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Astrocytes, spontaneity, and the developing thalamus
Journal of Physiology - Paris 96 (2002) 221–230 www.elsevier.com/locate/jphysparis
Astrocytes, spontaneity, and the developing thalamus H. Rheinallt Parri, Vincenzo Crunelli* School of Biosciences, Cardiff University, Museum Avenue, PO Box 911, Cardiff CF10 3US, Wales, UK
Abstract Recent studies in the ventrobasal (VB) thalamus have shown that astrocytes display spontaneous intracellular calcium [Ca2+]i oscillations early postnatally. [Ca2+]i oscillations are correlated in groups of up to five astrocytes, and propagate between cells. NMDA receptor-mediated, long lasting inward currents in thalamocortical (TC) neurons of the VB complex are correlated to [Ca2+]i increases in neighbouring astrocytes, and stimulation of astrocytic [Ca2+]i increases also lead to inward currents in neurons. These findings suggest that astrocytes are spontaneously active and can induce neuronal activity, a reversal of the previously held view of neuron–glia interactions in the central nervous system. This activity occurs at an important period in the development of the thalamus and therefore suggests a potential functional role in a variety of processes. Along with data on the neurotransmitter receptor repertoire of thalamic astrocytes these findings enlarge the body of knowledge on astrocytes in the thalamus, and further contribute to the emerging field of astrocyte–neuron and neuron–astrocyte interactions in the central nervous system. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Ventrobasal thalamus; Oscillations; Development
1. Introduction The acknowledged roles of glia in general, and of astrocytes in particular, in the functioning of the nervous system have increased dramatically in recent years. This increased consciousness of the physiological significance of astrocytes has seen them emerge from a stage where they were thought to be supportive housekeeping cells that express certain neurotransmitter receptors and channels, to a stage where they have assumed functions traditionally exclusively assigned to neurons, such as glutamate release [39]. From adopting some of the roles of neurons and being intimately associated with the synapse and synaptic transmission, it is now also known that in some adult brain areas, astrocytes actually give rise to new neurons [49]. A recent focus of astrocytic research has been the ways that astrocytes react to, and interact with, synaptic transmission. Astrocytes express many of the same neurotransmitter receptors as neurons and react to these neurotransmitters with increases in [Ca2+]i. Following such activation, astrocytes have been shown to modulate synaptic transmission [4,28].
A further step in the interaction of neurons and glia, however, has been seen in the developing thalamus, where astrocytes display spontaneous [Ca2+]i oscillations which are not dependent on previous neuronal activity [43]. These oscillations are correlated to NMDA receptor mediated currents recorded in neighbouring neurons, and stimulation of astrocytic [Ca2+]i increases also lead to inward currents in neighbouring neurons [43], indicating that activity arising in astrocytes can induce activity in neurons. These findings extend the repertoire of potential astrocytic functions and again assign a role to astrocytes previously reserved for neurons. Here, we will place these recent findings in the context of the developing thalamus. We first discuss spontaneous activity in the developing CNS, the evidence for astrocyte-neuron signalling and how this fits into the scheme of the developing thalamus. We will then review evidence on the neurotransmitter receptors that are present on VB astrocytes and discuss these potential astrocyte-neuron interactions.
2. Spontaneous activity in the CNS 2.1. Spontaneous neuronal activity during development
* Corresponding author. Tel.: +44-29-20874801; fax: +44-292087986. E-mail address: [email protected] (V. Crunelli).
Spontaneous activity is a key feature of the developing nervous system. Neuronal precursor cells exhibit
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spontaneous [Ca2+]i fluctuations in the ventricular zone of the neocortex [38]. This intracellular store-dependent activity persists until the cells migrate out into the other cortical layers. Prior to synapse formation in the spinal cord, neurons also display a variety of spontaneous [Ca2+]i oscillatory patterns [51], where calcium spikes occur at a frequency of 1–10/hour and involve voltagegated calcium channels which trigger calcium induced calcium release (CICR) from intracellular stores [21]. This activity has been implicated in the frequency dependent regulation of neurotransmitter-related gene transcription. [Ca2+]i transients seen in growth cones of the same neurons also involve CICR in their generation and regulate the rate of outgrowth of growth cones in a frequency-dependent manner [21]. Following synapse formation, spontaneous activity becomes important in the development of neural circuits and in the refinement of neuronal connections, and at this time spontaneous synchronous activity is seen in large groups of neurons. The retina, for example, exhibits spontaneous intercellular waves which propagate over large distances. These propagating waves seem dependent on action potential generation since they are blocked by tetrodotoxin (TTX) [56]. The generation of these waves is important in the development of cortical ocular dominance columns, since blocking the retinal waves with TTX affects the segregation of retinogeniculate afferents in the dorsal lateral geniculate nucleus (LGN) of the thalamus [50]. Another form of synchronous spontaneous activity occurring in neuronal groups has been described in developing neocortex. Here groups or ‘‘domains’’ of neurons can be identified on the basis that they exhibit coordinated [Ca2+]i increases [58]. This coordination is dependent on gap junctional coupling [27], and presents the possibility that electrical excitability and also metabolic factors and second messengers are distributed within cells in the same domain in ways which affect the developing cortical architecture and the plasticity of afferent synaptic inputs. Neuronal gap junctional communication occurs in many parts of the CNS during development and domains have also been reported to exist in many brain areas including, reportedly, in the thalamus [59]. 2.2. Spontaneous astrocytic activity Although radial glia are involved in CNS development, and have been extensively investigated, the description of spontaneous [Ca2+]i oscillations in glial cells during development has only been made recently [43]. Astrocytes in the VB thalamus of the rat were found to display spontaneous oscillations in [Ca2+]i in acute slices taken from neonatal rats (Fig. 1). The oscillations were seen between the ages of postnatal day 5 (P5) and P17, decreasing in frequency of occurrence with age, so suggesting a developmental expression.
Fig. 1. Spontaneous [Ca2+]i oscillations in VB astrocytes. A. Fluorescence image of a region of an acutely isolated VB slice, ringed areas a–d denote the positions of astrocytes that displayed spontaneous [Ca2+]i oscillations. Image was taken at 260 s after beginning of recording. B. Plots of F% over time for the astrocytes indicated in A. Astrocyte a displays increases that are independent of astrocytes b– d and also a transient that is correlated with the other astrocytes. (Reproduced from [43]).
Oscillations were observed in astrocytes throughout the VB slice and could be manifested as single transients or multiple [Ca2+]i fluctuations occurring during the 10 min recording period. The mode of the inter-oscillation duration distribution was 60–70 s [42] and interestingly, a subset (4%) of spontaneously active astrocytes, termed ‘‘pacemaker’’ astrocytes, displayed long lasting regular oscillations with a periodicity also of around 60 s. Oscillations were not dependent on neuronal activity since they were not blocked by TTX, and were not blocked by antagonists to the main thalamic neurotransmitters, ie glutamate and GABA, nor by suramin. Spontaneous astrocytic [Ca2+]i increases have been described previously in culture. Cortical astrocytes displayed spontaneous [Ca2+]i increases which propagated in confluent cultures. These oscillations were not dependent on extracellular calcium and differed from increases seen following agonist application [15]. In cultured suprachiasmatic nucleus astrocytes, cocultured with neurons, astrocytic [Ca2+]i oscillations were seen that had periodicities ranging from 7–20s, and were not blocked by TTX [53]. Another
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study indicating a potential pathological role for spontaneous astrocytic oscillations was reported in human cortical astrocytes cultured from a patient with intractable childhood epilepsy [30]. The oscillations in the latter study (ref. [30]) however had much slower kinetics than reported in other studies [15,53] with the mean time from initial elevation to return to baseline being about 5 min. The [Ca2+]i oscillations were also dependent on the presence of extracellular calcium. 2.3. Mechanism of spontaneous astrocytic [Ca2+]i oscillations Spontaneous oscillations in VB astrocytes were blocked by thapsigargin and cyclopiazonic acid, showing that the observed increases were due to [Ca2+]i release from intracellular stores, and removal of extracellular Ca2+ did not block the oscillations [43]. The L-type calcium channel antagonist nimodipine reduced the number of oscillations suggesting a role for Ca2+ entry via dihydropyridine sensitive channels in sustaining the oscillations. In line with other studies on calcium oscillations, it may be that inositol-1,4,5-trisphosphate (IP3) receptor activation is required to release [Ca2+]i from the astrocytic stores. This in turn would suggest a phospholipase C (PLC) coupled mechanism. The spontaneous nature of the activity, and the regularity of observed pacemaker oscillations could arise from long lasting action of an extracellular agonist or constitutive activation of PLC. This could perhaps be due to an inherent property of the membrane receptor or enzyme in the cascade, or even due to the presence of an as yet unknown agonist in the extracellular milieu at this time of development. Spontaneous activity seen in cultures of isolated astrocytes [15] suggests that astrocytes have an intrinsic ability to display [Ca2+]i oscillations without the presence of another cell type. However, even these types of studies do not rule out the possibility of a secreted agonist or factor activating surface receptors to induce oscillations. Bezzi and coworkers [5] recently showed a chemokine-prostaglandin pathway that evoked glutamate release from astrocytes. The pathway was suggested to be active physiologically, and also activated by a viral agent in pathological conditions. It is possible that such compounds or growth factors could effect astrocytic activity during development. Spontaneous activity therefore seems to be a property of astrocytes as well as of neurons in the CNS. In accordance with the situation of spontaneous neuronal activity during development, the findings in the VB thalamus also indicates a developmental role for spontaneous astrocytic activity: this is also supported by the fact that spontaneous astrocyte activity seen in culture studies [15] are from astrocytes cultured early postnatally.
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3. Astrocyte–astrocyte and astrocyte–neuron signalling Groups of astrocytes show coordinated increases in [Ca2+]i and [Ca2+]i waves are seen to propagate between astrocytes in the VB thalamus. Although propagating [Ca2+]i waves are a defining feature of astrocytes in culture, this developmental astrocytic thalamic wave is the first demonstration of spontaneous astrocytic [Ca2+]i propagation in situ in the CNS [43]. A large amount of work has been conducted under culture conditions on the propagation of astrocytic [Ca2+]i waves via gap junctions [18], probably via diffusion of IP3 [48]. Chemical transmission has however also been demonstrated, the two main candidates for this form of transmission being ATP and glutamate. In cultured cortical astrocytes, [Ca2+]i waves that propagated between physically isolated astrocytes [8], or were induced by stimulated astrocyte conditioned media [22], were blocked by the purinergic antagonist suramin or treatment with the ATP degrading enzyme apyrase, indicating transmission by ATP. In the retina, [Ca2+]i waves propagate between astrocytes and muller cells. Propagation between astrocytes seems mainly via gap junctions while propagation to, and between muller cells is blocked by apyrase treatment or suramin [36]. Glutamate has been shown to be released from astrocytes following stimulation which result in [Ca2+]i increases [39], and waves of glutamate release, concurrent with propagating [Ca2+]i waves in cultured astrocytes have been imaged [24]. Since glutamate seems to be the most abundant neurotransmitter in the CNS, much of the effort on exploring potential astrocyteneuron signalling has focused on the role that astrocytereleased glutamate may play. The release of glutamate from astrocytes is now well established [4,5,39]. Flash photolysis studies used to increase astrocytic [Ca2+]i levels in astrocyte -neuron cocultures show that raising astrocytic [Ca2+]i within physiological levels results in glutamate release and the generation of neuronal inward currents [40]. Other studies show the presence of the vesicle associated SNARE protein synaptobrevin in astrocytes [3], the cleavage of which by botulinum B neurotoxin blocks neuronal inward currents induced by astrocyte stimulation. Agonist stimulation also elicits glutamate release [39] and stimulation of metabotropic glutamate receptors (mGluRs) which induces cytosolic [Ca2+]i oscillations [44], also results in glutamate release suggesting that glutamate could act as a wave propagating ‘gliotransmitter’ and a transmitter to neurons. In the developing VB thalamus, adjacent spontaneously active astrocytes exhibit coordinated [Ca2+]i increases, with up to five astrocytes in a restricted area exhibiting coordinated activity (Fig. 2) [43]. Propagating [Ca2+]i waves between astrocytes can also be seen. These propagate within the range of velocities seen in culture studies [17]. The propagation mechanism did not
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Fig. 2. Propagation of spontaneous [Ca2+]i waves between astrocytes. A. Fluorescent image (taken 285 s into experiment shown in B) displaying position of the somas of astrocytes a–c in an acute VB slice. Scale bar 20 mm. B. Plots of fluorescence over time for somas of astrocytes a–c, showing that activity initiates in astrocyte a, then propagates to b and c. (Reproduced from [43]).
seem to involve gap junctions since filling of single astrocytes with Lucifer yellow or biocytin via patch pipettes failed to fill neighbouring astrocytes: this is consistent with observations in rat visual cortex where dye coupling between astrocytes is not seen until P11 [6]. The mechanism of spontaneous activity propagation would therefore seem to be chemical. Suramin and broad spectrum ionotropic glutamate antagonists did not reduce the number of astrocytes exhibiting spontaneous activity in the VB thalamus, but experiments were not performed to directly address the identity of a substance mediating inter-astrocytic [Ca2+]i wave propagation. In this study [43], the inference of chemically mediated propagation presented the possibility that as well as propagation between astrocytes a chemical messenger could also be affecting neighbouring neurons. Patch clamp recording revealed large long lasting inward currents in VB thalamocortical (TC) neurons which were correlated to spontaneous activity in neighbouring astrocytes in the slice. The currents were striking in their slow activation and slow decay kinetics (tact 330 ms, tdecay 2.7 s), resulting in inward currents that could last for several seconds. In this respect they are reminiscent of currents elicited in neurons by astrocytic glutamate release in culture studies [40, 44]. Spontaneously recorded currents in VB TC neurons were blocked by the NMDA receptor antagonist APV, indicating that spontaneous astrocytic activity leads to glutamate release and activation of TC neurons via NMDA receptor activation. Responses were seen in the presence of external Mg2+, indicating that at least at this developmental stage, prior TC neuronal depolarisation was
not required to enable the removal of NMDA-R Mg2+ block in order to obtain NMDA-R currents [43]. The simplest explanation for the results observed in the VB thalamus is that astrocytes release glutamate which directly activate neuronal NMDA receptors (Fig. 3A). However, there is no direct evidence so far for [Ca2+]i dependent glutamate release from astrocytes in the VB thalamus, and it is therefore equally possible that astrocytes are acting presynaptically to evoke glutamate release from glutamatergic afferents (Fig. 3B). Such presynaptic effects have been shown in culture and in slice preparations: astrocytic glutamate release has been shown to modulate neurotransmitter release differentially by activating metabotropic glutamate receptors [1] or NMDA [2] receptors presynaptically, and a presynaptic effect of astrocyte released glutamate would also explain the role of astrocytic activation in potentiating inhibitory transmission in the hippocampus [28]. The possibility also exists that the compound released by the astrocyte is not glutamate but another compound. This might be a substance such as ATP that could perhaps act presynaptically, or a glutamate analogue that could act either pre or postsynaptically. Such a feasible candidate is homocysteic acid (HCA). HCA activates both NMDA and non-NMDA ionotropic glutamate receptors with an EC50 of about 100 mM [60], is localized to glial cells and is also released by K+ induced depolarisation from brain slices [12]. Interestingly, in the rat LGN, another thalamic nucleus, HCA immunoreactivity is restricted to astrocytic end feet under normal conditions, while following cortical and retinal lesions HCA immunoreactivity increases in glial cell bodies in the LGN [20].
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Fig. 3. Two possible scenarios for the coupling of astrocytic [Ca2+]i increases and observed inward neuronal currents. A. Spontaneous [Ca2+]i increases in the astrocyte leads to glutamate (Glut.) release which then directly activates postsynaptic NMDA receptors (NMDA-R) on the TC neuron to cause Ca2+ entry and a depolarisation of membrane potential (Vm). B. Astrocytic [Ca2+]i increases lead to the release of a ‘gliotransmitter’ (GT) which activates presynaptic afferents which then release glutamate to activate postsynaptic NMDA receptors on the TC neuron.
4. Development of the thalamus 4.1. Glia The development of the neuronal architecture of the CNS is intimately involved, and dependent on, glial development and differentiation, with radial glial cells providing a scaffold for neuronal cell migration. Following neuronal migration radial glial cells transform into astrocytes, and recent work in the neocortex has shown that radial glial cells also give rise to clonally related groups of neurons [37]. In the thalamus, during prenatal development radial glial cells extend from the ventricular germinative zone to the pial surface [16], and by embryonic day 16 some thalamic nuclei are evident in stained sections. The radial glial cells stain for vimentin, their predominant structural protein. Neuronal staining shows that neurons migrate radially along the fibres, and that GABAergic cells migrate along glial fibres that are perpendicular to the radial glia. Radial glial cell processes are filled with microtubules, vacuoles and clear vesicles, and sometimes vesicles are grouped at large appositions between the processes and migrating neurons. Neuronal migration seems to be over by birth (P0) in the thalamus, and in the first postnatal week vimentin staining decreases markedly and is restricted mainly to some thin processes surrounding blood vessels and immature astrocytes which seem rich in organelles. Vimentin stained astrocytes become further reduced during the first and second postnatal weeks and are virtually absent in the adult thalamus. During the first 2
weeks postnatally, GFAP stained cells become visible in outlining some nuclei though in adult the principal thalamic nuclei also seem devoid of GFAP staining.
5. Neuronal development A discussion of the development of the neurons and connections of the thalamus must be done in the context of the thalamocortical loop. In the rat, the VB thalamus receives somatosensory input, with the arcuate subdivision receiving input from the vibrissae (whiskers). The VB TC neurons then project to the ‘‘barrel cortex’’ where, as indicated by the name, a somatotopic map of the whisker field is reproduced. A somatotopic ‘‘barreloid’’ map also exists in the VB thalamus. Like other thalamic nuclei, the VB complex also receives corticothalamic afferents from its cortical projection target (i.e. barrel cortex), so forming the thalamocortical loop. At birth, before sensory input has shaped the sensory pathway, dye injections in the barrel cortex retrogradely fill cells in the VB barreloids indicating that a rudimentary pathway exists. However, the first 2 weeks postnatally result in major developmental changes in the thalamus and the thalamocortical loop. At P7 there are few synaptic complexes in the VB thalamus but these increase threefold by P12, synapse formation then increases until P15 but after this, the rate of synaptogenesis is attenuated (Fig. 4) [31]. This coincides with a large proliferation in corticothalamic afferents between P8–15 [25].
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Fig. 4. Developmental context of spontaneous astrocytic [Ca2+]i oscillations in the VB. Horizontal bars represent the time courses of the indicated events from P0 to P20. The relative magnitude of the indicated process is expressed by the darkness of the bar at a particular age. Dotted lines in upper bar indicate that the level of spontaneous activity at these ages are unknown.
At this time the neuronal elements of the VB thalamus are also undergoing radical changes. At P7, TC neurons are characterized by short stubby irregular dendrites with distinct growth cones and fillipodia. By P12 there is dendritic elongation with secondary and tertiary branching and the appearance of spinous protrusions [31]. The matching of dendritic elongation and generation of secondary and tertiary branches with the proliferation of thalamocortical afferents agrees with the findings in the LGN that corticothalamic afferents synapse distally while sensory afferents synapse proximally on TC neurons [55]. During this postnatal period, the complement of receptors mediating synaptic transmission on TC neurons also change. Corticothalamic synaptic stimulation at early postnatal ages produces synaptic responses largely due to NMDA receptors, during maturation the duration of postsynaptic responses in the dLGN decreases and the proportion of the postsynaptic response due to AMPA/kainate receptor activation increases [19]. NMDA receptor expression is therefore changing during thalamic development; in this respect the NMDA receptor subtype NR2D displays an interesting developmental profile in the VB thalamus. High levels of NR2D mRNA are seen at birth which are maintained until about P10, while expression decreases between P10 and P21 when it is virtually absent [54]. The properties of NMDA receptors containing the NR2D subunit would seem to endow them for a role where the coordination of associative inputs was less
temporally constrained than for other NMDA-Rs. Receptors containing the NR2D subunit have a weaker Mg2+ block than NMDA receptors of differing subunit composition [34], and a very slow decay following glutamate activation [57], as well as a low EC50 for glutamate. Recombinant NMDA receptors containing NR2D subunit have current decay time constants of 4.4 s following a 1 ms glutamate application. Similar glutamate application on excised patches from extrasynaptic regions of purkinje neurons elicited currents with a decay time constant of 3s [33]. The extrasynaptic location of the receptors is supported by experiments that show that synaptic stimulation of purkinje neurons does not elicit such long lasting NMDA-R currents [33]. NR2D subunits are expressed in a developmentally regulated manner between P0 and P8 in cerebellar purkinje neurons. It would seem therefore that to have a functional role these developmentally expressed extrasynaptic receptors must therefore presumably need an extrasynaptic source of glutamate for activation.
6. Neurotransmission and thalamic astrocytes Although the central theme of this review is the spontaneous activity of thalamic astrocytes and their possible roles in a developmental context, astrocytes would also seem to have a role in the adult physiological functioning of the thalamus.
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Astrocytes express a vast array of neurotransmitter and neuromodulator receptors, and some of these are known to be activated during synaptic transmission [10,45]. The receptors present on thalamic astrocytes therefore indicate the further potential for communication between neurons and glia in the thalamus. Since thalamic nuclei not only receive sensory and cortical glutamatergic inputs but also gabaergic, cholinergic, serotonergic, adrenergic and histaminergic from within the CNS, the potential for astrocytic activation by synaptic activation is very large. Thalamic astrocytes in the rat express the AMPA receptor GluR1 subunit [52], while in monkey thalamus high expression of the NMDA subunit NR2B is seen in astrocytes in the LGN [26]. As mentioned, mGluRs are widely expressed in astrocytes, and addition of the mGluR agonist trans-ACPD elicit [Ca2+]i increases in VB astrocytes [43], possibly indicating the presence of Group I receptors which are coupled to IP3 production. Immunocytochemistry shows that astrocytes also express the Group II mGluR 2 and 3 receptors [32] on the glial processes that ensheath synapses in the VB thalamus. Activation of these receptors in the VB complex is known to inhibit GABAergic input from the rat nucleus reticularis thalami (nRT) [47]. Since Group II receptors are rare on presynaptic elements in the VB thalamus [32], the authors suggest that the inhibition of GABAergic signalling is due to an action on astrocytic mGluR receptors. The physiological scenario which they suggest is one whereby strong glutamatergic lemniscal input into the VB complex activates astrocytic Group II receptors and perhaps via upregulation of the GABA transporter 1 and GABA transporter 3 lead to a reduction of the synaptic effect of GABA. These transporters are also restricted to GABAergic synapse ensheathing astrocytic processes and are not present on GABAergic synaptic terminals [11]. VB astrocytes also seem to express functional GABAA receptors, at least early postnatally, since GABA application elicits [Ca2+]i increases while the GABAB agonist baclofen does not [41]. Neuronal modulation by nitric oxide (NO), which is synthesised following calcium influx from the activation of NMDA receptors, has been implicated in many physiological processes including long term potentiation and the patterning of synaptic inputs during development [7]. In the thalamus, this pathway has been implicated in a developmental and physiological context which might directly involve astrocytes. In the development of the ferret LGN, blocking neuronal NO synthase disrupts the segregation of retinal afferents [9]. In vivo recording of neuronal activity in the VB complex induced by stimulation of a vibrissa [13] showed that such activity was enhanced by iontophoresis of the amino acid arginine, the enhancement was inhibited by the NO synthase inhibitor L-NAME indicating that
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arginine was serving as a precursor to NO in its synthesis pathway, where the supply of arginine is usually rate limiting. Arginine levels in the VB thalamus also increased during vibrissae stimulation. Since arginine immunoreactivity is mainly confined to glial cells, and arginine is released from astrocytes following glutamate receptor activation, the hypothesis presented is that glutamate release during sensory stimulation induces glial arginine release which leads to NO production and enhancement of the sensory signal [14]. This suggests an intimate interaction between neurons and glia in the normal functioning of the VB complex and the thalamus. Other inputs into the thalamus release histamine, serotonin (5HT), acetylcholine and noradrenaline. Histaminergic input into the thalamus comes from the mammillary body of the hypothalamus, though histamine is also released from mast cells within the thalamus [29]. Astrocytes have histamine receptors, and VB astrocytes respond to histamine with [Ca2+]i increase [41]. The thalamus also receives serotonergic input from the raphe´ nucleus. 5HT increases cAMP in thalamic astrocytes via presumed 5HT7 receptors [23]. This is consistent with [Ca2+]i increases caused by 5HT in VB astrocytes [41]. There is not much evidence for the potential neuron-glia interactions concerning the cholinergic input from the parabrachial region nor the noradrenergic pathway from the locus coereleus, though acetylcholine and noradrenaline both elicit [Ca2+]i increases in VB astrocytes [41].
7. Perspectives and conclusions There is therefore accumulating evidence of the importance of astrocytes in the development and physiology of the thalamus. During the development of the thalamus vimentin positive radial glia seem to guide migrating neurons in the way that has been described for other CNS regions. Vimentin positive astrocytes, presumably derived from these radial glia then display spontaneous [Ca2+]i oscillations which underlie NMDA receptor mediated signalling to neurons. The role for this developmentally regulated activity is at present unknown: although spontaneous neuronal activity is recognised as an important mechanism in wiring the nervous system, an involvement of spontaneous astrocytic activity has not been considered. Essentially the astrocytes would seem to be providing a local excitatory signal to thalamic neurons, so the first possibility to consider is perhaps a local developmental signal. Due to the nature of Ca2+ as a pluripotent messenger, the role of this activity could be to provide one of many biochemical signals to developing TC neurons. The TC neuron Ca2+ entry is via NMDA receptors, and NMDA receptors have been implicated in the control of
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dendritic arborisation [46]. Since this is a time of dendritic development in the VB thalamus, such a role may also be implicated. Another function suggested by the involvement of NMDA receptors is in synaptic plasticity. The activation of NMDA receptors and the association of synaptic inputs are important steps in the strengthening of some synapses. By activating a neuron’s NMDA receptors, spontaneously acting astrocytes could therefore be facilitating synapse formation onto that neuron. As groups of astrocytes are involved in the spontaneous [Ca2+]i activity [43] does this mean that groups of neurons rather than individual neurons are activated? Such a scenario would be useful for coordinating clusters of neurons. The magnitude of the observed NMDA currents indicate that they would elicit action potential firing in TC neurons and thus lead to the excitation of cortical neurons. This would then be expected to feed back to the thalamus, via the thalamocortical loop, and due to the duration of the thalamic NMDA stimulation, would perhaps facilitate synapse formation onto the very same cell or group of cells that initiated the activity. Such a putative role, however, must be considered in relation to the large amount of sensory activity occurring at this time. An interesting feature of these findings is that the long lasting NMDA responses are reminiscent of NR2D receptor activation. Since these receptors are now thought to be situated extrasynaptically and not activated by synaptic stimulation it is interesting to speculate that astrocytic glutamate release might be ideally situated for their activation. Since the demonstration that neuronal activity can trigger [Ca2+]i wave propagation through astrocytes in organotypic slice cultures [10], there has been the suggestion that astrocytes form a network parallel to neurons that processes information on a slower time scale using calcium as a medium compared to fast neuronal electrical processing. Propagation between groups of astrocytes in the VB thalamus and subsequent signalling to neurons support the existence of such a parallel system, and demonstrate a way that astrocytes signal to neurons. The developmental expression of these spontaneous oscillations raises questions about the astrocytes that are involved in signalling to neurons. Glutamate release from astrocytes is well studied in culture but this type of robust glutamate signalling is not obvious in situ in the CNS. Do these data indicate that glutamate release is a feature of developing astrocytes in the VB thalamus? Immature astrocytes expressing vimentin have been implicated in the developmental plasticity of the visual cortex. Most notably, the study of Muller and Best [35] showed that when immature astrocytes were injected into the visual cortex of adult cats, the plasticity of ocular dominance columns was re-induced, suggesting
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Astrocytes, spontaneity, and the developing thalamus H. Rheinallt Parri, Vincenzo Crunelli* School of Biosciences, Cardiff University, Museum Avenue, PO Box 911, Cardiff CF10 3US, Wales, UK
Abstract Recent studies in the ventrobasal (VB) thalamus have shown that astrocytes display spontaneous intracellular calcium [Ca2+]i oscillations early postnatally. [Ca2+]i oscillations are correlated in groups of up to five astrocytes, and propagate between cells. NMDA receptor-mediated, long lasting inward currents in thalamocortical (TC) neurons of the VB complex are correlated to [Ca2+]i increases in neighbouring astrocytes, and stimulation of astrocytic [Ca2+]i increases also lead to inward currents in neurons. These findings suggest that astrocytes are spontaneously active and can induce neuronal activity, a reversal of the previously held view of neuron–glia interactions in the central nervous system. This activity occurs at an important period in the development of the thalamus and therefore suggests a potential functional role in a variety of processes. Along with data on the neurotransmitter receptor repertoire of thalamic astrocytes these findings enlarge the body of knowledge on astrocytes in the thalamus, and further contribute to the emerging field of astrocyte–neuron and neuron–astrocyte interactions in the central nervous system. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Ventrobasal thalamus; Oscillations; Development
1. Introduction The acknowledged roles of glia in general, and of astrocytes in particular, in the functioning of the nervous system have increased dramatically in recent years. This increased consciousness of the physiological significance of astrocytes has seen them emerge from a stage where they were thought to be supportive housekeeping cells that express certain neurotransmitter receptors and channels, to a stage where they have assumed functions traditionally exclusively assigned to neurons, such as glutamate release [39]. From adopting some of the roles of neurons and being intimately associated with the synapse and synaptic transmission, it is now also known that in some adult brain areas, astrocytes actually give rise to new neurons [49]. A recent focus of astrocytic research has been the ways that astrocytes react to, and interact with, synaptic transmission. Astrocytes express many of the same neurotransmitter receptors as neurons and react to these neurotransmitters with increases in [Ca2+]i. Following such activation, astrocytes have been shown to modulate synaptic transmission [4,28].
A further step in the interaction of neurons and glia, however, has been seen in the developing thalamus, where astrocytes display spontaneous [Ca2+]i oscillations which are not dependent on previous neuronal activity [43]. These oscillations are correlated to NMDA receptor mediated currents recorded in neighbouring neurons, and stimulation of astrocytic [Ca2+]i increases also lead to inward currents in neighbouring neurons [43], indicating that activity arising in astrocytes can induce activity in neurons. These findings extend the repertoire of potential astrocytic functions and again assign a role to astrocytes previously reserved for neurons. Here, we will place these recent findings in the context of the developing thalamus. We first discuss spontaneous activity in the developing CNS, the evidence for astrocyte-neuron signalling and how this fits into the scheme of the developing thalamus. We will then review evidence on the neurotransmitter receptors that are present on VB astrocytes and discuss these potential astrocyte-neuron interactions.
2. Spontaneous activity in the CNS 2.1. Spontaneous neuronal activity during development
* Corresponding author. Tel.: +44-29-20874801; fax: +44-292087986. E-mail address: [email protected] (V. Crunelli).
Spontaneous activity is a key feature of the developing nervous system. Neuronal precursor cells exhibit
0928-4257/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0928-4257(02)00009-8
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spontaneous [Ca2+]i fluctuations in the ventricular zone of the neocortex [38]. This intracellular store-dependent activity persists until the cells migrate out into the other cortical layers. Prior to synapse formation in the spinal cord, neurons also display a variety of spontaneous [Ca2+]i oscillatory patterns [51], where calcium spikes occur at a frequency of 1–10/hour and involve voltagegated calcium channels which trigger calcium induced calcium release (CICR) from intracellular stores [21]. This activity has been implicated in the frequency dependent regulation of neurotransmitter-related gene transcription. [Ca2+]i transients seen in growth cones of the same neurons also involve CICR in their generation and regulate the rate of outgrowth of growth cones in a frequency-dependent manner [21]. Following synapse formation, spontaneous activity becomes important in the development of neural circuits and in the refinement of neuronal connections, and at this time spontaneous synchronous activity is seen in large groups of neurons. The retina, for example, exhibits spontaneous intercellular waves which propagate over large distances. These propagating waves seem dependent on action potential generation since they are blocked by tetrodotoxin (TTX) [56]. The generation of these waves is important in the development of cortical ocular dominance columns, since blocking the retinal waves with TTX affects the segregation of retinogeniculate afferents in the dorsal lateral geniculate nucleus (LGN) of the thalamus [50]. Another form of synchronous spontaneous activity occurring in neuronal groups has been described in developing neocortex. Here groups or ‘‘domains’’ of neurons can be identified on the basis that they exhibit coordinated [Ca2+]i increases [58]. This coordination is dependent on gap junctional coupling [27], and presents the possibility that electrical excitability and also metabolic factors and second messengers are distributed within cells in the same domain in ways which affect the developing cortical architecture and the plasticity of afferent synaptic inputs. Neuronal gap junctional communication occurs in many parts of the CNS during development and domains have also been reported to exist in many brain areas including, reportedly, in the thalamus [59]. 2.2. Spontaneous astrocytic activity Although radial glia are involved in CNS development, and have been extensively investigated, the description of spontaneous [Ca2+]i oscillations in glial cells during development has only been made recently [43]. Astrocytes in the VB thalamus of the rat were found to display spontaneous oscillations in [Ca2+]i in acute slices taken from neonatal rats (Fig. 1). The oscillations were seen between the ages of postnatal day 5 (P5) and P17, decreasing in frequency of occurrence with age, so suggesting a developmental expression.
Fig. 1. Spontaneous [Ca2+]i oscillations in VB astrocytes. A. Fluorescence image of a region of an acutely isolated VB slice, ringed areas a–d denote the positions of astrocytes that displayed spontaneous [Ca2+]i oscillations. Image was taken at 260 s after beginning of recording. B. Plots of F% over time for the astrocytes indicated in A. Astrocyte a displays increases that are independent of astrocytes b– d and also a transient that is correlated with the other astrocytes. (Reproduced from [43]).
Oscillations were observed in astrocytes throughout the VB slice and could be manifested as single transients or multiple [Ca2+]i fluctuations occurring during the 10 min recording period. The mode of the inter-oscillation duration distribution was 60–70 s [42] and interestingly, a subset (4%) of spontaneously active astrocytes, termed ‘‘pacemaker’’ astrocytes, displayed long lasting regular oscillations with a periodicity also of around 60 s. Oscillations were not dependent on neuronal activity since they were not blocked by TTX, and were not blocked by antagonists to the main thalamic neurotransmitters, ie glutamate and GABA, nor by suramin. Spontaneous astrocytic [Ca2+]i increases have been described previously in culture. Cortical astrocytes displayed spontaneous [Ca2+]i increases which propagated in confluent cultures. These oscillations were not dependent on extracellular calcium and differed from increases seen following agonist application [15]. In cultured suprachiasmatic nucleus astrocytes, cocultured with neurons, astrocytic [Ca2+]i oscillations were seen that had periodicities ranging from 7–20s, and were not blocked by TTX [53]. Another
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study indicating a potential pathological role for spontaneous astrocytic oscillations was reported in human cortical astrocytes cultured from a patient with intractable childhood epilepsy [30]. The oscillations in the latter study (ref. [30]) however had much slower kinetics than reported in other studies [15,53] with the mean time from initial elevation to return to baseline being about 5 min. The [Ca2+]i oscillations were also dependent on the presence of extracellular calcium. 2.3. Mechanism of spontaneous astrocytic [Ca2+]i oscillations Spontaneous oscillations in VB astrocytes were blocked by thapsigargin and cyclopiazonic acid, showing that the observed increases were due to [Ca2+]i release from intracellular stores, and removal of extracellular Ca2+ did not block the oscillations [43]. The L-type calcium channel antagonist nimodipine reduced the number of oscillations suggesting a role for Ca2+ entry via dihydropyridine sensitive channels in sustaining the oscillations. In line with other studies on calcium oscillations, it may be that inositol-1,4,5-trisphosphate (IP3) receptor activation is required to release [Ca2+]i from the astrocytic stores. This in turn would suggest a phospholipase C (PLC) coupled mechanism. The spontaneous nature of the activity, and the regularity of observed pacemaker oscillations could arise from long lasting action of an extracellular agonist or constitutive activation of PLC. This could perhaps be due to an inherent property of the membrane receptor or enzyme in the cascade, or even due to the presence of an as yet unknown agonist in the extracellular milieu at this time of development. Spontaneous activity seen in cultures of isolated astrocytes [15] suggests that astrocytes have an intrinsic ability to display [Ca2+]i oscillations without the presence of another cell type. However, even these types of studies do not rule out the possibility of a secreted agonist or factor activating surface receptors to induce oscillations. Bezzi and coworkers [5] recently showed a chemokine-prostaglandin pathway that evoked glutamate release from astrocytes. The pathway was suggested to be active physiologically, and also activated by a viral agent in pathological conditions. It is possible that such compounds or growth factors could effect astrocytic activity during development. Spontaneous activity therefore seems to be a property of astrocytes as well as of neurons in the CNS. In accordance with the situation of spontaneous neuronal activity during development, the findings in the VB thalamus also indicates a developmental role for spontaneous astrocytic activity: this is also supported by the fact that spontaneous astrocyte activity seen in culture studies [15] are from astrocytes cultured early postnatally.
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3. Astrocyte–astrocyte and astrocyte–neuron signalling Groups of astrocytes show coordinated increases in [Ca2+]i and [Ca2+]i waves are seen to propagate between astrocytes in the VB thalamus. Although propagating [Ca2+]i waves are a defining feature of astrocytes in culture, this developmental astrocytic thalamic wave is the first demonstration of spontaneous astrocytic [Ca2+]i propagation in situ in the CNS [43]. A large amount of work has been conducted under culture conditions on the propagation of astrocytic [Ca2+]i waves via gap junctions [18], probably via diffusion of IP3 [48]. Chemical transmission has however also been demonstrated, the two main candidates for this form of transmission being ATP and glutamate. In cultured cortical astrocytes, [Ca2+]i waves that propagated between physically isolated astrocytes [8], or were induced by stimulated astrocyte conditioned media [22], were blocked by the purinergic antagonist suramin or treatment with the ATP degrading enzyme apyrase, indicating transmission by ATP. In the retina, [Ca2+]i waves propagate between astrocytes and muller cells. Propagation between astrocytes seems mainly via gap junctions while propagation to, and between muller cells is blocked by apyrase treatment or suramin [36]. Glutamate has been shown to be released from astrocytes following stimulation which result in [Ca2+]i increases [39], and waves of glutamate release, concurrent with propagating [Ca2+]i waves in cultured astrocytes have been imaged [24]. Since glutamate seems to be the most abundant neurotransmitter in the CNS, much of the effort on exploring potential astrocyteneuron signalling has focused on the role that astrocytereleased glutamate may play. The release of glutamate from astrocytes is now well established [4,5,39]. Flash photolysis studies used to increase astrocytic [Ca2+]i levels in astrocyte -neuron cocultures show that raising astrocytic [Ca2+]i within physiological levels results in glutamate release and the generation of neuronal inward currents [40]. Other studies show the presence of the vesicle associated SNARE protein synaptobrevin in astrocytes [3], the cleavage of which by botulinum B neurotoxin blocks neuronal inward currents induced by astrocyte stimulation. Agonist stimulation also elicits glutamate release [39] and stimulation of metabotropic glutamate receptors (mGluRs) which induces cytosolic [Ca2+]i oscillations [44], also results in glutamate release suggesting that glutamate could act as a wave propagating ‘gliotransmitter’ and a transmitter to neurons. In the developing VB thalamus, adjacent spontaneously active astrocytes exhibit coordinated [Ca2+]i increases, with up to five astrocytes in a restricted area exhibiting coordinated activity (Fig. 2) [43]. Propagating [Ca2+]i waves between astrocytes can also be seen. These propagate within the range of velocities seen in culture studies [17]. The propagation mechanism did not
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Fig. 2. Propagation of spontaneous [Ca2+]i waves between astrocytes. A. Fluorescent image (taken 285 s into experiment shown in B) displaying position of the somas of astrocytes a–c in an acute VB slice. Scale bar 20 mm. B. Plots of fluorescence over time for somas of astrocytes a–c, showing that activity initiates in astrocyte a, then propagates to b and c. (Reproduced from [43]).
seem to involve gap junctions since filling of single astrocytes with Lucifer yellow or biocytin via patch pipettes failed to fill neighbouring astrocytes: this is consistent with observations in rat visual cortex where dye coupling between astrocytes is not seen until P11 [6]. The mechanism of spontaneous activity propagation would therefore seem to be chemical. Suramin and broad spectrum ionotropic glutamate antagonists did not reduce the number of astrocytes exhibiting spontaneous activity in the VB thalamus, but experiments were not performed to directly address the identity of a substance mediating inter-astrocytic [Ca2+]i wave propagation. In this study [43], the inference of chemically mediated propagation presented the possibility that as well as propagation between astrocytes a chemical messenger could also be affecting neighbouring neurons. Patch clamp recording revealed large long lasting inward currents in VB thalamocortical (TC) neurons which were correlated to spontaneous activity in neighbouring astrocytes in the slice. The currents were striking in their slow activation and slow decay kinetics (tact 330 ms, tdecay 2.7 s), resulting in inward currents that could last for several seconds. In this respect they are reminiscent of currents elicited in neurons by astrocytic glutamate release in culture studies [40, 44]. Spontaneously recorded currents in VB TC neurons were blocked by the NMDA receptor antagonist APV, indicating that spontaneous astrocytic activity leads to glutamate release and activation of TC neurons via NMDA receptor activation. Responses were seen in the presence of external Mg2+, indicating that at least at this developmental stage, prior TC neuronal depolarisation was
not required to enable the removal of NMDA-R Mg2+ block in order to obtain NMDA-R currents [43]. The simplest explanation for the results observed in the VB thalamus is that astrocytes release glutamate which directly activate neuronal NMDA receptors (Fig. 3A). However, there is no direct evidence so far for [Ca2+]i dependent glutamate release from astrocytes in the VB thalamus, and it is therefore equally possible that astrocytes are acting presynaptically to evoke glutamate release from glutamatergic afferents (Fig. 3B). Such presynaptic effects have been shown in culture and in slice preparations: astrocytic glutamate release has been shown to modulate neurotransmitter release differentially by activating metabotropic glutamate receptors [1] or NMDA [2] receptors presynaptically, and a presynaptic effect of astrocyte released glutamate would also explain the role of astrocytic activation in potentiating inhibitory transmission in the hippocampus [28]. The possibility also exists that the compound released by the astrocyte is not glutamate but another compound. This might be a substance such as ATP that could perhaps act presynaptically, or a glutamate analogue that could act either pre or postsynaptically. Such a feasible candidate is homocysteic acid (HCA). HCA activates both NMDA and non-NMDA ionotropic glutamate receptors with an EC50 of about 100 mM [60], is localized to glial cells and is also released by K+ induced depolarisation from brain slices [12]. Interestingly, in the rat LGN, another thalamic nucleus, HCA immunoreactivity is restricted to astrocytic end feet under normal conditions, while following cortical and retinal lesions HCA immunoreactivity increases in glial cell bodies in the LGN [20].
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Fig. 3. Two possible scenarios for the coupling of astrocytic [Ca2+]i increases and observed inward neuronal currents. A. Spontaneous [Ca2+]i increases in the astrocyte leads to glutamate (Glut.) release which then directly activates postsynaptic NMDA receptors (NMDA-R) on the TC neuron to cause Ca2+ entry and a depolarisation of membrane potential (Vm). B. Astrocytic [Ca2+]i increases lead to the release of a ‘gliotransmitter’ (GT) which activates presynaptic afferents which then release glutamate to activate postsynaptic NMDA receptors on the TC neuron.
4. Development of the thalamus 4.1. Glia The development of the neuronal architecture of the CNS is intimately involved, and dependent on, glial development and differentiation, with radial glial cells providing a scaffold for neuronal cell migration. Following neuronal migration radial glial cells transform into astrocytes, and recent work in the neocortex has shown that radial glial cells also give rise to clonally related groups of neurons [37]. In the thalamus, during prenatal development radial glial cells extend from the ventricular germinative zone to the pial surface [16], and by embryonic day 16 some thalamic nuclei are evident in stained sections. The radial glial cells stain for vimentin, their predominant structural protein. Neuronal staining shows that neurons migrate radially along the fibres, and that GABAergic cells migrate along glial fibres that are perpendicular to the radial glia. Radial glial cell processes are filled with microtubules, vacuoles and clear vesicles, and sometimes vesicles are grouped at large appositions between the processes and migrating neurons. Neuronal migration seems to be over by birth (P0) in the thalamus, and in the first postnatal week vimentin staining decreases markedly and is restricted mainly to some thin processes surrounding blood vessels and immature astrocytes which seem rich in organelles. Vimentin stained astrocytes become further reduced during the first and second postnatal weeks and are virtually absent in the adult thalamus. During the first 2
weeks postnatally, GFAP stained cells become visible in outlining some nuclei though in adult the principal thalamic nuclei also seem devoid of GFAP staining.
5. Neuronal development A discussion of the development of the neurons and connections of the thalamus must be done in the context of the thalamocortical loop. In the rat, the VB thalamus receives somatosensory input, with the arcuate subdivision receiving input from the vibrissae (whiskers). The VB TC neurons then project to the ‘‘barrel cortex’’ where, as indicated by the name, a somatotopic map of the whisker field is reproduced. A somatotopic ‘‘barreloid’’ map also exists in the VB thalamus. Like other thalamic nuclei, the VB complex also receives corticothalamic afferents from its cortical projection target (i.e. barrel cortex), so forming the thalamocortical loop. At birth, before sensory input has shaped the sensory pathway, dye injections in the barrel cortex retrogradely fill cells in the VB barreloids indicating that a rudimentary pathway exists. However, the first 2 weeks postnatally result in major developmental changes in the thalamus and the thalamocortical loop. At P7 there are few synaptic complexes in the VB thalamus but these increase threefold by P12, synapse formation then increases until P15 but after this, the rate of synaptogenesis is attenuated (Fig. 4) [31]. This coincides with a large proliferation in corticothalamic afferents between P8–15 [25].
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Fig. 4. Developmental context of spontaneous astrocytic [Ca2+]i oscillations in the VB. Horizontal bars represent the time courses of the indicated events from P0 to P20. The relative magnitude of the indicated process is expressed by the darkness of the bar at a particular age. Dotted lines in upper bar indicate that the level of spontaneous activity at these ages are unknown.
At this time the neuronal elements of the VB thalamus are also undergoing radical changes. At P7, TC neurons are characterized by short stubby irregular dendrites with distinct growth cones and fillipodia. By P12 there is dendritic elongation with secondary and tertiary branching and the appearance of spinous protrusions [31]. The matching of dendritic elongation and generation of secondary and tertiary branches with the proliferation of thalamocortical afferents agrees with the findings in the LGN that corticothalamic afferents synapse distally while sensory afferents synapse proximally on TC neurons [55]. During this postnatal period, the complement of receptors mediating synaptic transmission on TC neurons also change. Corticothalamic synaptic stimulation at early postnatal ages produces synaptic responses largely due to NMDA receptors, during maturation the duration of postsynaptic responses in the dLGN decreases and the proportion of the postsynaptic response due to AMPA/kainate receptor activation increases [19]. NMDA receptor expression is therefore changing during thalamic development; in this respect the NMDA receptor subtype NR2D displays an interesting developmental profile in the VB thalamus. High levels of NR2D mRNA are seen at birth which are maintained until about P10, while expression decreases between P10 and P21 when it is virtually absent [54]. The properties of NMDA receptors containing the NR2D subunit would seem to endow them for a role where the coordination of associative inputs was less
temporally constrained than for other NMDA-Rs. Receptors containing the NR2D subunit have a weaker Mg2+ block than NMDA receptors of differing subunit composition [34], and a very slow decay following glutamate activation [57], as well as a low EC50 for glutamate. Recombinant NMDA receptors containing NR2D subunit have current decay time constants of 4.4 s following a 1 ms glutamate application. Similar glutamate application on excised patches from extrasynaptic regions of purkinje neurons elicited currents with a decay time constant of 3s [33]. The extrasynaptic location of the receptors is supported by experiments that show that synaptic stimulation of purkinje neurons does not elicit such long lasting NMDA-R currents [33]. NR2D subunits are expressed in a developmentally regulated manner between P0 and P8 in cerebellar purkinje neurons. It would seem therefore that to have a functional role these developmentally expressed extrasynaptic receptors must therefore presumably need an extrasynaptic source of glutamate for activation.
6. Neurotransmission and thalamic astrocytes Although the central theme of this review is the spontaneous activity of thalamic astrocytes and their possible roles in a developmental context, astrocytes would also seem to have a role in the adult physiological functioning of the thalamus.
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Astrocytes express a vast array of neurotransmitter and neuromodulator receptors, and some of these are known to be activated during synaptic transmission [10,45]. The receptors present on thalamic astrocytes therefore indicate the further potential for communication between neurons and glia in the thalamus. Since thalamic nuclei not only receive sensory and cortical glutamatergic inputs but also gabaergic, cholinergic, serotonergic, adrenergic and histaminergic from within the CNS, the potential for astrocytic activation by synaptic activation is very large. Thalamic astrocytes in the rat express the AMPA receptor GluR1 subunit [52], while in monkey thalamus high expression of the NMDA subunit NR2B is seen in astrocytes in the LGN [26]. As mentioned, mGluRs are widely expressed in astrocytes, and addition of the mGluR agonist trans-ACPD elicit [Ca2+]i increases in VB astrocytes [43], possibly indicating the presence of Group I receptors which are coupled to IP3 production. Immunocytochemistry shows that astrocytes also express the Group II mGluR 2 and 3 receptors [32] on the glial processes that ensheath synapses in the VB thalamus. Activation of these receptors in the VB complex is known to inhibit GABAergic input from the rat nucleus reticularis thalami (nRT) [47]. Since Group II receptors are rare on presynaptic elements in the VB thalamus [32], the authors suggest that the inhibition of GABAergic signalling is due to an action on astrocytic mGluR receptors. The physiological scenario which they suggest is one whereby strong glutamatergic lemniscal input into the VB complex activates astrocytic Group II receptors and perhaps via upregulation of the GABA transporter 1 and GABA transporter 3 lead to a reduction of the synaptic effect of GABA. These transporters are also restricted to GABAergic synapse ensheathing astrocytic processes and are not present on GABAergic synaptic terminals [11]. VB astrocytes also seem to express functional GABAA receptors, at least early postnatally, since GABA application elicits [Ca2+]i increases while the GABAB agonist baclofen does not [41]. Neuronal modulation by nitric oxide (NO), which is synthesised following calcium influx from the activation of NMDA receptors, has been implicated in many physiological processes including long term potentiation and the patterning of synaptic inputs during development [7]. In the thalamus, this pathway has been implicated in a developmental and physiological context which might directly involve astrocytes. In the development of the ferret LGN, blocking neuronal NO synthase disrupts the segregation of retinal afferents [9]. In vivo recording of neuronal activity in the VB complex induced by stimulation of a vibrissa [13] showed that such activity was enhanced by iontophoresis of the amino acid arginine, the enhancement was inhibited by the NO synthase inhibitor L-NAME indicating that
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arginine was serving as a precursor to NO in its synthesis pathway, where the supply of arginine is usually rate limiting. Arginine levels in the VB thalamus also increased during vibrissae stimulation. Since arginine immunoreactivity is mainly confined to glial cells, and arginine is released from astrocytes following glutamate receptor activation, the hypothesis presented is that glutamate release during sensory stimulation induces glial arginine release which leads to NO production and enhancement of the sensory signal [14]. This suggests an intimate interaction between neurons and glia in the normal functioning of the VB complex and the thalamus. Other inputs into the thalamus release histamine, serotonin (5HT), acetylcholine and noradrenaline. Histaminergic input into the thalamus comes from the mammillary body of the hypothalamus, though histamine is also released from mast cells within the thalamus [29]. Astrocytes have histamine receptors, and VB astrocytes respond to histamine with [Ca2+]i increase [41]. The thalamus also receives serotonergic input from the raphe´ nucleus. 5HT increases cAMP in thalamic astrocytes via presumed 5HT7 receptors [23]. This is consistent with [Ca2+]i increases caused by 5HT in VB astrocytes [41]. There is not much evidence for the potential neuron-glia interactions concerning the cholinergic input from the parabrachial region nor the noradrenergic pathway from the locus coereleus, though acetylcholine and noradrenaline both elicit [Ca2+]i increases in VB astrocytes [41].
7. Perspectives and conclusions There is therefore accumulating evidence of the importance of astrocytes in the development and physiology of the thalamus. During the development of the thalamus vimentin positive radial glia seem to guide migrating neurons in the way that has been described for other CNS regions. Vimentin positive astrocytes, presumably derived from these radial glia then display spontaneous [Ca2+]i oscillations which underlie NMDA receptor mediated signalling to neurons. The role for this developmentally regulated activity is at present unknown: although spontaneous neuronal activity is recognised as an important mechanism in wiring the nervous system, an involvement of spontaneous astrocytic activity has not been considered. Essentially the astrocytes would seem to be providing a local excitatory signal to thalamic neurons, so the first possibility to consider is perhaps a local developmental signal. Due to the nature of Ca2+ as a pluripotent messenger, the role of this activity could be to provide one of many biochemical signals to developing TC neurons. The TC neuron Ca2+ entry is via NMDA receptors, and NMDA receptors have been implicated in the control of
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dendritic arborisation [46]. Since this is a time of dendritic development in the VB thalamus, such a role may also be implicated. Another function suggested by the involvement of NMDA receptors is in synaptic plasticity. The activation of NMDA receptors and the association of synaptic inputs are important steps in the strengthening of some synapses. By activating a neuron’s NMDA receptors, spontaneously acting astrocytes could therefore be facilitating synapse formation onto that neuron. As groups of astrocytes are involved in the spontaneous [Ca2+]i activity [43] does this mean that groups of neurons rather than individual neurons are activated? Such a scenario would be useful for coordinating clusters of neurons. The magnitude of the observed NMDA currents indicate that they would elicit action potential firing in TC neurons and thus lead to the excitation of cortical neurons. This would then be expected to feed back to the thalamus, via the thalamocortical loop, and due to the duration of the thalamic NMDA stimulation, would perhaps facilitate synapse formation onto the very same cell or group of cells that initiated the activity. Such a putative role, however, must be considered in relation to the large amount of sensory activity occurring at this time. An interesting feature of these findings is that the long lasting NMDA responses are reminiscent of NR2D receptor activation. Since these receptors are now thought to be situated extrasynaptically and not activated by synaptic stimulation it is interesting to speculate that astrocytic glutamate release might be ideally situated for their activation. Since the demonstration that neuronal activity can trigger [Ca2+]i wave propagation through astrocytes in organotypic slice cultures [10], there has been the suggestion that astrocytes form a network parallel to neurons that processes information on a slower time scale using calcium as a medium compared to fast neuronal electrical processing. Propagation between groups of astrocytes in the VB thalamus and subsequent signalling to neurons support the existence of such a parallel system, and demonstrate a way that astrocytes signal to neurons. The developmental expression of these spontaneous oscillations raises questions about the astrocytes that are involved in signalling to neurons. Glutamate release from astrocytes is well studied in culture but this type of robust glutamate signalling is not obvious in situ in the CNS. Do these data indicate that glutamate release is a feature of developing astrocytes in the VB thalamus? Immature astrocytes expressing vimentin have been implicated in the developmental plasticity of the visual cortex. Most notably, the study of Muller and Best [35] showed that when immature astrocytes were injected into the visual cortex of adult cats, the plasticity of ocular dominance columns was re-induced, suggesting
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