Jeffrey G. Netzeband

The chemokine CCL2 modulates Ca2+ dynamics and electrophysiological properties of cultured cerebellar Purkinje neurons.

The chemokine CCL2 is produced at high levels in the central nervous system (CNS) during infection, injury, neuroinflammation and other pathological conditions. Cells of the CNS including neurons and glia express receptors for CCL2 and these receptors may contribute to a signaling system through which pathologic conditions in the CNS are communicated. However, our understanding of the consequences of activation of chemokine signaling in the CNS is limited, especially for neurons. In many cell types, chemokine signaling alters intracellular Ca2+ dynamics. Therefore, we investigated the potential involvement of this mechanism in neuronal signaling activated by CCL2. In addition, we examined the effects of CCL2 on neuronal excitability. The studies focused on the rat cerebellar Purkinje neuron, an identified CNS neuronal type reported to express both CCL2 and its receptor, CCR2. Immunohistochemical studies of Purkinje neurons in situ confirmed that they express CCR2 and CCL2. The effect of exogenous application on Purkinje neurons was studied in a cerebellar culture preparation. CCL2 was tested by micropressure or bath application, at high concentrations (13–100 nm) to simulate conditions during a pathologic state. Results show that Purkinje neurons express receptors for CCL2 and that activation of these receptors alters several neuronal properties. CCL2 increased resting Ca2+ levels, enhanced the Ca2+ response evoked by activation of metabotropic glutamate receptor 1 and depressed action potential generation in the cultured Purkinje neurons. Passive membrane properties were unaltered. These modulatory effects of CCL2 on neuronal properties are likely to contribute to the altered CNS function associated with CNS disease and injury.

Chronic interleukin‐6 exposure alters metabotropic glutamate receptor‐activated calcium signalling in cerebellar Purkinje neurons

Chronic central nervous system expression of the cytokine interleukin‐6 (IL‐6) is thought to contribute to the histopathological, pathophysiological, and cognitive deficits associated with various neurological disorders. However, the effects of chronic IL‐6 expression on neuronal function are largely unknown. Previous studies have shown that chronic IL‐6 exposure alters intrinsic electrophysiological properties and intracellular Ca2+ signalling evoked by ionotropic glutamate receptor activation in cerebellar Purkinje neurons. In the current study, using primary cultures of rat cerebellum, we investigated the effects of chronic IL‐6 exposure on metabotropic glutamate receptor (mGluR)‐activated Ca2+ signalling and release from intracellular Ca2+ stores. Chronic exposure (6–10 days) of Purkinje neurons to 500 units/mL IL‐6 resulted in elevated resting Ca2+ levels and increased intracellular Ca2+ signals evoked by the group I mGluR agonist (S)‐3,5‐dihydroxyphenylglycine (DHPG) compared to untreated control neurons. Chronic IL‐6 treatment also augmented Ca2+ signals evoked by brief 100 mm K+ depolarization, although to a lesser degree than responses evoked by DHPG. Depleting intracellular Ca2+ stores with sarcoplasmic‐endoplasmic reticulum ATPase inhibitors (thapsigargin or cyclopiazonic acid) or blocking ryanodine receptor‐dependent release from intracellular stores (using ryanodine) resulted in a greater reduction of DHPG‐ and K+‐evoked Ca2+ signals in chronic IL‐6‐treated neurons than in control neurons. The present data show that chronic exposure to elevated levels of IL‐6, such as occurs in various neurological diseases, alters Ca2+ signalling involving release from intracellular stores. The results support the hypothesis that chronic IL‐6 exposure disrupts neuronal function and thereby may contribute to the pathophysiology associated with many neurological diseases.

Chronic ethanol exposure enhances AMPA-elicited Ca2+ signals in the somatic and dendritic regions of cerebellar Purkinje neurons

Intracellular Ca2+ signals produced by the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA; 5 microM) were measured in the somatic and dendritic regions of cerebellar Purkinje neurons in mature cerebellar control cultures (> or = 20 days in vitro) and cultures chronically treated with 32 mM ethanol (146 mg%; 8-11 days). Recordings were made in physiological saline without ethanol. The mean peak amplitude of the Ca2+ signal elicited by AMPA (applied by brief 1-s microperfusion) in the somatic region was enhanced 38% in chronic ethanol-treated Purkinje neurons compared with control neurons. In contrast, Ca2+ signals evoked by AMPA in the dendritic region were similar in magnitude between control and chronic ethanol-treated Purkinje neurons. When tetrodotoxin (TTX; 500 nM) was included in the bath saline to block spike activity and synaptically-generated events, the mean peak amplitude of the Ca2+ signal elicited by AMPA was enhanced 60% in both the somatic and dendritic regions of chronic ethanol-treated Purkinje neurons compared with control neurons. Thus, TTX-sensitive mechanisms (i.e., spike or synaptic activity) appear to play a role in normalizing neuronal functions involved in Ca2+ signaling in the chronic ethanol-treated neurons. In parallel current clamp experiments, the resting membrane potential of chronic ethanol-treated neurons was slightly depolarized compared with control neurons. However, no differences were found between control and chronic ethanol-treated Purkinje neurons in input resistance or the peak amplitude or duration of the depolarizations or hyperpolarizations elicited by AMPA. AMPA receptors mediate fast excitatory neurotransmission in the majority of neurons in the central nervous system (CNS) and Ca2+ signals in response to AMPA receptor activation contribute to synaptic function. Thus, our results suggest that modulation of Ca2+ signals to AMPA receptor activation (or other cellular inputs) may provide an important mechanism contributing to the actions of prolonged ethanol exposure in the CNS.

Modulatory effects of acute ethanol on metabotropic glutamate responses in cultured Purkinje neurons

Ethanol has been shown to affect several transmitter- and voltage-gated channels in the brain, although little attention has focused on potential interactions between ethanol and metabotropic glutamate receptors (mGluRs). This is of interest as mGluRs are now recognized to be important components of synaptically mediated responses, including short- and long-term changes in the efficacy of neurotransmission. Cerebellar Purkinje neurons are sensitive to the effects of ethanol and express high levels of mGluRs. We made extracellular recordings from cerebellar Purkinje neurons at 21-37 days in culture to examine the effect of ethanol on mGluR-mediated responses. mGluRs were activated by pressure ejection of 300 microM (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), a selective agonist of mGluRs, or 5 microM quisqualate (Quis). As Quis activates both ionotropic and metabotropic glutamate receptors, 50 microM 6,7-dinitroquinoxaline-2,3-dione (DNQX) was used to block the ionotropic component of Quis-mediated responses. Both ACPD and Quis produced biphasic changes in firing rates consisting of an initial brief excitatory phase (5-20 s) followed by a prolonged inhibitory phase (10 s to 2.5 min), and induced the generation of bursts. Addition of 33 mM (150 mg%) ethanol to the recording medium had little effect on ACPD-mediated responses. In the presence of 66 mM (300 mg%) ethanol, however, ACPD-mediated responses exhibited an increase in the total response duration, with no change in the percent excitation or the induction of bursts as compared to controls. On the other hand, 66 mM ethanol decreased Quis-induced burst activity, while having no effect on the percent excitation or the total response duration.(ABSTRACT TRUNCATED AT 250 WORDS)

Somatic Ca2+ signaling in cerebellar Purkinje neurons

Activity‐driven Ca2+ signaling plays an important role in a number of neuronal functions, including neuronal growth, differentiation, and plasticity. Both cytosolic and nuclear Ca2+ has been implicated in these functions. In the current study, we investigated membrane‐to‐nucleus Ca2+ signaling in cerebellar Purkinje neurons in culture to gain insight into the pathways and mechanisms that can initiate nuclear Ca2+ signaling in this neuronal type. Purkinje neurons are known to express an abundance of Ca2+ signaling molecules such as voltage‐gated Ca2+ channels, ryanodine receptors, and IP3 receptors. Results show that membrane depolarization evoked by brief stimulation with K+ saline elicits a prominent Ca2+ signal in the cytosol and nucleus of the Purkinje neurons. Ca2+ influx through P/Q‐ and L‐type voltage‐gated Ca2+ channels and Ca2+‐induced Ca2+ release (CICR) from intracellular stores contributed to the Ca2+ signal, which spread from the plasma membrane to the nucleus. At strong K+ stimulations, the amplitude of the nuclear Ca2+ signal exceeded that of the cytosolic Ca2+ signal, suggesting the involvement of a nuclear amplification mechanism and/or differences in Ca2+ buffering in these two cellular compartments. An enhanced nuclear Ca2+ signal was more prominent for Ca2+ signals elicited by membrane depolarization than for Ca2+ signals elicited by activation of the metabotropic glutamate receptor pathway (mGluR1), which is linked to Ca2+ release from intracellular stores controlled by the IP3 receptor.

Contribution of L-type channels to Ca2+ regulation of neuronal properties in early developing purkinje neurons.

Activity driven Ca2+ signaling is an important regulator of neuronal development. Early developing Purkinje neurons (postnatal day 5–7) prior to the stage of dendritic development express a somatic Ca2+ signaling pathway that is electrically driven and communicates information from the cell membrane to the cytosol and nucleus. In the current studies, we examined the properties and potential functional role of this pathway using acutely isolated Purkinje neurons from postnatal day 5–7 rat pups and brief K+ stimulation to activate the pathway. Results show that the amplitude of the nuclear Ca2+ 1signal increases as a function of the cytosolic Ca2+ signal but is larger than the cytosolic Ca2+ signal at strong K+ stimulations. Both L-type and P-type Ca2+ channels contribute to the Ca2+ signal. We also show using semiquantitative immunohistochemical methods that activation of this Ca2+ signaling pathway results in activation the transcription factor CREB and that L-type Ca2+ channels play a prominent role in this effect. The level of cfos, a transcription factor whose expression is regulated by CREB, was also increased by K+ stimulation. K+ stimulation also altered the level of the Ca2+ binding protein calbindin, an effect that involved L-type Ca2+ channels. The relationship between increases in Ca2+ and calbindin expression was bell-shaped, with high levels of Ca2+ decreasing calbindin expression. The level of the transmitter GABA was also increased by K+ stimulation but this effect was not dependent on L-type Ca2+ channels. Taken together, these results support a role for L-type channels in the phenotypic expression of Purkinje neuron properties during early development and suggest that the different activity patterns of early developing Purkinje neurons could be one mechanism for signaling the induction of specific genes through differences in cytosolic or nuclear Ca2+.

Chronic ethanol treatment alters AMPA-induced calcium signals in developing Purkinje neurons

Cerebellar Purkinje neurons developing in culture were treated chronically with 30 mM (140 mg%; 3-11 days in vitro) ethanol to study the actions of prolonged ethanol exposure on responses to exogenous application of AMPA, a selective agonist at the AMPA subtype of ionotropic glutamate receptors. There was no consistent difference between control and chronic ethanol-treated neurons in resting membrane potential, input resistance, or the amplitude or duration of the membrane responses to AMPA (1 or 5 microM applied by brief microperfusion) as measured using the nystatin patch method of whole cell recording. In additional studies, the Ca2+ signal to AMPA was examined using the Ca2+ sensitive dye fura-2. The mean peak Ca2+ signal elicited by 5 microM AMPA was enhanced in the dendritic region (but not the somatic region) of chronic ethanol-treated Purkinje neurons compared to control neurons. In contrast, there was no difference between control and chronic ethanol-treated neurons in the peak amplitude of the Ca2+ signal to 1 microM AMPA, whereas the recovery of the Ca2+ signals was more rapid in both somatic and dendritic regions of ethanol-treated neurons. Resting Ca2+ levels in the somatic and dendritic regions were similar between control and ethanol-treated neurons. These data show that the membrane and Ca2+ responses to AMPA in Purkinje neurons are differentially affected by prolonged ethanol exposure during development. Moreover, chronic ethanol exposure produces a selective enhancement of AMPA-evoked dendritic Ca2+ signals under conditions reflecting intense activation (i.e., 5 microM AMPA), whereas both somatic and dendritic Ca2+ signals are attenuated with smaller levels of activation (i.e., 1 microM AMPA). Because Ca2+ is an important regulator of numerous intracellular functions, chronic ethanol exposure during development could produce widespread changes in the development and function of the cerebellum.

mGluR1 agonists elicit a Ca2+ signal and membrane hyperpolarization mediated by apamin‐sensitive potassium channels in immature rat purkinje neurons

The type 1 metabotropic glutamate receptor (mGluR1) plays an import role in the synaptic physiology and development of cerebellar Purkinje neurons. mGluR1 expression occurs early in the developmental program of Purkinje neurons, at an age that precedes expression of the dendritic structure. Few studies have investigated the physiological response produced by mGluR1 activation in early‐developing Purkinje neurons. To address this question, simultaneous recording of membrane potential and intracellular Ca2+ was performed in immature cultured Purkinje neurons coupled with exogenous application of mGluR1 agonists. Membrane potential was measured using the perforated patch method of whole‐cell recording, and intracellular Ca2+ was measured using fura‐2‐based Ca2+ imaging. Brief, 1‐sec micropressure application of the group I mGluR‐selective agonist (S)‐3,5‐dihydroxyphenylglycine (DHPG) evoked a prominent Ca2+ signal and coincident fast hyperpolarization in the immature neurons. The mGluR1‐selective antagonist 7‐(hydroxyimino)cyclopropa[b]chromen‐1a‐carboxylate ethyl ester blocked the Ca2+ signal and fast hyperpolarization, confirming the involvement of mGluR1s. Amplitude of the fast hyperpolarization varied as a function of membrane potential and intracellular Ca2+ and was blocked by apamin, an antagonist of the small‐conductance Ca2+‐activated K+ channel (SK), identifying this K+ channel as an underlying mechanism. In similar experiments with mature cultured Purkinje neurons, DHPG elicited a Ca2+ signal, but fast membrane hyperpolarization was not evident. These results suggest that mGluR1 activation and the resulting release of Ca2+ from intracellular stores and activation of SK channels may be a mechanism through which mGluR1 can modulate neuronal excitability of Purkinje neurons during early development.

Cannabinoids and Interleukin-6 Enhance the Response to Nmda in Developing CNS Neurons

Maternal cannabinoid use during pregnancy is harmful to the central nervous system (CNS) of the developing infant12. Cannabinoids pass easily from the mother to the fetus, where they can access neuronal receptors in the developing CNS. Cannabinoid receptors are known to be expressed by CNS neurons, although their role in normal brain functions has yet to be defined. It is likely that these receptors mediate some form of intercellular signaling, since biochemicals endogenous to the CNS (e.g., anandamide)5 have been shown to act at CNS cannabinoid receptors.4 Cannabinoid receptors are expressed during the main period of morphological and physiological development of the CNS,11 a correlation that may reflect a role in the developmental process. Hyperactivation of these receptors by maternal cannabinoid use could lead to a disruption of the normal signaling pattern and, consequently, altered development and permanent changes in CNS function.

L-type Ca2+ channels contribute to current-evoked spike firing and associated Ca2+ signals in cerebellar Purkinje neurons.

The physiological properties of Purkinje neurons play a central role in their ability to regulate information transfer through the cerebellum. A number of ion channels contribute to Purkinje neuron physiology including an abundance of P-type Ca2+ channels, particularly in the dendritic region. Purkinje neurons also express L-type Ca2+ channels both during development and in the mature state. However, a role for L-type channels in Purkinje neuron physiology has yet to be fully defined. In the current study we used physiological recordings from cultured Purkinje neurons and the L-type Ca2+ channel agonist S-(2)-Bay K to assess a potential role for L-type Ca2+ channels in spike firing. Results show that Bay K alters current-evoked spike firing in young, immature Purkinje neurons without dendritic structure and in older, more mature Purkinje neurons with dendritic structure. Bay K also enhanced Ca2+ signals associated with the current-evoked spike firing. The effect of Bay K was more prominent in the young Purkinje neurons than in the older Purkinje neurons, suggesting that L-type Ca2+ channels may be more important in the Purkinje neuron physiology during the early stages of development rather than at mature stages. In the older Purkinje neurons, immunohistochemical studies using antibodies to L-type Ca2+ channels showed more intense immunolabeling in the somatic region than in the dendritic region. This result suggests that L-type Ca2+ channels may play a more important role in somatic physiology than dendritic physiology, whereas P-type channels may play a more important role in dendritic physiology.