Astrocytes In Explant Cultures Research Articles

Colocalization of neurotransmitter receptors on astrocytes in explant cultures of rat CNS

In recent years evidence has accumulated that astrocytes express functional receptors for a variety of neurotransmitters/neuromodulators. By means of electrophysiological and combined autoradiographic and immunohistochemical methods we have demonstrated the colocalization of cholinergic, adrenergic and peptidergic receptors on astrocytes in explant cultures from various regions of rat central nervous system. A great number of biochemical and electrophysiological studies from other laboratories have shown that most of the neurotransmitters exert their effects on second messenger systems and on Ca 2+-activated K +-channels . Furthermore, certain neurotransmitters are involved in the regulation of energy metabolism by stimulating enzymatic breakdown of glycogen in astrocytes. It was suggested that there is a cross-talk between the various neurotransmitter receptors on the glial membrane and that these receptors act in a synergistic or antagonistic way. The coexistence of cholinergic and peptidergic receptors on astrocytes is of great interest since both neurotransmitter systems are involved in cognitive functions and are impaired in patients with Alzheimer’s dementia. The question is therefore raised whether not only neurones but also astrocytes might be involved in neurodegenerative disorders such as Alzheimer’s disease.

Histochemical and electrophysiological evidence for estrogen receptors on cultured astrocytes: colocalization with cholinergic receptors

By means of autoradiographic and immunohistochemical methods it was demonstrated that astrocytes in explant and primary cultures of rat neocortex, hippocampus, preoptic area and spinal cord express estrogen α- and β-receptors. Immunoreactivity was mainly distributed over the soma, the nuclei being more intensely stained. Combined autoradiographic and immunohistochemical studies as well as double-immunostaining revealed a colocalization of estrogen α- and β-receptors on many astrocytes. There was also a coexistence of estrogen receptors and cholinergic muscarinic and nicotinic sites. Electrophysiological investigations have shown that 17β-estradiol induced hyperpolarizations on the majority of astrocytes in explant cultures of hippocampus and spinal cord, providing evidence for the existence of functional estrogen receptors on these cells. Furthermore, on the same astrocytes, 17β-estradiol, muscarine and nicotine caused hyperpolarizations, suggesting a coexistence of receptors for estrogen and the cholinergic agonists on glial cells. The presence of glial estrogen receptors and their colocalization with cholinergic receptors is discussed with respect to the effects of these neurotransmitters/neuromodulators in development and maturation of the central nervous system, as well as to neurodegenerative events such as Alzheimer’s disease.

Action and binding of the endothelin antagonist bosentan on astrocytes of cultured rat central nervous system. Electrophysiological and autoradiographic studies

Our autoradiographic studies demonstrate that astrocytes in explant cultures of rat central nervous system possess binding sites for the first orally active, mixed, nonpeptide endothelin receptor antagonist [ 3H]bosentan. Binding of [ 3H]bosentan was inhibited by unlabelled bosentan and endothelin-1 at high concentrations, suggesting specific binding of the antagonist. Electrophysiological studies have revealed that bosentan reversibly blocked the depolarizations by endothelin but not by angiotensin II, indicating that the antagonist specifically antagonizes the action of endothelin on the glial membrane. This is consistent with biochemical studies from other laboratories demonstrating that bosentan did not interfere with binding of angiotensin II. The availability of bosentan, a potent and selective endothelin receptor antagonist should help to elucidate the role of endothelin on astrocyte function.

Autoradiographic studies on the uptake of 3H-noradrenaline and 3H-serotonin by neurones and astrocytes in explant and primary cultures of rat CNS: Effects of antidepressants

Autoradiographic studies were made on the uptake of 3H-noradrenaline and 3H-serotonin in explant cultures and primary astrocyte cultures from various regions of rat central nervous system (cortex, cerebellum, locus coeruleus, nucleus raphé, spinal cord). In explant cultures from locus coeruleus and nucleus raphé cell bodies and processes of many neurones revealed intense labelling by 3H-noradrenaline and 3H-serotonin, respectively. In cultures from cortex, cerebellum and spinal cord the cell bodies of neurones did not show labelling by the monoamines but many nerve fibres in the outgrowth zone had taken up 3H-noradrenaline and 3H-serotonin. Astrocytes in explant cultures did not take up 3H-noradrenaline and 3H-serotonin whereas astrocytes in primary cultures showed heavy uptake of both monoamines. In contrast, amino acid transmitters such as 3H-GABA and 3H-glutamate were accumulated by astrocytes in explant as well as in primary cultures. Uptake of both 3H-noradrenaline and 3H-serotonin by neurones and astrocytes was considerably reduced or inhibited in Na+-free incubation medium or at low temperature, suggesting an active uptake mechanism. Addition of the antidepressants maprotiline and (+)oxaprotiline inhibited the uptake of 3H-noradrenaline by neuronal cell bodies and fibres in explant cultures and by astrocytes in primary cultures. The uptake of 3H-serotonin by neurones and astrocytes was blocked by citalopram and paroxetine. Our studies demonstrate that astrocytes in primary cultures are able to actively take up 3H-noradrenaline and 3H-serotonin whereas there was no uptake of monoamines into astrocytes in explant cultures, suggesting that there is a difference between astrocytes in different culture systems (explant cultures vs primary cultures) with respect to the uptake of monoamine transmitters.

Coexistence of cholinergic and somatostatin receptors on astrocytes of rat CNS.

Electrophysiological studies have shown that somatostatin (SOM; 10(-8) and 10(-7) M) causes a hyperpolarization of the majority of astrocytes in explant cultures of rat spinal cord and cortex. When SOM and the cholinergic agonists muscarine and nicotine (10(-6) M) were tested on the same cell, all three compounds produced hyperpolarizations, suggesting a colocalization of functional cholinergic and SOM receptors on the glial membrane. Combined immunohistochemical and autoradiographic binding studies demonstrating that almost all astrocytes which were immunostained by the monoclonal muscarinic or nicotinic antibodies were also intensely labelled by 125I-SOM, provide further evidence for the coexistence of cholinergic and SOM receptors on astrocytes.

Binding of cholecystokinin, bombesin and muscarine to neurons and astrocytes in explant cultures of rat central nervous system: Autoradiographic and immunohistochemical studies

The cellular localization of binding sites for the gastrointestinal peptides [ 3H]cholecystokinin and [ 125Ibombesin as well as the cholecystokinin B-antagonist [ 3H]L-365,260 was investigated in expiant cultures of rat cortex, cerebellum, brainstem and spinal cord using autoradiographic techniques. Many neurons in cortical, brainstem and spinal cord cultures revealed intense labelling of the radioligands whereas cerebellar neurons showed only little binding. In addition to neurons, binding sites for these peptides were also observed on astrocytes. Labelling of glial cells in cerebellar cultures was usually weaker than in the other CNS areas studied, suggesting a certain specialization of astrocytes in various brain regions. By means of combined immunohistochemical and autoradiographic techniques it was demonstrated that many neurons and astrocytes which expressed binding sites for [ 3H]cholecystokinin, [ 3H]L-365,260 and [ 125I]bombesin were also immunostained by the monoclonal muscarinic receptor antibody M 35 providing evidence for a co-localization of peptidergic and cholinergic receptors on the membrane of these cells. Our autoradiographic findings suggesting the presence of receptors for cholecystokinin and bombesin on astrocytes are supported by electrophysiological studies demonstrating that both peptides induce a hyperpolarization of glial cells.

Electrophysiological evidence for the presence of receptors for cholecystokinin and bombesin on cultured astrocytes of rat central nervous system

The action of cholecystokinin (CCK) and bombesin (Bom) was studied on the membrane potential of astrocytes in explant cultures of rat cortex, cerebellum, brain stem and spinal cord. Both peptides (10 −8 and 10 −7 M) caused a hyperpolarization of most astrocytes studied. The hyperpolarization by CCK was markedly reduced or blocked by the CCK B-antagonist L-365,260 whereas addition of the Bom-antagonist [ d-Phe 12,Leu 14]-Bom antagonized the effects of Bom, suggesting a specific action of these peptides. When CCK and Bom were tested on the same cell, both peptides were effective, indicating a colocalization of receptors for CCK and Bom on the glial membrane. Our electrophysiological investigations provide strong evidence for the existence of functional CCK and Bom receptors on astrocytes.

Colocalization of cholinergic, adrenergic and peptidergic receptors on astrocytes

By means of combined immunohistochemical and auto-radiographic techniques we have studied the colocalization of cholinergic, adrenergic and peptidergic binding sites on astrocytes in explant cultures of rat spinal cord, brain stem and cerebellum. Many astrocytes which were immunostained by the monoclonal muscarinic receptor antibody M 35 were also intensely labelled by 3H-noradrenaline, the beta-adrenergic antagonist 3H-dihydroalprenolol and the peptides 125I-angiotensin II, 125I-neuropeptide Y and 3H-bradykinin. Electrophysiological studies demonstrating that muscarine, the adrenergic agonists noradrenaline and isoprenaline as well as angiotensin II, neuropeptide Y and bradykinin affect the membrane potential of the same astrocytes provide further evidence for the coexistence of cholinergic, adrenergic and peptidergic receptors on astrocytes.

Reactive astrocytes in explant cultures of goal scars derived from lesioned rat optic nerve: An ultrastructural study

Explant cultures of glial scars generated by surgical removal of the retina in 3–60-day-old rats were used to determine if reactive astrocytes survive in vitro and how closely reactive astrocytes in culture resemble their in vivo counterparts. Characterization of the composition of age matched glial scars in vivo and in vitro showed that reactive astrocytes survived in glial scar explants even after several weeks in culture. Reactive astrocytes in both neonatal and adult glial scars retained ultrastructural features characteristic of reactive astrocytes in vivo. However, fewer reactive astrocytes survived in culture when explants were prepared from adult rat glial scars. The results of this study demonstrate that tissue culture is a viable model for the study of reactive astrocytes. A critical factor in the survival of reactive astrocytes in culture was the complete removal of myelin debris prior to the establishment of the culture. This outcome suggests that it will be important to clarify why myelin debris persists in culture and how it affects the survival of reactive astrocytes.

Colocalization of receptors for vasoactive peptides on astrocytes of cultured rat spinal cord and brain stem: electrophysiological effects of atrial and brain natriuretic peptide, neuropeptide Y and bradykinin

Electrophysiological studies have been made of the actions of the vasoactive peptides atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), neuropeptide Y (NPY) and bradykinin (BK) on astrocytes in explant cultures of rat brain stem and spinal cord. Addition of ANP, BNP and NPY to the bathing solution at concentrations of 10 −8 and 10 −7 M caused depolarizations of the majority of astrocytes studied, some cells were hyperpolarized and on a small number of cells the peptides had no action. BK (10 −8 M) only induced depolarizations of almost all astrocytes studied. Our electrophysiological data provide strong evidence for the presence of receptors for these vasoactive peptides on astrocytes. When ANP, BNP, NPY and BK were tested on the same cell, all peptides were effective, suggesting a coexistence of peptidergic receptors on astrocytes.

Electrophysiological evidence for the existence of receptors for endothelin and vasopressin on cultured astrocytes of rat spinal cord and brainstem

By means of intracellular recordings we have studied the actions of the vasoactive peptides endothelin-1 and -3 (ET-1, ET-3) and arginine vasopressin (AVP) on the membrane potential of astrocytes in explant cultures of rat spinal cord and brainstem. Addition of ET-1, ET-3 or AVP to the bathing solution at concentrations of 10 −8–10 −6 M depolarized the membrane of the majority of glial cells studied. The AVP antagonist d(CH 2) 5 1, Tyr(Me) 2,Arg 8-vasopressin reversibly blocked the depolarizations by AVP. Our electrophysiological findings together with autoradiographic binding studies strongly suggest the existence of ET and AVP receptors on astrocytes.

Action of baclofen, GABA and antagonists on the membrane potential of cultured astrocytes of rat spinal cord

The action of γ-aminobutyric acid A (GABA A) and B (GABA B)-agonists has been studied on the membrane potential of astrocytes in explant cultures of rat spinal cord by means of intracellular microelectrode recordings. The GABA B-agonists (−)-baclofen and CGP 27 492 (3-aminopropyl phosphonous acid; 10 −6 to 10 −4 M) caused a hyperpolarization of the majority of astrocytes studied. On approximately 25% of the cells, the compounds had no effect. The hyperpolarization by baclofen (10 −4 M) was reversibly antagonized by the GABA B-antagonist 5-hydroxysaclofen (10 −4 M). GABA and the GABA A-agonist muscimol (10 −4 and 10 −3 M) depolarized approximately two thirds of the glial cells tested, whereas the remaining third remained unaffected. The GABA A-antagonist bicuculline (10 −4 and 10 −3 M) only reduced the depolarization by GABA (10 −4 M) but did not completely block it. On half of the cells tested, the depolarization by GABA was not affected by bicuculline, suggesting that the glial GABA A-receptor is different from the neuronal GABA A-receptor. Our electrophysiological investigations together with recent autoradiographic binding studies strongly suggest the existence of GABA B-receptors on astrocytes whereas there is less evidence for GABA A-sites on these cells.

Electrophysiological evidence for receptors for vasoactive intestinal peptide and angiotensin II on astrocytes of cultured rat central nervous system

Electrophysiological studies have been made of the actions of vasoactive intestinal peptide (VIP) and angiotensin II (Ang II) on astrocytes in explant cultures of rat brain stem and spinal cord. Addition of VIP to the bathing solution at concentrations of 10 −8 to 10 −6 M caused hyperpolarizations. Ang II at the same concentrations depolarized the majority of astrocytes tested. At 10 −9 M, both peptides had no action or only small effects. In approximately one third of the cells, the depolarizations by Ang II were accompanied by rhythmic oscillations. The Ang II antagonist saralasin reversibly blocked the depolarizations by Ang II. These findings, together with autoradiographic binding studies from our laboratory strongly suggest the existence of VIP and Ang II receptors on astrocytes.