JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 15369-376 (1990)

Synaptic Circuitry Identified by Intracellular Labeling With Horseradish Peroxidase JAMES E. HAMOS Department of Neurology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655

KEY WORDS

Lateral geniculate nucleus, Electron microscopy, HRP, Structure/function correlations, Identified neurons

ABSTRACT

During the past two decades new techniques have been developed to directly test the dogma that neuronal structure is correlated with neuronal function. In the earliest experiments, Procion yellow was injected into neurons after they had been characterized physiologically; these neurons were then viewed through the light microscope. Recent advances in the method generally employ horseradish peroxidase a s the dye which is injected since i t diffuses quite readily throughout the injected neuron and produces a stable reaction product for both light and electron microscopic studies. This review explores the utility of examining synaptic circuitry after physiologically recording from axons or neurons and then injecting horseradish peroxidase into them. As a model system, we studied the cat lateral geniculate nucleus and investigated, at the electron microscopic level, the synaptic contribution to this nucleus from retinogeniculate axons, from interneurons, and from the axon collaterals of neurons that project to visual cortex.

INTRODUCTION At a n ultrastructural level, injection with Procion yellow was of little practical benefit as the dye does not One of the key tenets of neuroanatomy is that neuproduce a n electron-dense product that would distinronal structure is intricately related to neuronal function. At the light microscopic level, therefore, anato- guish the labeled element from other components of the mists have described the morphological features of neuropil in conventional electron micrographs (howneurons in extensive detail, believing that classes or ever, see Purves and McMahan, 1972; Kellerth, 1973). types of neurons with distinct morphological properties For the purposes of studies of synaptic organization of will be ultimately associated with neuronal classes or physiologically identified neurons, two new compounds types classified according to physiological criteria. This gained appeal. Initially, cobaltous chloride was emconcept has been carried further to the electron micro- ployed as a n intracellular stain since it forms a n elecscopic level by investigators who have described ultra- tron-dense precipitate when processed with ammonium structural features of synaptic terminals deriving from sulfide (Gillette and Pomeranz, 1973; Pitman et al., afferent sources as reviewed in the first three chapters 1972; Szekely, 1976). However, this method is limited of this volume. Again, the belief is that by “mapping” by the toxicity of cobalt to neurons and by the tendency the input(s) from known sources onto specific locations of cobalt to block microelectrodes, thereby hampering of neurons, one will be able to discern the neurons’ one’s ability to record from neurons. Ultimately, intracellular injection with horseradish integrative and functional properties. peroxidase (HRP), together with appropriate hisDirect tests for these dogmas have become available only during the past two decades with the advent of tochemistry, became the method of choice for joint techniques in which both the morphological and phys- studies of structure and function. The earliest HRP iological attributes of neurons can be assessed in the studies produced light microscopic views of neurons same experiment. The earliest studies along these lines with as much and more detail t h a n Golgi studies or Procion yellow injections (Cullheim and involved the intracellular injection of Procion dyes analogous Kellerth, 1976; Jankowska et al., 1976; Kitai et al., :most commonly, Procion yellow) into neurons whose physiological properties had been determined (Remler 1976; reviewed in Bishop and King, 1982). Furthermore, when HRP is reacted through a histochemical ?t al., 1968; Stretton and Kravitz, 1968; for review, see protocol in which 3,3’-diaminobenzidine is used as a Kater and Nicholson, 1973); after appropriate histologsubstrate, the resultant reaction product becomes elec,cal processing, each neuron could then be viewed ;hrough a light microscope arranged with fluorescence tron dense. This facilitates electron microscopic studies nicroscopy. Since the same micropipette was used for of a n injected neuron since portions of the labeled neu30th physiological recording and intracellular injec;ion, these studies provided a method to study directly 30th the anatomical and functional features of single Received May 8, 1989; accepted in revised form August 8, 1989. neurons. Occasionally, the dye also diffused into the Address reprint requests to James E . Hamos, Department of Neurology, Uniixon of the injected neuron and permitted visualization versity of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, MA 01655. If that neuron’s local axon collaterals.

D 1990 WILEY-LISS, INC.

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For the purposes of visual field recording, the cat was placed in a stereotactic device and prepared for physiological testing. Initially, the pupils were dilated and nictitating membranes were retracted with topical applications of atropine sulphate and phenylephrine. Contact lenses were fitted on the corneas to focus the retinae on visual stimuli presented during the recording sessions. A hydraulically sealed chamber was placed over a craniotomy positioned above the stereotactic location of the LGN. Stimulating electrodes were inserted across the optic chiasm to enable orthodromic activation of input to LGN neurons or to directly activate optic tract axons. Neuronal elements studied included retinogeniculate axons, LGN interneurons, and the axon collaterals of LGN projection neurons. The activity of these elements were initially recorded through micropipettes that were filled with 3% HRP and 0.3 M KC1 in Tris buffer; the pipettes had been beveled to a final impedance of 120 megohms a t 100 Hz, which corresponds to a tip diameter of 0.2-0.5 pm. The sites for these recordings were either within the A laminae of the LGN for interneurons and projection neurons or within that portion of the optic tract immediately below the C laminae of the LGN for retinogeniculate axons. Extracellular responses of neurons or axons were characterized and the appropriate physiological class of the retinal or LGN neuron was determined by using standard criteMATERIALS AND METHODS ria (Cleland et al., 1971; Hochstein and Shapley, 1976; Roughly 40 years ago, it became practical to pull Hoffman e t al., 1972). We examined their responses to glass capillary tubes to a submicron diameter that, in a battery of tests including latency of response to optic turn, made i t possible to use the resulting micropipette chiasm shock, ocular dominance, receptive field center to impale neurons in anesthetized animals. If the pi- size, responses to modulated sine-wave gratings, and pette was filled with a suitable electrolyte (typically, tonic or phasic nature of the response to stimulation of KCl), i t was possible to record the intracellular re- the receptive field center. sponses of the neuron to a number of physiologically In these experiments,the neuron or axon was impaled relevant stimuli. Consequently, changing the solution after its extracellular responses to visual stimulation to one containing 3-6% HRP in 0.3-0.5 M KC1 did not were evaluated; after impalement, it was quickly posalter the recording properties of the electrode. More- sible to assess that the element was identical to that over, after physiological properties have been deter- which had been previously recorded, and only then was mined, the HRP could be iontophoresed into the neuron HRP iontophoresis initiated. For other studies, i t may by using brief depolarizing current. be appropriate to characterize a range of additional The results presented below derive from combined intracellular responses prior to iontophoresis. In any anatomical and physiological experiments performed case, iontophoresis was achieved by the direct adminin the laboratory of S.M. Sherman by using a protocol istration of brief depolarizing pulses through the electhat has been widely documented in previous publica- trode by using low current levels (2-10 nA). tions (Friedlander et al., 1981; Humphrey et al., After any single neuron or axon was injected, the 1985a,b; Sur and Sherman, 1982). The majority of this HRP-filled micropipette was withdrawn from the brain work was devoted to studies of the circuitry of the cat and repositioned to another region of the LGN; this lateral geniculate nucleus (LGN) and the role of this assured that there would be no overlap of stained strucnucleus in visual processing. All experiments were per- tures when viewed through either the light or electron formed on anesthetized and paralyzed cats which were microscope. The cat was maintained on anesthetics for artificially respirated. Anesthetics included halothane a n additional 10 hours after the final HRP injection in during the initial surgical procedure to expose the sur- order to allow the enzyme to diffuse throughout the face of the brain followed by a combination of NzOz and stained element’s axonal terminal field. At the complebarbiturates. Appropriate levels of anesthesia were tion of this waiting period, the cat was deeply anesthemaintained by monitoring the cat’s core temperature, tized with barbiturates and perfused transcardially expired CO,, and heart rate. While this review prima- with a n appropriate fixative for electron microscopic rily relates to circuitry of the LGN of adult cats, this studies (e.g., 1% paraformaldehyde and 2% glutaraldephysiological protocol, and subsequent HRP ionto- hyde in 0.15 M phosphate buffer with calcium chlophoresis, has also been adapted to studies of developing ride). After perfusion, the brain was removed and resystems (Sur et al., 1984) andior of experimental mod- frigerated overnight in 5% dextrose in phosphate buffer to rinse out the fixative. els (e.g., monocular deprivation; Sur et al., 1982).

ron are easily distinguished from other structures in the neuropil due to the injected H R P s concentrated electron density. Intracellular injection with HRP has a n additional benefit in that, under appropriate conditions, the dye easily diffuses within axons. Therefore, after injecting a neuron with HRP, one often fills the neuron’s entire local axonal arborization. Moreover, it is possible to record, through HRP-filled micropipettes, the electrophysiological properties of large and medium-sized axons whose cell bodies of origin are far removed from the site of impalement. With subsequent intracellular injection of the dye into the axon, the complete terminal field of the axon may be revealed. This review highlights the use of HRP to fill axonal fields and the ensuing electron microscopic identification of HRP-labeled, electron-dense synaptic terminals. The power of this technique lies in a n ability to directly correlate physiological properties with light and electron microscopic analyses of synaptic contacts of a labeled neuron or axon. When combined with three-dimensional reconstruction of neurons postsynaptic to electron-dense synaptic terminals, it is also possible to decipher elements of neuronal networks involving axonal terminal fields, the axon collaterals of projection neurons, or dendritic and axonal circuitry related to interneurons.

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In each experiment, a block of tissue containing the injected element and other adjacent regions of the LGN was sectioned at 50 pm on a Vibratome. The sections were processed for HRP histochemistry by using 3,3'diaminobenzidine as a substrate. An additional treatment with 1% cobaltous chloride intensified the resultant reaction product. We determined which sections contained the injected neuron or axon by examining, through the light microscope, sections mounted on slides in a phosphate buffer solution. Portions of the LGN containing the label were then osmicated, dehydrated, and embedded in plastic resin between two plastic sheets. After the resin had hardened, we made drawings of the injected elements through 1 0 0 ~oil Fig. 1. Electron micrograph of a labeled terminal, in the LGN, from immersion objectives and a drawing tube attached to a retinal Y-cell whose axon had been injected with HRP in the optic the light microscope. tract. This example of a labeled terminal is representative of the For electron microscopy, blocks of tissue were thin dense congregation of reaction product that accumulates within insectioned on a n ultramicrotome. A key feature for the jected neurons and axons. significantly, after HRP injection it is posstudies described below was our intent to reconstruct sible to determine sites at which labeled terminals formed synapses (arrowhead) onto unlabeled dendrites (D) by the accumulation of synportions of unlabeled neurons postsynaptic to HRP-la- aptic vesicles in the labeled terminal and by the presence of a postsynbeled structures. Therefore, we serially thin-sectioned aptic density in the dendrite. Scale bar, 1.0 ym. blocks of tissue containing the label and mounted the sections on consecutively maintained, Formvar-coated slot grids. For the findings presented in this review, we produced series of between 200 and 1,200 thin sections brane, and by synaptic thickenings within the postsynof different HRP-labeled neurons and axons. All sec- aptic structure. Of great importance was that all elections were further stained with lead citrate and uranyl tron-dense profiles within a single block prepared for acetate. electron microscopy derived from a single injected neuThin sections were viewed on a JEOL 100B electron ron or axon. This allowed for quantitative comparisons microscope at a magnification of x 3,250. At this mag- (see below) between labeled terminals and other, unlanification, electron dense synaptic terminals were eas- beled terminals whose morphological classifications ily identified and we produced micrographs with a final were easily determined by using the standard nomenmagnification of x 8,500. After labeled terminals were clature for the synaptic terminals within the cat LGN located, we identified the dendritic processes of neu- (Guillery, 1969). rons postsynaptic to the labeled profiles. Additional micrographs were made, throughout the series of serial Retinogeniculate axons thin sections, of the unlabeled dendrites so that they Morphological differences, as seen at the light microcould be reconstructed back to their cell body of origin. The procedures for reconstruction have been previously scope, have been previously identified in the terminal documented (Hamos et al., 1987) and form the basis for arborizations of the two predominant physiological vaa three-dimensional analysis of neurons innervated by rieties of retinal axons, both in the adult (Bowling and single, HRP-labeled elements. Moreover, utilizing in- Michael, 1984; Sur and Sherman, 1982) and in develformation related to the size, dendritic morphology, oping cat (Sur et al., 1984). At a n ultrastructural level, and circuitry of the reconstructed neurons, we have we have extensively analyzed retinogeniculate cirhypothesized their membership in a particular physio- cuitry devoted to the X-pathway and reconstructed postsynaptic targets of a single axon from a retinal logical and/or morphological class of LGN neuron. X-cell (Hamos et al., 1987). For the present review, I RESULTS will compare ultrastructural features of this and other The task of identifying portions of injected neurons axons from retinal X-cells with recent data gleaned or axons through the electron microscope was quite from the axon of a retinal Y-cell that was injected, in simple due to the intense electron density of the diami- the optic tract, with HRP and subsequently processed nobenzidine-HRP reaction product, even after thin sec- for electron microscopy (Fig. 2). Labeled terminals from the axons of both retinal Xtions were stained by lead citrate and uranyl acetate (e.g., Fig. 1). Within the labeled profiles, the reaction and Y -cells display morphological features identical to product diffused throughout the cytoplasm, obscuring LGN terminals that have been previously identified as intracellular components that were not membrane retinal (RLP) terminals by utilizing a variety of other bound, such as neurofilaments and presynaptic densi- techniques (Guillery, 1969; Szentagothai, 1973). That ties, while not penetrating other organelles, such as is, labeled terminals a) contained round synaptic vesimitochondria or synaptic vesicles. Sites where labeled cles, b) were among the larger profiles within the LGN, terminals made synaptic contacts (arrowhead, Fig. 1) and c) also contained pale mitochondria. Beyond this, were identified by a n aggregation of synaptic vesicles however, our analysis revealed features of synaptic cirwithin the HRP-filled terminal, by the apposition of cuitry that were distinctly different between physiologthe presynaptic membrane with a postsynaptic mem- ically different axons. Such distinctions could not have

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Fig. 2. Retinal Y-cell arbor. a: Light micrograph of a portion of the Y-cell’s arborization in the LGN. Arrow indicates region of swellings along the axon which resolved as synaptic terminals shown in b at the electron microscopic level. Scale bar, 20 pm. b: Electron micrograph of a labeled terminal forming a synapse (arrowhead) onto a largediameter dendrite (D). The same dendrite also received input from F-terminals (F)although their synapses were generally formed apart

from input from retinal terminals; this is very different than the participation of terminals from retinal X-cells in synaptic triads (see text). Note the density of reaction product in this labeled terminal is greater than the density within the terminal illustrated in Figure 1 although both terminals derive from the same injected axon; this likely represents different rates of diffusion of HRP through the axon’s arbor (see Discussion). Scale bar, 1.0 km.

been revealed without the benefit of intracellularly labeling physiologically identified axons. Labeled terminals from the axons of X-retinal cells were found to be associated strongly with a distinctive feature of LGN synaptic organization, the synaptic triad, in which a retinal terminal synapses onto a postsynaptic dendrite as well as onto another type of synaptic terminal containing flattened synaptic vesicles (F-terminal); the F-terminal, whose source is the dendrite of an LGN interneuron (Hamos et al., 1985; Montero, 19861, also synapses onto the same dendrite. For the previously reported retinal X-cell axon, 222 of 260 synapses from labeled terminals participated in such synaptic triads. Conversely, retinal Y-cell axons do not extensively enter into such synaptic triads, as evidenced by our sampling, where only 15 of 203 labeled terminals from one of these axons provided input to both dendrites and postsynaptic F terminals. The degree of involvement of retinal terminals in synaptic triads was highly correlated with the complexity of the synaptic neuropil related to geniculate Xand Y-cells. “Encapsulated synaptic zones” (Guillery, 1969; also termed a “glomerulus” by Famiglietti and Peters, 1972; Peters and Palay, 1966; Szentagothai, 1973) were regions in which labeled terminals from injected retinal X-cell axons formed extensive synaptic contacts, together with numerous F-terminals and RSD (round synaptic vesicles, small profiles, and dark mitochondria)-terminals, onto clusters of dendritic apDendapes from neniculate X-cells (see Figs. 7 and 10 in

Hamos et al., 1987). Conversely, terminals from an HRP-filled retinal Y-cell axon typically formed simple, isolated synaptic contacts onto dendritic shafts of geniculate Y-cells (Fig. 2). In several instances the Y-cell axon participated in a distinct variety of synaptic triad that did not utilize F-terminals as an intermediary; rather, this type of triad included a presynaptic dendritic shaft (Famiglietti, 1970; Hamos et al., 1985; Rapisardi and Miles, 1984). Such presynaptic dendrites are rarely found in the LGN. We reconstructed the postsynaptic targets of an injected axon from a retinal X-axon (Hamos et al., 1987) in a series of roughly 1,200 consecutive thin sections. In the region of the LGN containing the terminal arbor, we injected only a single axon in order to address several issues that could not be studied by other means. For example, we sought to determine how many neurons within the physical domain of the injected axon’s arbor were contacted by labeled terminals and found that only four of 43 neurons were repetitively contacted by this axon (Fig. 4 in Hamos et al., 19871, indicating a high degree of selectivity in circuitry for retinogeniculate axons. For these four neurons, we sought to determine what percent of each neuron’s total retinal input (synapses from unlabeled RLP terminals plus those from labeled terminals) derive from the single injected axon and found that this percentage varied; for one neuron all of its retinal input came from labeled terminals while for the other three neurons there were varying percentages (610, 3396, and 49%) of synapses

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Fig. 3. Circuitry of an interneuron. a and e: Electron micrographs of labeled terminals from a n injected interneuron that formed symmetrical synapses (arrows) onto the dendritic appendages iAppt of a presumed geniculate X-cell. Note that the same appendages also received converging input from unlabeled F terminals. In addition, the labeled terminals participated in synaptic triads k g . , c! by receiving input from RLP terminals that formed synapses on the appendages in other planes of section. Scale bar, 1.0 pm. b Three-dimensional reconstruction from serial thin sections of labeled dendritic terminals of

the interneuron (stippled) that derived from a single dendritic stem (D), and the unlabeled postsynaptic appendages (open) that derived from a single medium-sized dendrite (D). The interneuron formed nine synapses (arrows) onto this dendritic segment of the target neuron, which also received a n additional 40 synapses from unlabeled F-terminals, nine synapses from RLP-terminals, and three synapses from RSD-terminals. Scale bar, 1.0 pm. Reproduced from Hamos et al. 11985).

from labeled terminals relative to the total retinal input that was reconstructed. This later finding suggested diverse integrative properties of neurons in the LGN.

Electron microscopic data of HRP-labeled interneurons provide further conclusions regarding the Xpathway in the LGN. Within the confines of a single ‘cglomerulus~’ we reconstructed multiple synaptic contacts from an HRP-filled interneuron, whose physiological properties were that of an X-cell, onto the dendritic appendages of a postsynaptic target neuron (Fig. 3 ) . In all cases, the interneuron provided only a subset of the synapses from F-terminals within a glomerulus. For example, in reconstructing the three glomeruli that comprised the synaptic interrelationships between an injected interneuron, unlabeled retinal terminals, and a neuron presumed to be an X-cell, we found nine synapses from labeled terminals onto the X-cell as compared to 40 synapses from unlabeled F-terminals (Fig. 3). These findings suggest that convergence from inhibitory interneurons is a key feature of local circuitry devoted to the X-pathway in the cat LGN. Additional data from the injected interneuron suggested that cells of this class may operate in two states. First, we found evidence that the interneu~onprovided outputs to the geniculate neuropil via two separate mechanisms, a predominant source being dendritic terminals and a less prominent source being a locally ramifying axon (Hamos et al., 1985). Based on morphometric measurements of the interneuron’s dendrites and a passive electrical model of postsynaptic membranes, we calculated that regions of the cell emitting dendritic terminals were electrically isolated from the cell body and, thus, from its axon. This suggested that clusters of dendritic terminals operated as local microcircuits while the axon was activated only when a volley of inputs summed a t the cell body. Second, we found that retinal input to the interneuron was focused to two different regions. Once again, a predominant retinal input reached the dendritic terminals, where many participated in synaptic triads; a few additional retinal

LGN interneurons Definitive evidence for interneurons in the cat LGN was difficult to achieve for many years, although it had long been hypothesized that neurons with a distinctive morphological appearance (“Class 3” neurons; Guillery, 1966) were the source of many F-terminals in the geniculate neuropif (Famiglietti and Peters, 1972). Furthermore, in the early years of the intracellular HRP technique, efforts to inject HRP into neurons with these morphological features failed, leading to the postulate that either a) interneurons did not display conventional physiological responses and so their responses would have been ignored in physiological recordings, or b) that the size or membrane properties of interneurons were somehow different and so they could not be injected. However, in recent years, we successfully injected HRP into neurons whose morphological appearances were identical to those of Class 3 neurons, and produced conclusive evidence, a t the electron microscopic level, that interneurons provide dendritic terminals within synaptic triads of the X-pathway (Hamos et al., 1985; supported by Golgi-EM studies of Montero, 1986); these dendritic terminals resembled one variety of F-terminal and formed only symmetrical synapses. In a more recent study, performed a t the light microscopic level, all interneurons that were successfully injected with HRP and recovered displayed physiological responses which were indistinguishable from the responses of relay X-cells (Sherman and Friedlander, 1988). At this time, however, there is no evidence for genicufate interneurons devoted to local circuitry in the Y-pathway.

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ployed with great success in a number of other regions of the central nervous system including the spinal cord (Conradi et al., 1983; Cullheim et al., 1977; Fyffe and Light, 1984; Gobel and Falls, 1979; Gobel et al., 1980; Lagerback et al., 1981; Maxwell et al., 1983; Ralston et al., 1984; Rastad et al., 1977; Semba et al., 1984,1985), the cuneate nucleus (Fyffe et al., 19861, the dorsal cochlear nucleus (Smith and Rhode, 19851,the substantia nigra (Chang et al., 1984), and the cerebral cortex (Freund et al., 1985a,b; Kisvarday et al., 1986, 1987; McGuire et al., 1984).

Fig. 4. Electron micrograph of a labeled terminal (arrowheadf, in the LGN, from the axon collateral of a relay X-cell that projected its major axon to the visual cortex. In the LGN, this neuron’s synaptic terminals resembled RSD-terminals and formed asymmetrical synapses on medium- and small-sized dendrites (D). Scale bar, 1.0 pm.

terminals (only four in our prior sample) directly synapsed on the cell body. This synaptic pattern is very different from the retinal innervation to geniculate relay cells whose retinal input is focused generally onto the region of the first 100 pm of primary and secondary dendrites {Wilson et al., 1984).

Axon collaterals While it is impossible to predict when a geniculate relay cell that is recorded from and injected with HRP will contribute a local axon collateral to the geniculate neuropil, we have succeeded in recovering several such axons deriving from geniculate X- and Y-cells whose major axon projected to visual cortex and have uncovered details regarding the contribution of the axonal collateral to local LGN synaptic organization. For the most part, synaptic terminals from these collaterals displayed ultrastructural features of RSD-terminals and provided asymmetrical synapses to medium- and small-sized dendrites (Fig. 4; see also, Wilson et al., 1984). However, in a single case, the terminals of an axon from an X-cell formed symmetrical synaptic contacts, indicative of one variety of F-terminal, within a complex glomeruli; this suggested that a t least some relay cells participate in complex synaptic arrangements within the confines of the LGN itself (Van Horn et al., 1986).

DISCUSSION The technique of intracellular injection of neurons with HRP has become a logical successor to the Golgi method as the means of revealing circuitry related to single axonal and dendritic elements in a complex neuropil (see Somogyi, this volume). We used the HRP protocol in the LGN of the cat to discern different synaptic relationships of axons from retinal X- and Y-cells and to provide conclusive evidence for a role of interneurons and local axon collaterals within geniculate synaptic organization. A similar strategy has been em-

Limitations of the HRP technique While we have employed the combination of electron microscopy with intracellular HRP injection to great benefit, we recognize several limitations to the technique. First, and foremost, one cannot record from and intracellularly impale all types of neurons and axons in the central nervous system. Using fine-tipped micropipettes, filled with KC1 alone or with a low percentage solution of HRP, in physiological recording sessions, selection biases seem to prevent the isolation of all varieties of neuronal elements. These biases may be related to size, specific physiological properties of the element, or some unknown cause. As described previously, such biases hampered our ability to identify interneurons in the lateral geniculate nucleus for many years; only recently have enough interneurons been recovered to reveal significant insights into the relationship of structure and function in this class of cell (Hamos et al., 1985; Sherman and Friedlander, 1988). Extremely fine axons are also difficult to impale intracellularly. Therefore, while X- and Y-retinogeniculate axons are easily recovered, we have failed a t attempts to inject the axons of retinal ganglion cells devoted to the W-pathway or corticogeniculate axons which provide a feedback mechanism from cells in layer VI of primary visual cortex to the lateral geniculate nucleus. Other techniques should be employed to establish the synaptic connections of such fine axons (e.g., see papers by Ralston and by Cucchiaro and Uhlrich in this volume). Second, one cannot be assured that HRP has diffused throughout the entire neuron or axon that was injected. Much of this limitation seems to be related to the overall size of the terminal arborization. For example, the terminals of axons from retinal X-cells, whose terminal arbors average 140 pm in width (Sur and Sherman, 19821, are uniformly labeled after HRP has been injected into the parent axon (Hamos et al., 1987). Conversely, the terminals of axons from retinal Y-cells, whose terminal arbors average over 290 pm in width, have different electron densities due to a variable amount of HRP (compare density of the label in Figs. 1 and 2). Such variability in the amount of HRP staining has also been noted in other studies where a large axonal terminal field has been recovered (e.g., Fig. 4 in McGuire et al., 1984). For such widely distributed axons, it is very likely that HRP has not diffused into all of the axon’s synaptic terminals. Third, the technique is labor-intensive and time-consuming. In any single experiment, there is typically a

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low yield in the number of neurons andior axons that have been recorded from and impaled. It is generally possible to iontophorese HRP into only a few of the impaled cells and, finally, some subset of the labeled neurons is recovered for light and electron microscopy. Careful orientation is required to ensure that stained terminals noted in the light microscope can then be sectioned and identified in t,he electron microscope (Fig. 2). Finally, studies in which one attempts to reconstruct the postsynaptic targetis) of an HRP-labeled element (e.g., Hamos et al., 1987) require a great deal of time to complete. It is possible t.o increase the number of elements stained in a single experiment by using a variant of the intracellular HRP labeling method, i.e., bulk filling axons within a defined tract with HRP. This method has been successfully used in a variety of regions (see, for example, Beattie et al., 1978: Falls, 1984; Mawe et al., 1984; Robson and Mason, 1979) but suffers in that the direct correlation between morphology and physiological properties is lost.

Benefits of the H R P technique The major benefit of this method is that it allows for the direct correlation of structure with function. Therefore, in our studies of the terminals of retinogeniculate axons, whose morphological features are the same in most respects (i.e., the RLP terminal variety), we noted significantly different synaptic relationships of terminals among the two major physiological pathways. The terminals of retinal Y-cells largely formed simple synaptic inputs to the dendrites of geniculate Y-cells whereas the terminals of retinal X-cells participated in complex synaptic circuits, synaptic triads, and glomeruli with local inhibitory neurons and geniculate X-cells that project their axons to visual cortex. These synaptic differences must reflect differences in the manner in which retinal inputs are integrated by geniculate neurons. Additional data, derived from a n intracellularly labeled int,erneuron with the physiological properties of an X-cell, confirmed and extended our knowledge of these mechanisms. Intracellular staining with HRP and subsequent recovery for electron microscopy extend the variety of data attainable by other methods. In comparison with the Golgi method (see Somogyi, this volume), intracellular labeling greatly increases the amount of a neuron that is visualized, suggesting that impregnation with the Golgi method is incomplete. Moreover, one may recover injected axons and axon collaterals, neuronal elements that are rarely recovered when the Golgi method is applied to adult tissue. Finally, when combined with extensive reconstruction of both the stained element and its unlabeled postsynaptic targets, the method provides a wealth of additional data. Such data depend on the ability to inject only a single clement and then compare its circuitry to that of the larger population of t.erminals. Thus, we identified varying, but limited, amounts of convergence from retinal X-axons onto geniculate cells after injecting a single retinogeniculate axon; the axon provided 100% of the input. t,o one cell and varying amounts, ranging from 6%to 49%, of the total retinal input to three other cells (Hamos et al., 1987). Our

preliminary data from an interneuron indicated that i converges with other inhibitory inputs onto the samc terminal dendritic segments of geniculate X-cell: (Hamos et al., 1985). In summary, the HRP met.hod provides a powerfu tool in the arsenal of anatomists and physiologists foi direct correlations between structure and function When applied at the electron microscopic level, thit technique provides information related to the synaptic organization of a neuropil, the synaptic region that de. fines the physiological properties of its neurons.

ACKNOWLEDGMENTS For these studies, I a m indebted to S.M.Sherman and colleagues in his laboratory, including S.A. Bloomfield, A.L. Humphrey, D. Raczkowski, and D.J. Uhlrich, who provided much of the expertise in physiological techniques required for this work. I also gratefully acknowledge t.he help of S.C. Van Horn, who collaborated with me on the electron microscopic studies.

REFERENCES Beattie. M.S., Bresnahan. . (1985h) Innervation nf cat visual areas 17 and 18 by physiologically ident.ified Xand Y-tvpe thalamic afferents. I. Arborization oattcrns and nimn-

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