Cytoplasmic morphology weaver (wv) mouse cerebellar neurons at the culture substratum

The purpose of this study was to determine the structural basis for the hypermotility and impaired growth cone elongation of the homozygous weaver (wv/wv) mouse cerebellar granule cell neurons in culture. Two-day cultures of dissociated week-old normal (+/+) and wv/wv cerebellum were processed for electron microscopy of intact cells and cytoskeleton. Serial sections parallel to and starting from the substrate were examined. Fine-caliber neurites of normal granule cells are packed with parallel arrays of microtubules at all levels. Microfilament-packed microspikes are present at substrate level emanating from a cortical microfilament lattice at the terminus of neurites of varying length. Homozygous weaver granule cells at substrate level have lateral cytoplasmic extensions along the neurite. Microtubules that curve throughout the neurite are separated by cytoplasm. The lateral extensions and growth cone cytoplasmic projections contain microfilaments and occasionally microtubules. Microfilament-packed microspikes are not observed. Immunofluorescent detection of actin confirms the ultrastructural picture. A hallmark of the wv/wv cytopathology is the presence of large numbers of coated vesicles throughout the neurite shaft at the cell-substratum interface. These are rare at similar locations in +/+ neurites. We hypothesize that reduced tension in the growth cone and neurite owing to the presence of lateral extensions and absence of stable microspikes are responsible for the impaired elongation and hypermotility of mutant neurons.     

Age-related changes in the spinal cord microglial and astrocytic response profile to nerve injury

Neuropathic pain, arising from nerve injury or secondary to other diseases, occurs in young children as well as adults but little is known about its postnatal development. Neonatal rat pups do not display mechanical allodynia following nerve injury and young rats recover faster from spinal nerve damage. Since both spinal microglia and astrocytes are strongly implicated in the maintenance of persistent pain, we hypothesized that the magnitude and time course of spinal cord glial activation following nerve injury change throughout postnatal development. To test this, we have compared the time course and intensity of the microglial and astrocytic response in the spinal cord dorsal horn at various times following spared nerve injury in postnatal day 3, 10, 21 and adult rats. The levels of the microglial markers OX-42 and IBA-1 and of the astrocytic marker GFAP were analysed using immunohistochemistry and Western blots. We show that in the adult SNI evokes clear dorsal horn microglial activation at 5 days and astrocytic activation at 7 days post surgery. In contrast, SNI in young animals evokes a weak microglial response but a robust astrocytic response with an early onset at day 1 that is not observed in adults, followed by a second activation at day 7. These results highlight the differential development of the glial response to nerve injury which may explain the lack of neuropathic allodynia in young animals.     

Alterations in dopamine and serotonin uptake systems in the striatum of the weaver mutant mouse

Summary In the striatum of the homozygous weaver mutant mouse (wv/wv), dopamine content, uptake and tyrosine hydroxylase activity are decreased compared to wild-type (+/+) mice. In mice heterozygous for the weaver gene (wv/+), these dopaminergic parameters exhibit only minor reductions compared to +/+ mice. Thewv/wv striatum has recently been shown to have an increase in serotonin content. In the present study, the serotonin uptake system of the weaver striatum was investigated. Synaptosomal uptake of [³H] serotonin was determined in the dorsal portion ofwv/wv and +/+ striatum, and serotonin uptake sites were examined by the binding of [³H] citalopram in the striatum ofwv/wv, wv/+ and +/+ mice. The dopamine uptake system was also investigated in all three genotypes via the binding of [³H] mazindol. Synaptosomal uptake of [³H] serotonin was increased by 79% in the dorsal portion of thewv/wv striatum compared to that seen in the +/+ striatum. The binding of [³H] citalopram was increased by 62% in the dorsolateral and by 111% in the dorsomedial portions of thewv/wv striatum compared to +/+. [³H] Citalopram binding in thewv/+ striatum was also higher than +/+, but this increase did not reach statistical significance. Within thewv/wv striatum, [³H] mazindol binding was almost completely absent (88–89% reduction) in the dorsal portion and severely reduced in the other striatal areas. These data support the notion that the dorsal portion of thewv/wv striatum, which has the severest reduction in dopamine uptake, is hyperinnervated by serotonin fibers.     

Serotonin content is elevated in the dopamine deficient striatum of the weaver mutant mouse

In the present study, we measured the striatal serotonin content of weaver and control mice at different ages. Overall, weaver mutant mice exhibited 50% more striatal serotonin than controls. Neither a rostrocaudal gradient nor an age effect was found for either genotype. An analysis of serotonin content across the dorsoventral extent of the striatum revealed that in the dorsal striatum of the weaver, serotonin content was increased 200%, and in the ventral striatum, the increase amounted to 50% relative to control mice. Serotonin immunocytochemistry also revealed an increase in the dorsal striata of weaver mice. The major increase in striatal serotonin content seen in the weaver striatum occurs in the same region that exhibits the severest dopamine depletion. This observation is consistent with the notion that the increase in serotonin levels may be secondary to the decrease in dopamine content and may play an adaptive or compensatory role.     

Anatomical, physiological and biochemical studies of the cerebellum from mutant mice. II. Morphological study of cerebellar cortical neurons and circuits in the weaver mouse.

The vermis of the homozygous weaver mice has been examined with Golgi and electron microscopic techniques. In addition to the findings already reported by previous authors 12, 29, new cytological features concerning all the cerebellar neuronal types and the synaptic reorganization of the cerebellar circuitry are described. As in other agranular cerebella, Purkinje cells do not develop spiny branchlets and have a randomly oriented dendritic tree. By contrast, their thick dendrites are studded with spines; according to their size and shape these were classified into: (a) small stubby spines which are the normal postsynaptic targets for climbing fibers; (b) tertiary-like spines, most of which are free of axonal contacts; (c) dolichoderus spines; (d) branching spines; and (e) hypertrophic spines. The last 3 types do not exist in normal cerebellum. Postsynaptic-like differentiations are frequently undercoating the smooth surface of the Purkinje dendrites. As it happens in the case of the free spines, free postsynaptic sites in the shafts of the dendrites develop an extracellular material similar to the material present in synaptic clefts. Basket and stellate cells also develop postsynaptic-like differentiations undercoating the somatic and dendritic plasma membranes. These free postsynaptic sites can reach a gigantic size, being longer than 3 mum in length. The rare postmigrative granule cells which persist in wv exhibit claw-endings not only at the dendritc terminal segments, but at the proximal dendritic stems as well. Some of these granule cells, besides having fully achieved migration, undergo a degenerative process indicating that they are probably directly affected by the mutation. Concerning the cerebellar circuitry, and despite the great number of free postsynaptic sites, the large majority of the synaptic contacts keep their specificity. However, some quantitative variations have been disclosed. The surface density of climbing varicosities is increased, whereas that of mossy rosettes is decreased. Stellate and basket fibers are present and their density also decreased. Furthermore, the pinceau formation around the initial segment of the Purkinje cell axon is missing. In addition to all normal synapt iccontacts (with the exception of the'parallel fiber-omnicellularsystem') present in weaver, heterologous synapses have also been encountered, mainly concerning the Purkinje dendritic spines, which can be contacted by mossy rosettes, granule cell bodies and/or dendrites. Morphological signs of partial innervation of the free postsynaptic sites on the smooth surface of Purknje dendrites and the perikarya and dendrites of interneurons have also been observed. These results confirm the existence of synaptic remodeling in wv cerebellum     

The weaver mutation reverses the function of dopamine and GABA in mouse dopaminergic neurons.

In the present study, we characterized the intrinsic electrophysiological properties and the membrane currents activated by dopamine (DA) D(2) and GABA(B) receptors in midbrain dopaminergic neurons, maintained in vitro in a slice preparation, from wild-type and homozygous weaver (wv/wv) mice. By using patch-clamp techniques, we found that membrane potential, apparent input resistance, and spontaneous firing of wv/wv dopaminergic neurons were similar to those of dopamine-containing cells recorded from nonaffected (+/+) animals. More interestingly, the wv/wv neurons were excited rather than inhibited by dopamine and the GABA(B) agonist baclofen. This neurotransmitter-mediated excitation was attributable to the activation of a G-protein-gated inward current that reversed polarity at a membrane potential of approximately -30 mV. We suggest that the altered behavior of the receptor-operated wv G-protein-gated inwardly rectifying K(+) channel 2 (GIRK2) might be related to the selective degeneration of the dopaminergic neurons. In addition, the wv GIRK2 would not only suppress the autoreceptor-mediated feedback inhibition of DA release but could also establish a feedforward mechanism of DA release in the terminal fields.     

Weaver mouse cerebellar granule neurons fail to migrate on wild-type astroglial processes in vitro

To study the regulation of glial-guided neuronal migration, we have analyzed the behavior of cerebellar granule neurons purified from the homozygous weaver (wv/wv) B6CBA-w mouse, an autosomal recessive genetic mutation that suffers a failure of granule cell migration along Bergmann glial processes (Rakic and Sidman, 1973a, b; Rezai and Yoon, 1972), on the processes of astroglia purified from homozygous normal B6CBA-Aw-J-wv (+/+) mouse cerebella. When co-cultured with normal astroglia, weaver granule neurons failed to form neuron-glia contacts characteristic of migrating neurons and impaired normal astroglial morphological differentiation. Normal astroglial cells co-cultured with weaver granule cells had enlarged cell somata with stunted processes and enlarged endfeet compared to normal astroglia co-cultured with normal granule cells. In contrast, normal neurons associated with weaver astroglia, forming tight appositions seen for migrating neurons in vivo, and enhanced weaver astroglial morphological differentiation. Weaver astroglia co-cultured with normal granule cells contained a more normal complement of glial filaments and had a smaller perikaryon with longer, more tapered processes than their counterparts co-cultured with weaver neurons. These results suggest, in agreement with the study of Goldowitz and Mullen (1982) on heterozygous mutant chimeras, that the granule neuron is a primary site of action of the weaver gene, and further support our previous findings that neuron-glia interactions regulate astroglial morphological differentiation (Hatten, 1985).     

Noradrenergic neurons from the locus ceruleus in dissociated cell culture: culture methods, morphology, and electrophysiology

We have developed a dissociated primary cell culture of noradrenergic neurons from the locus ceruleus of postnatal (1- to 5-d-old) mice or rats. Slices of the brain stem were made on a Vibratome. Then the region of locus ceruleus, which was identified by observing the slices under a dissecting microscope, was dissected out from the slices. The removed fragments of brain slices were dissociated and cultured up to 3 weeks on a non-neuronal feeder layer, which consisted predominantly of astroglial cells, or on a fibronectin-treated collagen substratum. After 2 weeks of culture, about 70% of total neuronlike cells revealed positive catecholamine histofluorescence, indicating that they were probably noradrenergic neurons. About 98% of large- and medium-sized cultured neurons (soma diameter greater than or equal to 20 microns) was histofluorescence positive. The fluorescence-positive cells had long processes rich in varicosities, and the shape of their soma was either multipolar or fusiform. Electron microscopy using permanganate fixation revealed that the varicosities along their processes had small granular vesicles, which may contain norepinephrine. Physiological properties of these noradrenergic neurons were investigated with intracellular microelectrodes or with the whole-cell version of the patch clamp. We observed that many cells were producing spontaneous firing. Many of these spontaneously firing cells had no obvious contact with neighboring cells. The neurons were depolarized when glutamate was applied by pressure ejection. They also responded to GABA and glycine with either hyperpolarization or depolarization, and these responses were antagonized by picrotoxin and strychnine. Application of substance P generally produced depolarization with an increase in input resistance. The neurons responded with hyperpolarization to somatostatin, beta-endorphin, and enkephalin. This culture system will become a useful tool for elucidating the cellular and molecular properties of the central noradrenergic neurons.     

Dopamine-depleting effects of MPTP and reserpine in weaver mutant mice

The number of nigral dopamine neurons and striatal dopamine levels are reduced by 70% in the adult weaver mutant mouse (wv/wv), whereas these parameters are essentially unchanged in the heterozygote (wv/+). We hypothesized that the remaining nigral dopamine neurons and/or striatal dopamine levels in the weaver would be less sensitive to neurotoxic or dopamine-depleting agents and that nigral neurons in the heterozygote would be more vulnerable. Mice were treated with the dopaminergic neurotoxin MPTP using different injection schedules and also with reserpine. There was a similar percent decrease in striatal DA in weavers and heterozygotes compared to normal mice after these treatments. We did observe a gene-dose-related lethality to the highest dose treatment with MPTP. These results suggest that the remaining dopaminergic neurons in the weaver are not different from those in normal mice in their capacity to respond to MPTP and reserpine.     

Apparent dopamine D1 and D2 receptors in the weaver mutant mouse: receptor binding and coupling to adenylyl cyclase

Summary. Weaver mutant mice have a selective degeneration of the nigrostriatal dopamine pathway arising between 7–21 days after birth. The goal of this study was to investigate the effects of this mutation on different parameters of the nigrostriatal and mesolimbic dopamine system: apparent D1 and D2 receptor binding sites as well as their signal transduction pathway. Using quantitative autoradiography of ligands for dopamine D1, D2 receptors and the dopamine uptake site, we found a significant loss in apparent D1 receptor binding sites throughout the neostriatum, significant increase of apparent D2 receptor binding in the dorsal aspect of the neostriatum, and almost complete loss of DA uptake sites in these regions of the weaver mouse. In contrast to the neostriatum, the density of dopamine receptors and uptake sites in the nucleus accumbens of the weaver mouse did not differ from controls. Despite alterations in the binding of apparent D1 and D2 receptors, there was no significant difference in either basal, DA stimulated or GTPγS stimulated cAMP production. These findings suggest the down-regulation of apparent D1 receptor binding sites reported in this model, probably does not reflect an important physiological mechanism through which these animals compensate for loss of dopamine innervation. Received July 21, 1998; accepted November 11, 1998     

Cell body response to injury in motoneurons and primary sensory neurons of a mutant mouse,ola (Wld), in which wallerian degeneration is delayed

We examined the response to axon injury in the facial motoneurons and dorsal root ganglion (DRG) neurons of C57BL/Ola (Wld) mice, compared with the responses of C57BL/6J mice. The peripheral nerves of Ola mutants undergo remarkably slowed and muted Wallerian degeneration after injury. The increase in GAP-43 mRNA levels in facial motoneurons and DRG neurons was similar in both strains of mice, as was the initial decrease in medium-weight neurofilament (NFM) mRNA in facial motoneurons, and the increase in JUN immunoreactivity in both types of neurons. However, the subsequent recovery to normal low levels of JUN and GAP-43 mRNA expression and high levels of NFM mRNA was delayed in Ola motoneurons. We ascribe this delay to the slow regeneration and target reinnervation of facial axons in the Ola mice. These results show that absence of rapid Wallerian degeneration does not affect the initial cell body response to axonal injury. They also provide further evidence that restoration of normal levels of expression of GAP-43 and NFM mRNAs is dependent on target reinnervation and/or trophic factors provided by the distal nerve, Impaired regeneration in the Ola mouse does not seem to be a consequence of a defective cell body response to injury, and our results illustrate the general principle that, even if there is a vigorous cell body response to injury, normal axonal regeneration requires the additional provision of a favorable environment for growth. © 1995 Wiley-Liss, Inc.     

Cerebellar histamine-H1 receptor distribution: An autoradiographic study of purkinje cell degeneration, staggerer, weaver and reeler mutant mouse strains

The distribution of 3H-mepyramine binding sites in cerebellae of normal mice and Purkinje cell degeneration, staggerer, weaver and reeler mutant mice was studied by light microscopic autoradiography. The binding of 3H-mepyramine to 20 micron coronal sections through the cerebellum and medulla had the characteristics expected of histamine-H1 receptor labeling. In the cerebellar cortex of normal mice, a high density of 3H-mepyramine binding was observed over the molecular layer and an intermediate density over the Purkinje cell layer, while the granule cell layer and white matter were almost devoid of labeling. The deep cerebellar nuclei were labeled to an intermediate density. In the 54 day old Purkinje cell degeneration mutant cerebellum, which is depleted of Purkinje cells, a greatly reduced labeling of the cerebellar cortex was observed. Labeling in the deep cerebellar nuclei was unaffected. In the 27 day old staggerer cerebellum, a mutation characterized by Purkinje cells which are almost devoid of spines and which do not form synaptic contacts with granule cells, a higher than normal grain density was seen over the cerebellar cortex, while normal grain density was observed over the deep cerebellar nuclei. The cerebellar cortex of 81 day old weaver mice, which is almost devoid of granule cells, had a high grain density over medial regions of the cortex, while the portion of the granule cell layer which remained relatively unaffected in the lateral parts of the cerebellum was unlabeled. The deep cerebellar nuclei had grain densities similar to littermate controls. In the 29 day old reeler cerebellae, which contain malpositioned Purkinje cells, high grain density regions corresponding to the heterotopically located Purkinje cells were observed. The present observations suggest that cerebellar cortical histamine-H1 receptors are associated predominantly with Purkinje cells. Furthermore, the expression of these H1 receptors appears not to be adversely affected by several alterations in the Purkinje cell environment, which have previously been shown to dramatically influence Purkinje cell morphology.     

Cell proliferation in response to GABA in postnatal hippocampal slice culture

Recent studies have implicated the inhibitory neurotransmitter GABA in the regulation of cell migration and proliferation in the embryonic brain. Herein, we examine the possibility that these effects are maintained postnatally. Using 5 days postnatal hippocampus, we tested GABA effects on growth into an incision made in CA1 and the possibility that this response to GABA is mediated via GABA(A)-receptor. Our data shows that GABA promotes neurite growth, cell proliferation and migration up to day 6 in vitro. GABA(A)-receptor is not involved in the trophic response to either exogenous or endogenous GABA. Moreover, GABA induced a 20% increase in NGF secretion to the growth medium, in a similar time frame to its effect on growth. Elevated NGF secretion was suppressed by the inhibition of cell proliferation. These results suggest that GABA can promote growth in postnatal hippocampal tissue. The effect involves cell proliferation and NGF secretion and does not depend on GABA(A)-receptor activation.     

Organization of cerebellar cortex secondary to deficit of granule cells in weaver mutant mice

This study deals with some consequences of the early postnatal abnormalities of cerebellar Bergmann glial fibers and granule cell neurons. (1) Cerebellar size is mildly reduced in heterozygous weaver (+/wv) mice and markedly reduced in homozygotes (wv/wv), but the pattern of fissures is essentially normal. Comparison with other mutants displaying small cerebella suggests that cell proliferation rate in the external granular layer is a key deteminant of cerebellar cortical folding. (2) Mossy fiber terminals differentiate on schedule despite the reduced number and abnormal positions of granule cells. However, many of them enter the modified molecular layer, and as noted especially in noninbred wv/wv mice one to two years old, form synapses with dendrites of aberrant granule cells. Where granule cells are absent, mossy fibers form more than the normal number of synapses with dendrites of Golgi type II neurons. (3) Purkinje cells are only mildly affected by the disorder of neighbouring cells. Their dendrites grow abnormally into the territory occupied by external granule cells, reach the external surface, and may turn inward. They form few tertiary branches. Dendritic spines are present in profusion and show membrane thickenings akin to normal postsynaptic elements. Although they receive no axonal contacts, the spines persist, enveloped by glial processes, for at least two years. Apart from the absence of parallel fiber contacts, afferent and intrinsic axons form the normal classes of synaptic connections with Purkinje cells. (4) Interneurons of the molecular layer are generated on schedule. At the time of their earliest recognition, they reside in the external granular layer, where they receive synaptic contacts from climbing fibers and other interneurons. In the absence of parallel fibers, interneurons differentiate in situ but their dendrites are abortive and randomly oriented. Growth of their dendrites, in contrast to that of Purkinje cell dendrites, appears to be markedly influenced by the organization of the local cellular milieu.     

Somatostatin,cholecystokinin and neuropeptide Y mRNAs in normal and weaver mouse brain

Summary. The distribution of mRNAs encoding for somatostatin, cholecystokinin and neuropeptide Y was determined by in situ hybridization histochemistry in the weaver (wv/wv) mouse, a model of dopamine deficiency as well as in normal (+/+) controls. Weaver mutants did not show any appreciable departure from the normal pattern of expression for mRNA encoding for neuropeptide Y. In contrast, an 82% increase in mRNA encoding for somatostatin was observed in the reticular thalamic nucleus, whereas increases in the order of 20–87% were observed in different hypothalamic nuclei of the weaver brain. In addition, a 47–103% increase of the hybridization signal encoding for choleocystokinin was observed in the cerebral cortex, hippocampus and thalamus of the weaver brain. It can be assumed that the elevated and region-specific somatostatin and choleocystokinin levels observed in the weaver brain may be due to a secondary or compensatory response under conditions of altered neurotransmitter levels. Received November 15, 2001; accepted March 21, 2002 Published online July 26, 2002 Acknowledgements This work was supported by a “Career award in Greece to Greek-speaking researchers living abroad” to K.Z. from the General Secreteriat of Research and Technology of Greece. Authors' address: Prof. E. Kouvelas, Department of Physiology, Faculty of Medicine, University of Patras, 26500 Patras, Greece, e-mail: kouvelas@med.upatras.gr     

Distribution and morphology of tyrosine hydroxylase-immunoreactive neurons in the developing mouse retina

An antibody to tyrosine hydroxylase (TH), the rate-limiting enzyme in the production of catecholamines, was used to examine the morphology and distribution of catecholaminergic neurons in whole-mounted retinae of the developing and adult mouse. At adulthood TH-labeled cell bodies were located in the inner nuclear layer, stratifying mainly at the border to the inner plexiform layer (IPL). Few processes were found in the middle of the IPL. The majority of all TH-labeled cells also extended processes towards the outer plexiform layer and thus are interplexiform cells; the rest were considered to be amacrine cells. The TH-positive neurons were regularly distributed throughout the adult mouse retina. During development, the first TH-immunoreactive cells were observed by postnatal day 6 (P6) and most of them were present after the third postnatal week. The dendrites in the IPL only acquired varicosities after the eyes opened at P15. Biochemical measurements of the endogenous catecholamine content showed that at all developmental stages only dopamine was detectable, suggesting that the TH-labeled cells represent dopaminergic neurons. The content of dopamine was low before P6 and continuously increased during the following days. A strong increase in dopamine was observed during the time when varicosities formed.     

Rat cortical neurons in cell culture: Culture methods, cell morphology, electrophysiology, and synapse formation

Rat cortical neurons from 15 day embryos are grown in dissociated cell culture and maintained in vitro for 8--12 weeks. The neurons develop into forms which resemble mature cortical neurons in situ, stain with silver and exhibit passive and active electrophysiological properties similar to those of cortical neurons. Extensive chemical excitatory and inhibitory synapses develop de novo. These cultures can provide a model for future studies of mammalian CNS neuronal physiology, transmitter pharmacology, pathophysiology and mechanisms of drug action.     

Fibronectin and collagens modulate the proliferation and morphology of astroglial cells in culture

The proliferation and morphology of astroglia derived from neonatal rat cortex and cultured in serum-free medium on either untreated, or fibronectin-, or collagen I-, or collagen IV-treated substrates were investigated using tritiated thymidine autoradiography and immunocytochemical staining of glial fibrillary acidic protein (GFAP) and actin. Modification of culture substratum with fibronectin enhanced the rate of proliferation of astroglial cells and increased the proportion of process-bearing astroglial cells. The distribution of actin and patterns of adhesion observed were typical for motile cells. Both types of collagen decreased the proportion of astroglial cells undergoing mitosis. Many of the astroglial cells exhibited a flat morphology and displayed prominent stress fibres in the cell body and processes. The data suggest that specific interactions with the substratum modulate the proliferation and morphological behaviour of astroglial cells.     

Identified postnatal mesolimbic dopamine neurons in culture: morphology and electrophysiology.

To examine the intrinsic properties of postnatal mesolimbic dopamine (DA) neurons, we dissociated the ventral tegmental area (VTA) from postnatal rats, enriched for DA neurons by microdissection or gradient purification, and grew the cells in culture. In these cultures, up to 50% of neurons were dopaminergic. DA neurons resembled their in vivo counterparts in soma shapes, and in showing two levels of tyrosine hydroxylase (TH) expression, axodendritic differentiation, two sizes of synaptic vesicles, nest-like synaptic arrangements with non-DA cells, and synaptic specializations. Electrophysiologically, however, they could not be distinguished from non-DA cells, which could be consistent with heterogeneity in cell properties. To examine a functional subset of VTA DA neurons, we retrogradely labeled VTA neurons projecting to the nucleus accumbens. These mesoaccumbens neurons were 86% TH positive, 56% cholecystokinin positive, and 0% neurotensin positive; they also displayed the soma shapes characteristic of DA neurons more generally and two levels of TH expression. Like their in vivo counterparts, mesoaccumbens cells generally fired single broad spikes that were triggered by slow depolarizations and had robust spike afterhyperpolarizations, low- and high-threshold Ca2+ spikes, rapid accommodation of firing, time-dependent anomalous rectification, and hyperpolarizing autoreceptor responses. Strikingly, the expression of these active properties did not change with time in culture. Mesoaccumbens DA cells could be identified by a distinctive subset of properties that made up an electrophysiological signature; however, unlike their in vivo counterparts, they were less often spontaneously active and never fired in bursts. These results suggest that most DA cell properties are intrinsic to the cells, including a significant heterogeneity that is maintained in postnatal culture; their level and mode of activity, however, appear to require afferent input. Culturing identified postnatal VTA DA neurons now makes possible examination of the impact of their individual properties on synaptic function.