Michael Belenky

Glutamate‐like Immunoreactivity in Retinal Terminals of the Mouse Suprachiasmatic Nucleus

With a view to identifying the neurotransmitter content of retinal terminals within the mouse suprachiasmatic nucleus, a highly specific antiserum to glutaraldehyde‐coupled glutamate was used in a postembedding immunogold procedure at the ultrastructural level. Retinal terminals were identified by cholera toxin–horseradish peroxidase transported anterogradely from the retina and reacted with tetramethyl benzidine/tungstate/H2O2, or by their characteristically pale and distended mitochondria with irregular cristae. Controls included model ultrathin sections containing high concentrations of various amino acids. Alternate serial sections were labelled with anti‐glutamate and anti‐γ‐aminobutyric acid (GABA). Data were analysed by computer‐assisted image analysis. Density of glutamate labelling (gold particles per μm2) on whole retinal terminals was > 3 times higher than that on postsynaptic dendrites, and > 5 times higher than that on miscellaneous non‐retinal non‐glutamatergic terminals in the suprachiasmatic nucleus. The overall density of gold particles over retinal terminals was ∼ 3 times higher than that over GABAergic terminals, in which glutamate‐like immunoreactivity was mainly mitochondrial. Labelling of vesicles in retinal terminals was almost 5 times greater than the apparent labelling of vesicles in GABAergic terminals, underscoring the location of transmitter glutamate within synaptic vesicles in retinal terminals. In the retino‐recipient region of the suprachiasmatic nucleus there was also a small population of non‐retinal glutamatergic terminals. Their overall immunoreactivity was similar to or exceeded that of retinal terminals, but morphological features clearly distinguished between these two types of glutamate‐containing terminals. The present results indicate that the vast majority of retinal terminals may use glutamate as a transmitter, in keeping with electrophysiological and neuropharmacological data from other sources. The possibility of cotransmitters within retinal terminals, suggested by the presence of dense‐core vesicles among the glutamate‐containing synaptic vesicles, has still to be addressed.

Structural basis of sympathetic-sensory coupling in rat and human dorsal root ganglia following peripheral nerve injury

Tyrosine hydroxylase immunocytochemistry was used to reveal the sympathetic postganglionic axons that sprout to form basket-like skeins around the somata of some primary sensory neurons in dorsal root ganglia (DRGs) following sciatic nerve injury. Ultrastructural observations in rats revealed that these sprouts grow on the surface of glial lamellae that form on the neurons. Sciatic nerve injury triggers glial cell proliferation in the DRG, and the formation of multilamellar pericellular onion bulb sheaths, primarily around large diameter DRG neurons. We infer that these glia participate in the sprouting process by releasing neurotrophins and expressing growth supportive cell surface molecules. Many DRG cell somata, and their axons in intact nerves and nerve end neuromas, express α2A adrenoreceptors intracytoplasmically and on their membrane surface. However, sympathetic axons never make direct contacts with the soma membrane. The functional coupling known to occur between sympathetic efferents and DRG neurons must therefore be mediated by the diffusion of neurotransmitter molecules in the extracellular space. Sympathetic basket-skeins were observed in DRGs removed from human neuropathic pain patients, but the possibility of a functional relation between these structures and sensory symptoms remains speculative.

Heterogeneous expression of γ‐aminobutyric acid and γ‐aminobutyric acid‐associated receptors and transporters in the rat suprachiasmatic nucleus

The hypothalamic suprachiasmatic nucleus (SCN) is the primary mammalian circadian clock that regulates rhythmic physiology and behavior. The SCN is composed of a diverse set of neurons arranged in a tight intrinsic network. In the rat, vasoactive intestinal peptide (VIP)‐ and gastrin‐releasing peptide (GRP)‐containing neurons are the dominant cell phenotypes of the ventral SCN, and these cells receive photic information from the retina and the intergeniculate leaflet. Neurons expressing vasopressin (VP) are concentrated in the dorsal and medial aspects of the SCN. Although the VIP/GRP and VP cell groups are concentrated in different regions of the SCN, the separation of these cell groups is not absolute. The inhibitory neurotransmitter γ‐aminobutyric acid (GABA) is expressed in most SCN neurons irrespective of their location or peptidergic phenotype. In the present study, immunoperoxidase labeling, immunofluorescence confocal microscopy, and ultrastructural immunocytochemistry were used to examine the spatial distribution of several markers associated with SCN GABAergic neurons. Glutamate decarboxylase, a marker of GABA synthesis, and vesicular GABA transporter were more prominently observed in the ventral SCN. KCC2, a K+/Cl– cotransporter, was highly expressed in the ventral SCN in association with VIP‐ and GRP‐producing neurons, whereas VP neurons in the dorsal SCN were devoid of KCC2. On the other hand, GABAB receptors were observed predominantly in VPergic neurons dorsally, whereas, in the ventral SCN, GABAB receptors were associated almost exclusively with retinal afferent fibers and terminals. The differential expression of GABAergic markers within the SCN suggests that GABA may play dissimilar roles in different SCN neuronal phenotypes. J. Comp. Neurol. 506:708–732, 2008.

Light‐induced c‐Fos Expression in the Mouse Suprachiasmatic Nucleus: Immunoelectron Microscopy Reveals Co‐localization in Multiple Cell Types

Although light is known to regulate the level of c‐fos gene expression in the suprachiasmatic nucleus (SCN), the site of an endogenous circadian clock, little is known about the identities of the photically activated cells. We used light‐microscopic immunocytochemistry and immunoelectron microscopy to detect c‐Fos protein in the SCN of Sabra mice exposed to brief nocturnal tight pulses at zeitgeber time 15–16. Stimulation with light pulses that saturated the phase‐shifting response of the circadian locomotor rhythm revealed an upper limit to the number of photo‐inducible c‐Fos cells at about one‐fifth of the estimated total SCN cell population. This functionally defined set was morphologically and phenotypically heterogeneous. About 24% could be labelled for vasoactive intestinal polypeptide, 13% for vasopressin‐neurophysin, and 7% for glial fibrillary acidic protein. The remaining 56% of c‐Fos‐positive cells were largely of unknown phenotype, although many were presumptive interneurons, some of which were immunoreactive for nitric oxide synthase.

Non-synaptic and dendritic exocytosis from dense-cored vesicles in the suprachiasmatic nucleus.

The sites of exocytosis by dense-cored vesicles (DCVs) from neurones in the rat suprachiasmatic nucleus (SCN) were studied at the ultrastructural level. The tannic acid procedure, which stabilizes extruded proteinaceous substances, was used to demonstrate exocytosis of DCV cores. Fresh brain slices containing the SCN were incubated in media containing high levels of potassium (56 mM) or glutamate (10 mM) in the presence of tannic acid. Long-term slice explant cultures of the SCN were similarly treated. Exocytosis from DCVs occurred from axonal terminals, from dendrites, and occasionally from somata. The sites of DCV exocytosis were generally non-synaptic or para-synaptic, including release immediately adjacent to axo-spinous synaptic densities. These observations show that, in the suprachiasmatic nucleus, neuroactive substances contained in DCVs do not necessarily function at synaptic contact zones, but could also act as neuromodulators at non-synaptic sites.

Melanopsin Mediates Retrograde Visual Signaling in the Retina

The canonical flow of visual signals proceeds from outer to inner retina (photoreceptors→bipolar cells→ganglion cells). However, melanopsin-expressing ganglion cells are photosensitive and functional sustained light signaling to retinal dopaminergic interneurons persists in the absence of rods and cones. Here we show that the sustained-type light response of retinal dopamine neurons requires melanopsin and that the response is mediated by AMPA-type glutamate receptors, defining a retrograde retinal visual signaling pathway that fully reverses the usual flow of light signals in retinal circuits.

The suprachiasmatic nucleus in stationary organotypic culture

Suprachiasmatic nuclei, derived from neonate rats, were maintained for several weeks in stationary organotypic culture. Hypothalamic slice explants, supported by Millicell filters and incubated in Petri dishes containing serum-based medium, flattened appreciably, yet preserved the organization of the suprachiasmatic nucleus and the surrounding hypothalamic tissue. After two to three weeks, cultures were fixed, and three neuronal sub-populations were identified as vasopressinergic, vasoactive intestinal peptide-containing, or GABA-containing. The GABAergic component of the cultured suprachiasmatic nucleus was particularly profuse, projecting extensively into the hypothalamic slice. Unilateral ablation of the nucleus in the explant dramatically reduced ipsilateral GABA-immunoreactivity in the slice. Explants in which an incision separated the bilateral suprachiasmatic nucleus from the paraventricular nucleus, deprived the latter of its fine-caliber GABA-immunoreactive input. Extra- or intra-cellular electrophysiological recordings from the suprachiasmatic nucleus were obtained in 51 of 58 cultures. The electrical properties of the long-term cultured suprachiasmatic nucleus were similar to those recorded in acute slices from adult rats. In six cultures recordings were extended for up to 10-24 h. Within long-term stationary organotypic cultures of the suprachiasmatic nucleus, sub-populations of neurons, intrinsic to the nucleus in vivo, were identified immunocytochemically. Lesion studies supported the observation that the main source of the GABAergic innervation within the entire hypothalamic slice explant appeared to be the suprachiasmatic nucleus. Electrophysiological studies confirmed the viability of the long-term cultured nucleus and revealed changes in spontaneous electrical activity that may indicate circadian fluctuation.

Presynaptic and postsynaptic GABAA receptors in rat suprachiasmatic nucleus

The mammalian suprachiasmatic nucleus (SCN), the brains circadian clock, is composed mainly of GABAergic neurons, that are interconnected via synapses with GABA(A) receptors. Here we report on the subcellular localization of these receptors in the SCN, as revealed by an extensively characterized antibody to the alpha 3 subunit of GABA(A) receptors in conjunction with pre- and postembedding electron microscopic immunocytochemistry. GABA(A) receptor immunoreactivity was observed in neuronal perikarya, dendritic processes and axonal terminals. In perikarya and proximal dendrites, GABA(A) receptor immunoreactivity was expressed mainly in endoplasmic reticulum and Golgi complexes, while in the distal part of dendrites, immunoreaction product was associated with postsynaptic plasma membrane. Many GABAergic axonal terminals, as revealed by postembedding immunogold labeling, displayed GABA(A) receptor immunoreactivity, associated mainly with the extrasynaptic portion of their plasma membrane. The function of these receptors was studied in hypothalamic slices using whole-cell patch-clamp recording of the responses to minimal stimulation of an area dorsal to the SCN. Analysis of the evoked inhibitory postsynaptic currents showed that either bath or local application of 100 microM of GABA decreased GABAergic transmission, manifested as a two-fold increase in failure rate. This presynaptic effect, which was detected in the presence of the glutamate receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione and the selective GABA(B) receptor blocker CGP55845A, appears to be mediated via activation of GABA(A) receptors. Our results thus show that GABA(A) receptors are widely distributed in the SCN and may subserve both pre- and postsynaptic roles in controlling the mammalian circadian clock.

Cell-type Specific Distribution of Chloride Transporters in the Rat Suprachiasmatic Nucleus

The suprachiasmatic nucleus (SCN) is a circadian oscillator and biological clock. Cell-to-cell communication is important for synchronization among SCN neuronal oscillators and the great majority of SCN neurons use GABA as a neurotransmitter, the principal inhibitory neurotransmitter in the adult CNS. Acting via the ionotropic GABA(A) receptor, a chloride ion channel, GABA typically evokes inhibitory responses in neurons via Cl(-) influx. Within the SCN GABA evokes both inhibitory and excitatory responses although the mechanism underlying GABA-evoked excitation in the SCN is unknown. GABA-evoked depolarization in immature neurons in several regions of the brain is a function of intracellular chloride concentration, regulated largely by the cation-chloride cotransporters NKCC1 (sodium/potassium/chloride cotransporter for chloride entry) and KCC1-4 (potassium/chloride cotransporters for chloride egress). It is well established that changes in the expression of the cation-chloride cotransporters through development determines the polarity of the response to GABA. To understand the mechanisms underlying GABA-evoked excitation in the SCN, we examined the SCN expression of cation-chloride cotransporters. Previously we reported that the K(+)/Cl(-) cotransporter KCC2, a neuron-specific chloride extruder conferring GABAs more typical inhibitory effects, is expressed exclusively in vasoactive intestinal peptide (VIP) and gastrin-releasing peptide (GRP) neurons in the SCN. Here we report that the K(+)/Cl(-) cotransporter isoforms KCC4 and KCC3 are expressed solely in vasopressin (VP) neurons in the rat SCN whereas KCC1 is expressed in VIP neurons, similar to KCC2. NKCC1 is expressed in VIP, GRP and VP neurons in the SCN as is WNK3, a chloride-sensitive neuron-specific with no serine-threonine kinase which modulates intracellular chloride concentration via opposing actions on NKCC and KCC cotransporters. The heterogeneous distribution of cation-chloride cotransporters in the SCN suggests that Cl(-) levels are differentially regulated within VIP/GRP and VP neurons. We suggest that GABAs excitatory action is more likely to be evoked in VP neurons that express KCC4.

Ultrastructural immunolocalization of rat oxytocin-neurophysin in transgenic mice expressing the rat oxytocin gene

Cell-specific expression of the rat oxytocin (OT)-neurophysin transgene in mice was achieved using a construct containing both OT and vasopressin genes (Young III, W.S., Reynolds, K., Shepard, E.A., Gainer, H. and Castel, M., Cell-specific expression of the rat oxytocin gene in transgenic mice, J. Neuroendocrinol., 2 (1990) 1-9). The present study describes the distribution of the protein products of these genes in various regions of the cell, and determines whether the transgenic rat and endogenous mouse OT-neurophysins are colocalized within the same neurosecretory granules. Two monoclonal antibodies against OT-neurophysins were used: PS38 which can react with both rat and mouse OT-neurophysin (pan-specific), and PS67 which is specific for rat OT-neurophysin only. Various approaches to double immunolabeling at the ultrastructural level were employed; these included: (1) pre-embedding immunoperoxidase followed by post-embedding immunogold; (2) post-embedding immunolabeling using gold particles of different sizes; and (3) labeling of consecutive ultrathin sections with different antibodies. Results from each of these approaches showed that both in the transgenic mouse and in the rat (used as control), immunocytochemical labeling for both PS38 and PS67 occurred in the same OT-ergic neurosecretory granules. In the control mouse, only PS38 elicited labeling. Hence, it may be concluded that the protein and peptide products of the transgene and the endogenous gene for OT-neurophysin are being processed similarly in the cell and finally concentrated together in the same neurosecretory granules.