be spontaneously active, electrically excitable and sensitive to a variety of transmitter candidates including serotonin (5-HT) and gamma-aminobutyric acid. Several different responses to 5-HT have been observed. These include a depolarization accompanied by an increase in membrane resistance, a depolarization accompanied by a decrease in membrane resistance and a hyperpolarization accompanied by an increase in membrane resistance. As established by others, DRG cells grown in isolation were always quiescent. The application of 5-HT produced no effect on membrane potential, resistance or excitability. In the brainstem-DRG cocultures 52% of DRG cells exhibited synaptic activity and occasional spontaneous spiking, both of which were abolished in the presence of tetrodotoxin or low-Ca2+/high-Mg2+ solution. Transmission electron microscopy confirmed the presence of synapses on the DRG cells. The spontaneously active DRG cells were also found to be sensitive to the application of 5-HT. Thus it appears that a source of 5-HT nerve terminals may regulate the development and pharmacological sensitivity of primary afferent neurones in culture.

Acute membrane responses to viral action.

The effects of Sendai, a paramyxovirus, on the functional activity of 3 cell types, have been studied in vitro to establish whether a virus alone can cause pathophysiological changes. Neuronal cells are depolarized and suffer a loss of excitability which was attributed to an increase in membrane conductance. Spontaneously beating cardiac cells initially stop beating and then beat more rapidly and asynchronously. Anterior pituitary cells release hormones. In all 3 cases the effects are transient and the cells recover completely.

Effect of reserpine, phenoxybenzamine and cold stress on the neuropeptide Y content of the rat peripheral nervous system

The effect of reserpine treatment on the neuropeptide Y content of the rat adrenal gland, heart, kidney and vasculature was studied using a specific radioimmunoassay. One hour after reserpine administration (5 mg/kg) the neuropeptide Y concentration in the adrenal gland was significantly reduced and after 4 h a similar reduction was seen in the heart and kidney. After 48 h, neuropeptide Y concentrations were reduced in all tissues. The greatest reduction occurred in the cardiac septum (77%) and the least in the inferior vena cava (25%). Phenoxybenzamine (2 mg/kg) also caused a reduction in neuropeptide Y concentrations which was less marked than after reserpine, except in the adrenal gland where it was similar. Cold stress caused no change in neuropeptide Y concentrations. The neuropeptide Y depletion induced by reserpine was compared to that following 6-hydroxydopamine. In the heart and pial arteries both drugs caused a similar neuropeptide Y depletion whilst in the pineal gland and renal artery 6-hydroxydopamine had more effect than reserpine. The implications of these results on NPY storage sites are discussed.