Herpes Simplex Virus as a Transneuronal Tracer ROBERT B. NORGREN JR a , * AND MICHAEL N. LEHMAN b a Department of Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE 68198, U.S.A. b Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati College of Medicine, Cincinnati, OH 45267, U.S.A. NORGREN, R.B. AND M.N. LEHMAN. Herpes simplex virus as a transneuronal tracer. NEUROSCI BIOBEHAV REV 22 (6) 695– 708, 1998. Determining the connections of neural systems is critical for determining how they function. In this review, we focus on the use of HSV-1 and HSV-2 as transneuronal tracers. Using HSV to examine neural circuits is technically simple. HSV is injected into the area of interest, and after several days, the animals are perfused and processed for immunohistochemistry with antibodies to HSV proteins. Variables which influence HSV infection include species of host, age of host, titre of virus, strain of virus and phenotype of infected cell. The choice of strain of HSV is critically important. Several strains of HSV-1 and HSV-2 have been utilized for purposes of transneuronal tract-tracing. HSV has been used successfully to study neuronal circuitry in a variety of different neuroanatomical systems including the somatosensory, olfactory, visual, motor, autonomic and limbic systems.  1998 Elsevier Science Ltd. All rights reserved Herpes simplex virus HSV Tracer Transneuronal Somatosensory Olfactory Visual Autonomic Limbic Retina Hypothalamus Amygdala TRACING TRANSNEURONAL PATHWAYS DETERMINING THE connections of neural systems is critical for determining how they function. Typically, this is accomplished by injecting a nucleus with a tracer substance. If this substance is transported in the retrograde direction, all neurons which project to the injected nucleus will be labeled. One may follow the circuit backward by injecting the tracer in the afferent nuclei of the first nucleus injected. This process can be continued ad infinitum. If an anterograde tracer is used, neural circuits may be followed in the opposite direction. Transganglionic transport involves transfer of the tracer from the periphery to the central nervous system. Since a pseudounipolar neuron is involved, this method of transport can be considered to involve both retrograde (to the cell body) and anterograde (to the axon terminal in the central nervous system) transport. A considerable amount of information regarding neural circuitry has been obtained by neuroanatomists using these techniques. However, there are major limitations to this approach. A schematic diagram (Fig. 1) depicts two simple circuits and can be used to illustrate the problems with conventional tracers. In circuit A, neuron A1 receives input from neuron A2. Neuron A2 receives input from neuron A3. The same relationships obtain for circuit B. If these two circuits were completely separated in space, then sequential injections with conventional tracers would work well. However, neuron A2 and B2 are immediately adjacent to each other in the same nucleus. Thus, injecting this nucleus with a retrograde tracer would label both neuron A3 and neuron B3, even though only neuron A3 is part of the A circuit. Therefore, specific information regarding neural circuitry would be lost. This may be addressed by making smaller injections, but there are physical limitations on how small and how accurate injections can be made. In order to understand neural circuitry at fine resolution, the ideal solution would be to inject a tracer which is transported not only to neurons which project to the nucleus one is injecting, but also to the third-order neurons which project to the neurons which are afferent to the injection site. Thus, a single injection of a nucleus containing neuron A1 would result in labeling of neuron A2 and neuron A3, but not neuron B3. In order for this to occur, the transport of the tracer must be transneuronal. A variety of con- ventional tracers have been used in this way including: tritiated amino acids (32,72,163), wheat germ agglutinin (38,46,64,87,132,149), fragment C of tetanus toxin (16,41,97), rhodamine isothiocyanate (108,109), neuro- biotin (63,92) and DiI (14). However, there are limitations to the use of these molecules as they are diluted at each synapse (80,141). One way to overcome this problem is to use a tracer which multiplies itself in every new neuron to which it is transported, e.g. a virus (11,20,80,133,141,153). A variety of neurotropic viruses have been shown to be useful as transneuronal tracers. These include rabies (4,152), vesicular stomatitis virus (91), pseudorabies type 1 (PRV) (65,101) (Card; Enquist; Loewy; Miselis, this issue) and herpes simplex virus, type 1 (HSV-1) and herpes simplex virus, type 2 (HSV-2) (5,6,10–12,28,35,48,62,74,80,83), (88–90,95,105,110,111, 116,117,119,144,145,148,151,154,155,161,164,166,167). Neuroscience and Biobehavioral Reviews, Vol. 22, No. 6, pp. 695–708, 1998  1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0149-7634/98 $32.00 + .00 PII: S0149-7634(98)00008-6 Pergamon 695 * To whom requests for reprints should be addressed.